QTLs associated with and methods for identifying shatter resistance in canola

FIELD OF THE INVENTION

The present invention relates generally to plant molecular biology. More specifically, it relates to quantitative trait loci (QTLs) associated with resistance or tolerance to pod shatter in Brassica, and use of those QTLs to identify such resistance or tolerance in Brassica.

BACKGROUND OF THE INVENTION

Brassica napus (commonly referred to as canola or oilseed rape), which is grown in temperate climates of the northern and southern hemispheres, is an important cultivated oilseed crop species. While herbicide resistance has provided enhanced crop value, B. napus remains vulnerable to siliqua or pod shatter, resulting in significant seed loss, especially under adverse weather and harvest conditions. In crops with dry, dehiscent fruits, such as B. napus, the siliques or pods naturally release their seeds through a process called fruit dehiscence. When this process occurs prematurely, such as during adverse weather conditions (i.e., wind storm), seed recovery is reduced. This is especially problematic in crops where the oil from the seeds is desired. B. napus yield losses due to shatter fall within the range of 10%-25%, with increased losses observed as much as 50% when adverse climate conditions delay harvesting. Shatter can also result in the growth of volunteer plants or weeds.

Many plant species, including B. napus, disperse seed through the natural process of fruit dehiscence. In these species, siliques or pods are formed by two carpels that are separated by a thin replum. The dehiscence zone (DZ) is where the valve (fruit wall) margin connects to the replum. As the pod matures late in fruit development, the valve margin detaches from the replum, leading to seed dispersal. The DZ demarcates the precise location where the valves detach.

Several factors have been described to contribute to siliqua shatter resistance, including the morphology, anatomy and biochemistry of siliqua development and physiology, as well as environmental factors. Assessment of B. napus accessions for shatter resistance identified two resistant lines (Wen et al, 2008, Acta Agronomic Sinica 34: 163-166). Other studies of B. napus indicated limited genetic variation. Brassica rapa vars Yellow Sarson and Brown Sarson showed genetic variation in increased siliqua strength resulting in shatter resistance. Improved resistance to shatter was seen upon introgression of the trait from these Brassica types and B. juncea.

Several genes have been identified with putative roles in shatter resistance, including genes involved in dehiscence zone differentiation and their regulatory genes (see review by Hossain et al., 2012, in Plant Breeding, Dr. Ibrokhim Abdurakhmonov (Ed), ISBN: 978-953-307-932-5, InTech at URL intechopen.com/books/plant-breeding/breeding-brassica-napus-for-shatter-resistance). WO 2012/084742 A1 discloses Brassica plants comprising mutant ALCATRAZ (ALC) genes, ALC nucleic acid sequences and proteins that confer increased pod shatter resistance and reduction or delay of seed shatter, as well as methods for generating and identifying the resistant plants and alleles. US 2012/0023603 A1 discloses plants that comprise at least two IND genes, whereby the plants comprise in their genome either two partial knock-out mutant IND alleles or two partial and two full knock-out mutant IND alleles, and confer reduced shattering while simultaneously maintaining an agronomically acceptable pod threshability. Many other genes with numerous putative functions are described in WO 2012/084742 and US 2012/0023603. It is evident from these disclosures that shatter is controlled by numerous and diverse genetic factors, which are additive and/or interrelated in their effect.

Early methods for evaluating shatter resistance were based on imprecise, subjective visual measurements and manual testing, using field observations, crude mechanical tests and anatomical tests (Hossain et al., 2012, supra). Subsequent mechanical testing methods were developed that demonstrated greater accuracy. Means of measuring the level of resistance to pod shatter tendency are known in the art and include, but are not limited to, the pendulum-based test, cantilever test, manual bending test, microfracture test (MFT), siliqua twisting, ‘Ripping’ method and Random Impact Test (RIT) (See Hossain, 2012, supra for review). U.S. Pat. No. 7,412,880 B2 describes a device and method for screening crop plants, including Brassica, for stalk strength, root lodging, and/or other wind damage resistance traits by selectively applying wind forces to stands of plants in an agricultural environment. Current methods employed to reduce shattering include windrowing (swathing) and spraying desiccants, resulting in increased costs and less flexible farming practices (see Hossain, 2012, supra, for review)

What is needed in the art and industry is a means to identify genes or germplasm conferring resistance to shatter using molecular markers. These markers can then be used to tag the favorable alleles of these genes in segregating populations and then employed to make selection for resistance more effective, and to combine several resistance sources in a single genotype that has a high level of shattering resistance. The present invention provides these and other advantages.

SUMMARY OF THE INVENTION

The present invention provides methods and markers for identifying Quantitative Trait Loci (“QTLs”) associated with resistance to shatter in Brassica.

One aspect of the invention features a method of identifying a Brassica plant or germplasm that exhibits resistance to shatter. The method comprises detecting in the plant or germplasm at least one allele of at least one quantitative trait locus (QTL) that is associated with the shatter resistance, wherein the QTL is localized to a linkage group selected from N1, N3, N4, N6, N7, N9, N13, N14, N15, N18 or N19, wherein each said linkage group comprises at least one marker that is associated with the resistance to shatter with a statistical significance of p≤0.01, wherein the QTL is localized to a chromosomal interval selected from the group consisting of: (a) an interval flanked by and including markers N20003-001-Q001 and N23426-001-Q001 on linkage group N1; (b) an interval flanked by and including markers N05671-1-Q1 and N12643-001-Q001 on linkage group N3; (c) an interval flanked by and including markers N05943-1-Q1 and N88537-001-K001 on linkage group N4; (d) an interval flanked by and including markers N07541-1-Q1 and N14649-001-Q001 on linkage group N6; (e) one or more intervals flanked by and including: (i) markers N23310-001-Q001 and N23409-001-Q001 on linkage group N7, or (ii) markers N07278-1-Q1 and N23417-001-Q001 on linkage group N7; (f) one or more intervals flanked by and including: (i) markers N23119-001-Q001 and N20380-001-Q001 on linkage group N9, or (ii) markers NO5490-1-Q1 and N20834-001-Q001 on linkage group N9; (g) one or more intervals flanked by and including: (i) markers N21144-001-Q001 and N09862-001-Q001 on linkage group N13, or (ii) markers N22903-001-Q001 and N12902-001-Q001 on linkage group N13; (h) one or more intervals flanked by and including: (i) markers N23033-001-Q001 and N22724-001-Q001 on linkage group N14, or (ii) markers N23033-001-Q001 and N22802-001-Q001 on linkage group N14; (i) an interval flanked by and including markers N12785-001-Q001 and N19296-001-Q001 on linkage group N15; (j) one or more intervals flanked by and including: (i) markers N05205-1-Q1 and N22925-001-Q001 on linkage group N18, or (ii) markers N22803-001-Q001 and N18401-001-Q001 on linkage group N18; and (k) an interval flanked by and including markers N05656-1-Q1 and N16006-001-Q001 on linkage group N19; wherein each said linkage group comprises at least one marker that is associated with the shatter resistance with a statistical significance of p≤0.01, thereby identifying the Brassica plant or germplasm that will exhibit shatter resistance. More particularly, the QTL is localized to a chromosomal interval selected from the group consisting of: (a) an interval flanked by and including markers N10336-001-Q001 and N23426-001-Q001 on linkage group N1; (b) one or more intervals flanked by and including (i) markers N88514-001-K001 and N88537-001-K001 on linkage group N4, or (ii) markers N05943-1-Q1 and N06675-1-Q1 on linkage group N4; and (c) one or more intervals flanked by and including (i) markers N001RWT-001-Q001 and N20834-001-Q001 on linkage group N9, or (ii) markers N04807-1-Q1 and N17314-001-Q001 on linkage group N9.

In the method, the marker comprises a polymorphism that identifies the at least one allele of the at least one quantitative trait locus (QTL) as being associated with the shatter resistance, and the detecting comprises identifying the polymorphism. In certain embodiments, the polymorphism is a single nucleotide polymorphism (SNP) or a simple sequence repeat (SSR).

In certain embodiments, the detecting comprises detecting at least one marker selected from the group consisting of: N20003-001-Q001 (SEQ ID NO: 1); N03491-1-Q1 (SEQ ID NO:2); N0017NR-001-Q001 (SEQ ID NO:3); N10336-001-Q001 (SEQ ID NO:4); N23133-001-Q001 (SEQ ID NO:5); N16487-001-Q001 (SEQ ID NO:6); N23426-001-Q001 (SEQ ID NO:7); N05671-1-Q1 (SEQ ID NO:8); N12643-001-Q001 (SEQ ID NO:9); N05943-1-Q1 (SEQ ID NO:10); N06007-1-Q1 (SEQ ID NO: 11); N10105-001-Q001 (SEQ ID NO:12); N08181-1-Q1 (SEQ ID NO:13); N06675-1-Q1 (SEQ ID NO:14); N001KH2-001-Q001 (SEQ ID NO:15); N29313-001-Q001 (SEQ ID NO:16); N88512-001-K001 (SEQ ID NO:17); N88514-001-K001 (SEQ ID NO:18); N88515-001-K001 (SEQ ID NO:19); N88516-001-K001 (SEQ ID NO:20); N88517-001-K001 (SEQ ID NO:21); N88518-001-K001 (SEQ ID NO:22); N88519-001-K001 (SEQ ID NO:23); N88520-001-K001 (SEQ ID NO:24); N88521-001-K001 (SEQ ID NO:25); N001KFE-001-Q001 (SEQ ID NO:26); N88522-001-K001 (SEQ ID NO:27); N88523-001-K001 (SEQ ID NO:28); N88524-001-K001 (SEQ ID NO:29); N88525-001-K001 (SEQ ID NO:30); N88529-001-K001 (SEQ ID NO:31); N88530-001-K001 (SEQ ID NO:32); N88531-001-K001 (SEQ ID NO:33); N88533-001-K001 (SEQ ID NO:34); N88535-001-K001 (SEQ ID NO:35); N88536-001-K001 (SEQ ID NO:36); N88537-001-K001 (SEQ ID NO:37); N07541-1-Q1 (SEQ ID NO:38); N23413-001-Q001 (SEQ ID NO:39); N08344-1-Q1 (SEQ ID NO:40); N23533-001-Q011 (SEQ ID NO:41); N14649-001-Q001 (SEQ ID NO:42); N23310-001-Q001 (SEQ ID NO:43); N10526-001-Q001 (SEQ ID NO:44); N23373-001-Q001 (SEQ ID NO:45); N23353-001-Q001 (SEQ ID NO:46); N23206-001-Q001 (SEQ ID NO:47); N11025-001-Q001 (SEQ ID NO:48); N09969-001-Q001 (SEQ ID NO:49); N09882-001-Q001 (SEQ ID NO:50); N10389-001-Q001 (SEQ ID NO:51); N09940-001-Q001 (SEQ ID NO:52); N23409-001-Q001 (SEQ ID NO:53); N23119-001-Q001 (SEQ ID NO:54); N09861-001-Q001 (SEQ ID NO:55); N04807-1-Q1 (SEQ ID NO:56); N06778-1-Q1 (SEQ ID NO:57); N09897-001-Q001 (SEQ ID NO:58); N10499-001-Q001 (SEQ ID NO:59); N23447-001-Q001 (SEQ ID NO:60); N19834-001-Q001 (SEQ ID NO:61); N23362-001-Q001 (SEQ ID NO:62); N23266-001-Q001 (SEQ ID NO:63); N19862-001-Q001 (SEQ ID NO:64); N22187-001-Q001 (SEQ ID NO:65); N08651-1-Q1 (SEQ ID NO:66); N23296-001-Q001 (SEQ ID NO:67); N17314-001-Q001 (SEQ ID NO:68); N20380-001-Q001 (SEQ ID NO:69); N05490-1-Q1 (SEQ ID NO:70); N18849-001-Q001 (SEQ ID NO:71); N08200-1-Q1 (SEQ ID NO:72); N19827-001-Q001 (SEQ ID NO:73); N001R9W-001-Q001 (SEQ ID NO:74); N08264-1-Q1 (SEQ ID NO:75); N23132-001-Q001 (SEQ ID NO:76); N03615-1-Q1 (SEQ ID NO:77); N001RWT-001-Q001 (SEQ ID NO:78); N08465-1-Q1 (SEQ ID NO:79); N10774-001-Q001 (SEQ ID NO:80); N17035-001-Q001 (SEQ ID NO:81); N20834-001-Q001 (SEQ ID NO:82); N22903-001-Q001 (SEQ ID NO:83); N09920-001-Q001 (SEQ ID NO:84); N22822-001-Q001 (SEQ ID NO:85); N22688-001-Q001 (SEQ ID NO:86); N10074-001-Q001 (SEQ ID NO:87); N10057-001-Q001 (SEQ ID NO:88); N10086-001-Q001 (SEQ ID NO:89); N11084-001-Q001 (SEQ ID NO:90); N22814-001-Q001 (SEQ ID NO:91); N01564-2-Q1 (SEQ ID NO:92); N12902-001-Q001 (SEQ ID NO:93); N21144-001-Q001 (SEQ ID NO:94); N07534-1-Q1 (SEQ ID NO:95); N22993-001-Q001 (SEQ ID NO:96); N09963-001-Q001 (SEQ ID NO:97); N11542-001-Q001 (SEQ ID NO:98); N14681-001-Q001 (SEQ ID NO:99); N11636-001-Q001 (SEQ ID NO:100); N13732-001-Q001 (SEQ ID NO: 101); N11255-001-Q001 (SEQ ID NO: 102); N15511-001-Q001 (SEQ ID NO: 103); N10536-001-Q001 (SEQ ID NO:104); N09862-001-Q001 (SEQ ID NO:105); N23033-001-Q001 (SEQ ID NO:106); N06039-1-Q1 (SEQ ID NO:107); N10016-001-Q001 (SEQ ID NO:108); N22743-001-Q001 (SEQ ID NO:109); N22953-001-Q001 (SEQ ID NO: 110); N09987-001-Q001 (SEQ ID NO:111); N10092-001-Q001 (SEQ ID NO: 112); N10096-001-Q001 (SEQ ID NO: 113); N22728-001-Q001 (SEQ ID NO: 114); N22747-001-Q001 (SEQ ID NO:115); N22840-001-Q001 (SEQ ID NO:116); N23027-001-Q001 (SEQ ID NO:117); N22777-001-Q001 (SEQ ID NO:118); N09636-001-Q001 (SEQ ID NO: 119); N09879-001-Q001 (SEQ ID NO: 120); N10123-001-Q001 (SEQ ID NO:121); N10316-001-Q001 (SEQ ID NO: 122); N10507-001-Q001 (SEQ ID NO: 123); N09834-001-Q001 (SEQ ID NO:124); N22934-001-Q001 (SEQ ID NO:125); N22700-001-Q001 (SEQ ID NO: 126); N22725-001-Q001 (SEQ ID NO: 127); N22881-001-Q001 (SEQ ID NO: 128); N23032-001-Q001 (SEQ ID NO:129); N22786-001-Q001 (SEQ ID NO:130); N23014-001-Q001 (SEQ ID NO:131); N10471-001-Q001 (SEQ ID NO:132); N11419-001-Q001 (SEQ ID NO: 133); N22724-001-Q001 (SEQ ID NO: 134); N12785-001-Q001 (SEQ ID NO: 135); N09910-001-Q001 (SEQ ID NO:136); N21146-001-Q001 (SEQ ID NO:137); N17618-001-Q001 (SEQ ID NO:138); N09776-001-Q001 (SEQ ID NO:139); N19296-001-Q001 (SEQ ID NO:140); N05205-1-Q1 (SEQ ID NO:141); N10406-001-Q001 (SEQ ID NO:142); N22941-001-Q001 (SEQ ID NO:143); N22875-001-Q001 (SEQ ID NO:144); N13286-001-Q001 (SEQ ID NO:145); N04503-1-Q1 (SEQ ID NO:146); N22925-001-Q001 (SEQ ID NO: 147); N05656-1-Q1 (SEQ ID NO: 148); N17581-001-Q001 (SEQ ID NO: 149); N001NVH-001-Q001 (SEQ ID NO: 150); N22928-001-Q001 (SEQ ID NO: 151); N08219-1-Q001 (SEQ ID NO:152); N05710-1-Q1 (SEQ ID NO: 153); N15338-001-Q001 (SEQ ID NO: 154); N10424-001-Q001 (SEQ ID NO: 155); N16006-001-Q001 (SEQ ID NO: 156), N07278-1-Q1 (SEQ ID NO: 761); N16343-001-Q001 (SEQ ID NO: 762); N23417-001-Q001 (SEQ ID NO: 763); N22902-001-Q001 (SEQ ID NO: 764); N23063-001-Q001 (SEQ ID NO: 765); N22723-001-Q001 (SEQ ID NO: 766); N23049-001-Q001 (SEQ ID NO: 767); N10321-001-Q001 (SEQ ID NO: 768); N15374-001-Q001 (SEQ ID NO: 769); N22802-001-Q001 (SEQ ID NO: 770), N22803-001-Q001 (SEQ ID NO: 771), N18929-001-Q001 (SEQ ID NO: 772); N16041-001-Q001 (SEQ ID NO: 773); and N18401-001-Q001 (SEQ ID NO: 774).

More particularly, the detecting comprises detecting at least one marker selected from the group consisting of: N88514-001-K001 (SEQ ID NO:18); N88515-001-K001 (SEQ ID NO: 19); N88516-001-K001 (SEQ ID NO:20); N88517-001-K001 (SEQ ID NO:21); N88518-001-K001 (SEQ ID NO:22); N88519-001-K001 (SEQ ID NO:23); N88520-001-K001 (SEQ ID NO:24); N88521-001-K001 (SEQ ID NO:25); N001KFE-001-Q001 (SEQ ID NO:26); N88522-001-K001 (SEQ ID NO:27); N88523-001-K001 (SEQ ID NO:28); N88524-001-K001 (SEQ ID NO:29); N88525-001-K001 (SEQ ID NO:30); N88529-001-K001 (SEQ ID NO:31); N88530-001-K001 (SEQ ID NO:32); N88531-001-K001 (SEQ ID NO:33); N88533-001-K001 (SEQ ID NO:34); N88535-001-K001 (SEQ ID NO:35); N88536-001-K001 (SEQ ID NO:36); and N88537-001-K001 (SEQ ID NO:37).

In certain embodiments, the method comprises detecting two or more markers located in two or more different linkage groups. The detecting can involve amplifying the marker from genomic DNA of the plant or germplasm and determining if the marker comprises the polymorphism associated with the shatter resistance.

The Brassica plant to which the method is applied can be Brassica napus; Brassica juncea; Brassica rapa; Brassica oleracea; or Brassica carinata. In particular, the plant is Brassica napus (canola), and can be spring canola, winter canola or semi-winter canola.

Another aspect of the invention features a method of introgressing shatter resistance in a second plant by cross pollinating the identified plant or a progeny thereof of claim 1 with a second plant, wherein the second plant lacks the at least one allele of the at least one QTL detected in the identified plant.

Yet another aspect of the invention features a method of producing an F1 hybrid seed, wherein the F1 hybrid plant derived from the F1 hybrid seed exhibits shatter resistance, the method comprising cross pollinating the identified plant or progeny thereof of claim 1 with a second plant, wherein the second plant lacks the at least one allele of the at least one QTL detected in the identified plant.

Still another aspect of the invention features a method of positional cloning of a nucleic acid comprising a quantitative trait locus (QTL) associated with shatter resistance. The method comprises: (a) providing a nucleic acid from a plant comprising a marker that is associated with shatter resistance with a statistical significance of p≤0.01, wherein the QTL is localized to a linkage group selected from N1, N3, N4, N6, N7, N9, N13, N14, N15, N18 or N19 and intervals therein as set forth in the method described above, and wherein the linkage group comprises the marker; and (b) cloning the nucleic acid comprising a quantitative trait locus (QTL) associated with shatter resistance.

Yet another aspect of the invention features a method of making a transgenic dicot comprising a quantitative trait locus (QTL) associated with shatter resistance. The method comprises the steps of: (a) introducing a nucleic acid cloned according to the above-described cloning method into a dicot cell; and (b) growing the cell under cell growth conditions.

Still another aspect of the invention features a method of identifying a candidate nucleic acid comprising a QTL associated with shatter resistance from a dicot. This method comprises: (a) providing a nucleic acid cloned according to the above-described method; and (b) identifying a homolog of the nucleic acid in a dicot.

Another aspect of the invention features a method of marker assisted selection (MAS) of a quantitative trait locus (QTL) associated with shatter resistance in Brassica. This method comprises the steps of: (a) obtaining a first Brassica plant having at least one allele of a marker locus associated with the shatter resistance with a statistical significance of p≤0.01 as described above; (b) crossing the first Brassica plant with a second Brassica plant; (c) evaluating the progeny for the allele associated with the shatter resistance; and (d) selecting progeny plants that possess the allele. In one embodiment, the plant is a member of a segregating population. In certain embodiments, the marker assisted selection is performed using high throughput screening.

Another aspect of the invention features a Brassica plant identified by the marker assisted breeding method described above. Progeny of that Brassica plant are also provided, particularly F1, F2, and/or F3 progeny.

Another aspect of the invention features an isolated or recombinant nucleic acid comprising a polynucleotide selected from: (a) a sequence selected from any one of marker sequences: N20003-001-Q001 (SEQ ID NO: 1); N03491-1-Q1 (SEQ ID NO:2); N10336-001-Q001 (SEQ ID NO:4); N23133-001-Q001 (SEQ ID NO:5); N16487-001-Q001 (SEQ ID NO:6); N23426-001-Q001 (SEQ ID NO:7); N05671-1-Q1 (SEQ ID NO:8); N12643-001-Q001 (SEQ ID NO:9); N05943-1-Q1 (SEQ ID NO:10); N06007-1-Q1 (SEQ ID NO: 11); N10105-001-Q001 (SEQ ID NO:12); N08181-1-Q1 (SEQ ID NO:13); N06675-1-Q1 (SEQ ID NO:14); N29313-001-Q001 (SEQ ID NO:16); N88512-001-K001 (SEQ ID NO: 17); N88514-001-K001 (SEQ ID NO: 18); N88515-001-K001 (SEQ ID NO: 19); N88516-001-K001 (SEQ ID NO:20); N88517-001-K001 (SEQ ID NO:21); N88518-001-K001 (SEQ ID NO:22); N88519-001-K001 (SEQ ID NO:23); N88520-001-K001 (SEQ ID NO:24); N88521-001-K001 (SEQ ID NO:25); N88522-001-K001 (SEQ ID NO:27); N88523-001-K001 (SEQ ID NO:28); N88524-001-K001 (SEQ ID NO:29); N88525-001-K001 (SEQ ID NO:30); N88529-001-K001 (SEQ ID NO:31); N88530-001-K001 (SEQ ID NO:32); N88531-001-K001 (SEQ ID NO:33); N88533-001-K001 (SEQ ID NO:34); N88535-001-K001 (SEQ ID NO:35); N88536-001-K001 (SEQ ID NO:36); N88537-001-K001 (SEQ ID NO:37); N07541-1-Q1 (SEQ ID NO:38); N23413-001-Q001 (SEQ ID NO:39); N08344-1-Q1 (SEQ ID NO:40); N23533-001-Q011 (SEQ ID NO:41); N14649-001-Q001 (SEQ ID NO:42); N23310-001-Q001 (SEQ ID NO:43); N10526-001-Q001 (SEQ ID NO:44); N23373-001-Q001 (SEQ ID NO:45); N23353-001-Q001 (SEQ ID NO:46); N23206-001-Q001 (SEQ ID NO:47); N11025-001-Q001 (SEQ ID NO:48); N09969-001-Q001 (SEQ ID NO:49); N09882-001-Q001 (SEQ ID NO:50); N10389-001-Q001 (SEQ ID NO:51); N09940-001-Q001 (SEQ ID NO:52); N23409-001-Q001 (SEQ ID NO:53); N23119-001-Q001 (SEQ ID NO:54); N09861-001-Q001 (SEQ ID NO:55); N04807-1-Q1 (SEQ ID NO:56); N06778-1-Q1 (SEQ ID NO:57); N09897-001-Q001 (SEQ ID NO:58); N10499-001-Q001 (SEQ ID NO:59); N23447-001-Q001 (SEQ ID NO:60); N19834-001-Q001 (SEQ ID NO:61); N23362-001-Q001 (SEQ ID NO:62); N23266-001-Q001 (SEQ ID NO:63); N19862-001-Q001 (SEQ ID NO:64); N22187-001-Q001 (SEQ ID NO:65); N08651-1-Q1 (SEQ ID NO:66); N23296-001-Q001 (SEQ ID NO:67); N17314-001-Q001 (SEQ ID NO:68); N20380-001-Q001 (SEQ ID NO:69); N05490-1-Q1 (SEQ ID NO:70); N18849-001-Q001 (SEQ ID NO:71); N08200-1-Q1 (SEQ ID NO:72); N19827-001-Q001 (SEQ ID NO:73); N08264-1-Q1 (SEQ ID NO:75); N23132-001-Q001 (SEQ ID NO:76); N03615-1-Q1 (SEQ ID NO:77); N08465-1-Q1 (SEQ ID NO:79); N10774-001-Q001 (SEQ ID NO:80); N17035-001-Q001 (SEQ ID NO:81); N20834-001-Q001 (SEQ ID NO:82); N22903-001-Q001 (SEQ ID NO:83); N09920-001-Q001 (SEQ ID NO:84); N22822-001-Q001 (SEQ ID NO:85); N22688-001-Q001 (SEQ ID NO:86); N10074-001-Q001 (SEQ ID NO:87); N10057-001-Q001 (SEQ ID NO:88); N10086-001-Q001 (SEQ ID NO:89); N11084-001-Q001 (SEQ ID NO:90); N22814-001-Q001 (SEQ ID NO:91); N01564-2-Q1 (SEQ ID NO:92); N12902-001-Q001 (SEQ ID NO:93); N21144-001-Q001 (SEQ ID NO:94); N07534-1-Q1 (SEQ ID NO:95); N22993-001-Q001 (SEQ ID NO:96); N09963-001-Q001 (SEQ ID NO:97); N11542-001-Q001 (SEQ ID NO:98); N14681-001-Q001 (SEQ ID NO:99); N11636-001-Q001 (SEQ ID NO: 100); N13732-001-Q001 (SEQ ID NO: 101); N11255-001-Q001 (SEQ ID NO: 102); N15511-001-Q001 (SEQ ID NO: 103); N10536-001-Q001 (SEQ ID NO: 104); N09862-001-Q001 (SEQ ID NO:105); N23033-001-Q001 (SEQ ID NO:106); N06039-1-Q1 (SEQ ID NO:107); N10016-001-Q001 (SEQ ID NO:108); N22743-001-Q001 (SEQ ID NO: 109); N22953-001-Q001 (SEQ ID NO: 110); N09987-001-Q001 (SEQ ID NO: 111); N10092-001-Q001 (SEQ ID NO: 112); N10096-001-Q001 (SEQ ID NO: 113); N22728-001-Q001 (SEQ ID NO: 114); N22747-001-Q001 (SEQ ID NO: 115); N22840-001-Q001 (SEQ ID NO: 116); N23027-001-Q001 (SEQ ID NO: 117); N22777-001-Q001 (SEQ ID NO: 118); N09636-001-Q001 (SEQ ID NO:119); N09879-001-Q001 (SEQ ID NO:120); N10123-001-Q001 (SEQ ID NO:121); N10316-001-Q001 (SEQ ID NO:122); N10507-001-Q001 (SEQ ID NO:123); N09834-001-Q001 (SEQ ID NO:124); N22934-001-Q001 (SEQ ID NO: 125); N22700-001-Q001 (SEQ ID NO:126); N22725-001-Q001 (SEQ ID NO:127); N22881-001-Q001 (SEQ ID NO:128); N23032-001-Q001 (SEQ ID NO:129); N22786-001-Q001 (SEQ ID NO:130); N23014-001-Q001 (SEQ ID NO:131); N10471-001-Q001 (SEQ ID NO: 132); N11419-001-Q001 (SEQ ID NO:133); N22724-001-Q001 (SEQ ID NO:134); N12785-001-Q001 (SEQ ID NO:135); N09910-001-Q001 (SEQ ID NO:136); N21146-001-Q001 (SEQ ID NO:137); N17618-001-Q001 (SEQ ID NO:138); N09776-001-Q001 (SEQ ID NO:139); N19296-001-Q001 (SEQ ID NO: 140); N05205-1-Q1 (SEQ ID NO:141); N10406-001-Q001 (SEQ ID NO:142); N22941-001-Q001 (SEQ ID NO:143); N22875-001-Q001 (SEQ ID NO:144); N13286-001-Q001 (SEQ ID NO:145); N04503-1-Q1 (SEQ ID NO: 146); N22925-001-Q001 (SEQ ID NO: 147); N05656-1-Q1 (SEQ ID NO: 148); N17581-001-Q001 (SEQ ID NO: 149); N22928-001-Q001 (SEQ ID NO:151); N08219-1-Q001 (SEQ ID NO:152); N05710-1-Q1 (SEQ ID NO: 153); N15338-001-Q001 (SEQ ID NO: 154); N10424-001-Q001 (SEQ ID NO: 155); N16006-001-Q001 (SEQ ID NO: 156); N07278-1-Q1 (SEQ ID NO: 761); N16343-001-Q001 (SEQ ID NO: 762); N23417-001-Q001 (SEQ ID NO: 763); N22902-001-Q001 (SEQ ID NO: 764); N23063-001-Q001 (SEQ ID NO: 765); N22723-001-Q001 (SEQ ID NO: 766); N23049-001-Q001 (SEQ ID NO: 767); N10321-001-Q001 (SEQ ID NO: 768); N15374-001-Q001 (SEQ ID NO: 769); N22802-001-Q001 (SEQ ID NO: 770), N22803-001-Q001 (SEQ ID NO: 771), N18929-001-Q001 (SEQ ID NO: 772); N16041-001-Q001 (SEQ ID NO: 773); and N18401-001-Q001 (SEQ ID NO: 774),

(b) a polynucleotide sequence with at least 70% sequence identity to the polynucleotide of (a); and (c) a polynucleotide sequence complementary to the sequence of (a) or (b). In particular, the isolated or recombinant nucleic acid comprises a polynucleotide selected from: (a) a sequence selected from any one of marker sequences: N88514-001-K001 (SEQ ID NO:18); N88515-001-K001 (SEQ ID NO:19); N88516-001-K001 (SEQ ID NO:20); N88517-001-K001 (SEQ ID NO:21); N88518-001-K001 (SEQ ID NO:22); N88519-001-K001 (SEQ ID NO:23); N88520-001-K001 (SEQ ID NO:24); N88521-001-K001 (SEQ ID NO:25); N88522-001-K001 (SEQ ID NO:27); N88523-001-K001 (SEQ ID NO:28); N88524-001-K001 (SEQ ID NO:29); N88525-001-K001 (SEQ ID NO:30); N88529-001-K001 (SEQ ID NO:31); N88530-001-K001 (SEQ ID NO:32); N88531-001-K001 (SEQ ID NO:33); N88533-001-K001 (SEQ ID NO:34); N88535-001-K001 (SEQ ID NO:35); N88536-001-K001 (SEQ ID NO:36); and N88537-001-K001 (SEQ ID NO:37); (b) a polynucleotide sequence with at least 70% sequence identity to the polynucleotide of (a); and (c) a polynucleotide sequence complementary to the sequence of (a) or (b). In certain embodiments, the above-described isolated or recombinant nucleic acid is associated with shatter resistance in Brassica.

Another aspect of the invention features a synthetic and/or chemically modified nucleic acid molecule that detects a polymorphism in a Brassica plant DNA associated with shatter resistance, wherein the nucleic acid molecule comprises at least 10 nucleotides and is identical to a sequence of the same number of consecutive nucleotides in either strand of the plant DNA where the polymorphism is located, wherein the nucleic acid molecule comprises a sequence that is at least 70% identical to a marker sequence or a fragment of a marker sequence selected from the group consisting of: N20003-001-Q001 (SEQ ID NO: 1); N03491-1-Q1 (SEQ ID NO:2); N0017NR-001-Q001 (SEQ ID NO:3); N10336-001-Q001 (SEQ ID NO:4); N23133-001-Q001 (SEQ ID NO:5); N16487-001-Q001 (SEQ ID NO:6); N23426-001-Q001 (SEQ ID NO:7); N05671-1-Q1 (SEQ ID NO:8); N12643-001-Q001 (SEQ ID NO:9); N05943-1-Q1 (SEQ ID NO:10); N06007-1-Q1 (SEQ ID NO: 11); N10105-001-Q001 (SEQ ID NO:12); N08181-1-Q1 (SEQ ID NO:13); N06675-1-Q1 (SEQ ID NO:14); N001KH2-001-Q001 (SEQ ID NO:15); N29313-001-Q001 (SEQ ID NO:16); N88512-001-K001 (SEQ ID NO: 17); N88514-001-K001 (SEQ ID NO: 18); N88515-001-K001 (SEQ ID NO:19); N88516-001-K001 (SEQ ID NO:20); N88517-001-K001 (SEQ ID NO:21); N88518-001-K001 (SEQ ID NO:22); N88519-001-K001 (SEQ ID NO:23); N88520-001-K001 (SEQ ID NO:24); N88521-001-K001 (SEQ ID NO:25); N001KFE-001-Q001 (SEQ ID NO:26); N88522-001-K001 (SEQ ID NO:27); N88523-001-K001 (SEQ ID NO:28); N88524-001-K001 (SEQ ID NO:29); N88525-001-K001 (SEQ ID NO:30); N88529-001-K001 (SEQ ID NO:31); N88530-001-K001 (SEQ ID NO:32); N88531-001-K001 (SEQ ID NO:33); N88533-001-K001 (SEQ ID NO:34); N88535-001-K001 (SEQ ID NO:35); N88536-001-K001 (SEQ ID NO:36); N88537-001-K001 (SEQ ID NO:37); N07541-1-Q1 (SEQ ID NO:38); N23413-001-Q001 (SEQ ID NO:39); N08344-1-Q1 (SEQ ID NO:40); N23533-001-Q011 (SEQ ID NO:41); N14649-001-Q001 (SEQ ID NO:42); N23310-001-Q001 (SEQ ID NO:43); N10526-001-Q001 (SEQ ID NO:44); N23373-001-Q001 (SEQ ID NO:45); N23353-001-Q001 (SEQ ID NO:46); N23206-001-Q001 (SEQ ID NO:47); N11025-001-Q001 (SEQ ID NO:48); N09969-001-Q001 (SEQ ID NO:49); N09882-001-Q001 (SEQ ID NO:50); N10389-001-Q001 (SEQ ID NO:51); N09940-001-Q001 (SEQ ID NO:52); N23409-001-Q001 (SEQ ID NO:53); N23119-001-Q001 (SEQ ID NO:54); N09861-001-Q001 (SEQ ID NO:55); N04807-1-Q1 (SEQ ID NO:56); N06778-1-Q1 (SEQ ID NO:57); N09897-001-Q001 (SEQ ID NO:58); N10499-001-Q001 (SEQ ID NO:59); N23447-001-Q001 (SEQ ID NO:60); N19834-001-Q001 (SEQ ID NO:61); N23362-001-Q001 (SEQ ID NO:62); N23266-001-Q001 (SEQ ID NO:63); N19862-001-Q001 (SEQ ID NO:64); N22187-001-Q001 (SEQ ID NO:65); N08651-1-Q1 (SEQ ID NO:66); N23296-001-Q001 (SEQ ID NO:67); N17314-001-Q001 (SEQ ID NO:68); N20380-001-Q001 (SEQ ID NO:69); N05490-1-Q1 (SEQ ID NO:70); N18849-001-Q001 (SEQ ID NO:71); N08200-1-Q1 (SEQ ID NO:72); N19827-001-Q001 (SEQ ID NO:73); N001R9W-001-Q001 (SEQ ID NO:74); N08264-1-Q1 (SEQ ID NO:75); N23132-001-Q001 (SEQ ID NO:76); N03615-1-Q1 (SEQ ID NO:77); N001RWT-001-Q001 (SEQ ID NO:78); N08465-1-Q1 (SEQ ID NO:79); N10774-001-Q001 (SEQ ID NO:80); N17035-001-Q001 (SEQ ID NO:81); N20834-001-Q001 (SEQ ID NO:82); N22903-001-Q001 (SEQ ID NO:83); N09920-001-Q001 (SEQ ID NO:84); N22822-001-Q001 (SEQ ID NO:85); N22688-001-Q001 (SEQ ID NO:86); N10074-001-Q001 (SEQ ID NO:87); N10057-001-Q001 (SEQ ID NO:88); N10086-001-Q001 (SEQ ID NO:89); N11084-001-Q001 (SEQ ID NO:90); N22814-001-Q001 (SEQ ID NO:91); N01564-2-Q1 (SEQ ID NO:92); N12902-001-Q001 (SEQ ID NO:93); N21144-001-Q001 (SEQ ID NO:94); N07534-1-Q1 (SEQ ID NO:95); N22993-001-Q001 (SEQ ID NO:96); N09963-001-Q001 (SEQ ID NO:97); N11542-001-Q001 (SEQ ID NO:98); N14681-001-Q001 (SEQ ID NO:99); N11636-001-Q001 (SEQ ID NO:100); N13732-001-Q001 (SEQ ID NO:101); N11255-001-Q001 (SEQ ID NO: 102); N15511-001-Q001 (SEQ ID NO: 103); N10536-001-Q001 (SEQ ID NO: 104); N09862-001-Q001 (SEQ ID NO:105); N23033-001-Q001 (SEQ ID NO:106); N06039-1-Q1 (SEQ ID NO:107); N10016-001-Q001 (SEQ ID NO:108); N22743-001-Q001 (SEQ ID NO:109); N22953-001-Q001 (SEQ ID NO: 110); N09987-001-Q001 (SEQ ID NO: 111); N10092-001-Q001 (SEQ ID NO: 112); N10096-001-Q001 (SEQ ID NO: 113); N22728-001-Q001 (SEQ ID NO: 114); N22747-001-Q001 (SEQ ID NO:115); N22840-001-Q001 (SEQ ID NO:116); N23027-001-Q001 (SEQ ID NO:117); N22777-001-Q001 (SEQ ID NO:118); N09636-001-Q001 (SEQ ID NO: 119); N09879-001-Q001 (SEQ ID NO: 120); N10123-001-Q001 (SEQ ID NO:121); N10316-001-Q001 (SEQ ID NO:122); N10507-001-Q001 (SEQ ID NO: 123); N09834-001-Q001 (SEQ ID NO: 124); N22934-001-Q001 (SEQ ID NO: 125); N22700-001-Q001 (SEQ ID NO:126); N22725-001-Q001 (SEQ ID NO:127); N22881-001-Q001 (SEQ ID NO:128); N23032-001-Q001 (SEQ ID NO:129); N22786-001-Q001 (SEQ ID NO:130); N23014-001-Q001 (SEQ ID NO:131); N10471-001-Q001 (SEQ ID NO:132); N11419-001-Q001 (SEQ ID NO: 133); N22724-001-Q001 (SEQ ID NO: 134); N12785-001-Q001 (SEQ ID NO:135); N09910-001-Q001 (SEQ ID NO:136); N21146-001-Q001 (SEQ ID NO:137); N17618-001-Q001 (SEQ ID NO:138); N09776-001-Q001 (SEQ ID NO:139); N19296-001-Q001 (SEQ ID NO: 140); N05205-1-Q1 (SEQ ID NO: 141); N10406-001-Q001 (SEQ ID NO:142); N22941-001-Q001 (SEQ ID NO:143); N22875-001-Q001 (SEQ ID NO: 144); N13286-001-Q001 (SEQ ID NO: 145); N04503-1-Q1 (SEQ ID NO: 146); N22925-001-Q001 (SEQ ID NO:147); N05656-1-Q1 (SEQ ID NO:148); N17581-001-Q001 (SEQ ID NO: 149); N001NVH-001-Q001 (SEQ ID NO: 150); N22928-001-Q001 (SEQ ID NO: 151); N08219-1-Q001 (SEQ ID NO: 152); N05710-1-Q1 (SEQ ID NO: 153); N15338-001-Q001 (SEQ ID NO:154); N10424-001-Q001 (SEQ ID NO:155); N16006-001-Q001 (SEQ ID NO:156); N07278-1-Q1 (SEQ ID NO: 761); N16343-001-Q001 (SEQ ID NO: 762); N23417-001-Q001 (SEQ ID NO: 763); N22902-001-Q001 (SEQ ID NO: 764); N23063-001-Q001 (SEQ ID NO: 765); N22723-001-Q001 (SEQ ID NO: 766); N23049-001-Q001 (SEQ ID NO: 767); N10321-001-Q001 (SEQ ID NO: 768); N15374-001-Q001 (SEQ ID NO: 769); N22802-001-Q001 (SEQ ID NO: 770), N22803-001-Q001 (SEQ ID NO: 771), N18929-001-Q001 (SEQ ID NO: 772); N16041-001-Q001 (SEQ ID NO: 773); and N18401-001-Q001 (SEQ ID NO: 774).

In particular embodiments, the synthetic nucleic acid molecule is selected from any one of SEQ ID NOs: 157-760 and SEQ ID NOs: 775-830.

In certain embodiments, the synthetic nucleic acid molecule is associated with a marker sequence selected from any one of: N88514-001-K001 (SEQ ID NO:18); N88515-001-K001 (SEQ ID NO: 19); N88516-001-K001 (SEQ ID NO:20); N88517-001-K001 (SEQ ID NO:21); N88518-001-K001 (SEQ ID NO:22); N88519-001-K001 (SEQ ID NO:23); N88520-001-K001 (SEQ ID NO:24); N88521-001-K001 (SEQ ID NO:25); N001KFE-001-Q001 (SEQ ID NO:26); N88522-001-K001 (SEQ ID NO:27); N88523-001-K001 (SEQ ID NO:28); N88524-001-K001 (SEQ ID NO:29); N88525-001-K001 (SEQ ID NO:30); N88529-001-K001 (SEQ ID NO:31); N88530-001-K001 (SEQ ID NO:32); N88531-001-K001 (SEQ ID NO:33); N88533-001-K001 (SEQ ID NO:34); N88535-001-K001 (SEQ ID NO:35); N88536-001-K001 (SEQ ID NO:36); and N88537-001-K001 (SEQ ID NO:37). In particular embodiments, the synthetic nucleic acid molecule is selected from any one of SEQ ID NOs: 236-285.

Another aspect of the invention features a kit for screening a plant or germplasm for a QTL associated with shatter resistance. The kit includes a container in which is contained: (a) a plurality of synthetic and/or chemically modified nucleic acid molecules that detect polymorphism in Brassica plant DNA associated with shatter resistance, wherein each nucleic acid molecule comprises at least 10 nucleotides and is identical to a sequence of the same number of consecutive nucleotides in either strand of the plant DNA where the polymorphism is located, wherein the nucleic acid molecule comprises a sequence that is at least 70% identical to a marker sequence or a fragment of a marker sequence selected from SEQ ID NOs: 1-156 and SEQ ID Ns: 761-774; and (b) instructions for screening a Brassica plant for the QTL associated with shatter resistance.

In certain kits, the marker sequence or fragment of marker sequence is selected from SEQ ID NOs: 18-37. In certain embodiments, the kits contain at least one component for high throughput screening the plant or germplasm for the QTL.

Kits in some embodiments may include synthetic and/or chemically modified nucleic acids for detecting ten or more polymorphisms in Brassica plant DNA associated with shatter resistance. In other embodiments, the kits may include synthetic and/or chemically modified nucleic acids for detecting 20 or more polymorphisms in Brassica plant DNA associated with shatter resistance. In still other embodiments, the kits may include synthetic and/or chemically modified nucleic acids for detecting 30 or more polymorphisms in Brassica plant DNA associated with shatter resistance.

Another aspect of the invention features a Brassica plant that exhibits shatter resistance, comprising alleles favorable for shatter resistance in at least one QTL localized to a linkage group selected from N1, N3, N4, N6, N7, N9, N13, N14, N15, N18 or N19, wherein each said linkage group comprises at least one marker that is associated with the resistance to shatter with a statistical significance of p≤0.01, wherein the QTL is localized to a chromosomal interval selected from the group consisting of: (a) an interval flanked by and including markers N20003-001-Q001 and N23426-001-Q001 on linkage group N1; (b) an interval flanked by and including markers N05671-1-Q1 and N12643-001-Q001 on linkage group N3; (c) an interval flanked by and including markers N05943-1-Q1 and N88537-001-K001 on linkage group N4; (d) an interval flanked by and including markers N07541-1-Q1 and N14649-001-Q001 on linkage group N6; (e) one or more intervals flanked by and including: (i) markers N23310-001-Q001 and N23409-001-Q001 on linkage group N7, or (ii) markers N07278-1-Q1 and N23417-001-Q001 on linkage group N7; (f) one or more intervals flanked by and including: (i) markers N23119-001-Q001 and N20380-001-Q001; or (ii) NO5490-1-Q1 and N20834-001-Q001 on linkage group N9; (g) one or more intervals flanked by and including (i) markers N21144-001-Q001 and N09862-001-Q001 on linkage group N13, or (ii) markers N22903-001-Q001 and N12902-001-Q001 on linkage group N13; (h) one or more intervals flanked by and including: (i) markers N23033-001-Q001 and N22724-001-Q001 on linkage group N14, or (ii) markers N23033-001-Q001 and N22802-001-Q001 on linkage group N14; (i) an interval flanked by and including markers N12785-001-Q001 and N19296-001-Q001 on linkage group N15; (j) one or more intervals flanked by and including (i) markers N05205-1-Q1 and N22925-001-Q001 on linkage group N18, or (ii) markers N22803-001-Q001 and N18401-001-Q001 on linkage group N18; and (k) an interval flanked by and including markers N05656-1-Q1 and N16006-001-Q001 on linkage group N19; wherein each said linkage group comprises at least one marker that is associated with the shatter resistance with a statistical significance of p≤0.01. In particular embodiments, the QTL is localized to a chromosomal interval selected from the group consisting of: (a) an interval flanked by and including markers N10336-001-Q001 and N23426-001-Q001 on linkage group N1; (b) one or more intervals flanked by and including (i) markers N88514-001-K001 and N88537-001-K001, or (ii) markers N05943-1-Q1 and N06675-1-Q1 on linkage group N4; and (c) one or more intervals flanked by and including (i) markers N001RWT-001-Q001 and N20834-001-Q001, or (ii) markers N04807-1-Q1 and N17314-001-Q001 on linkage group N9.

In certain embodiments, the marker comprises a polymorphism that identifies the favorable allele. The polymorphism can be a single nucleotide polymorphism (SNP) or a simple sequence repeat (SSR).

In certain embodiments of the Brassica plant, the favorable allele is associated with a marker selected from the group consisting of: N20003-001-Q001 (SEQ ID NO: 1); N03491-1-Q1 (SEQ ID NO:2); N0017NR-001-Q001 (SEQ ID NO:3); N10336-001-Q001 (SEQ ID NO:4); N23133-001-Q001 (SEQ ID NO:5); N16487-001-Q001 (SEQ ID NO:6); N23426-001-Q001 (SEQ ID NO:7); N05671-1-Q1 (SEQ ID NO:8); N12643-001-Q001 (SEQ ID NO:9); N05943-1-Q1 (SEQ ID NO:10); N06007-1-Q1 (SEQ ID NO: 11); N10105-001-Q001 (SEQ ID NO:12); N08181-1-Q1 (SEQ ID NO:13); N06675-1-Q1 (SEQ ID NO:14); N001KH2-001-Q001 (SEQ ID NO:15); N29313-001-Q001 (SEQ ID NO:16); N88512-001-K001 (SEQ ID NO:17); N88514-001-K001 (SEQ ID NO:18); N88515-001-K001 (SEQ ID NO:19); N88516-001-K001 (SEQ ID NO:20); N88517-001-K001 (SEQ ID NO:21); N88518-001-K001 (SEQ ID NO:22); N88519-001-K001 (SEQ ID NO:23); N88520-001-K001 (SEQ ID NO:24); N88521-001-K001 (SEQ ID NO:25); N001KFE-001-Q001 (SEQ ID NO:26); N88522-001-K001 (SEQ ID NO:27); N88523-001-K001 (SEQ ID NO:28); N88524-001-K001 (SEQ ID NO:29); N88525-001-K001 (SEQ ID NO:30); N88529-001-K001 (SEQ ID NO:31); N88530-001-K001 (SEQ ID NO:32); N88531-001-K001 (SEQ ID NO:33); N88533-001-K001 (SEQ ID NO:34); N88535-001-K001 (SEQ ID NO:35); N88536-001-K001 (SEQ ID NO:36); N88537-001-K001 (SEQ ID NO:37); N07541-1-Q1 (SEQ ID NO:38); N23413-001-Q001 (SEQ ID NO:39); N08344-1-Q1 (SEQ ID NO:40); N23533-001-Q011 (SEQ ID NO:41); N14649-001-Q001 (SEQ ID NO:42); N23310-001-Q001 (SEQ ID NO:43); N10526-001-Q001 (SEQ ID NO:44); N23373-001-Q001 (SEQ ID NO:45); N23353-001-Q001 (SEQ ID NO:46); N23206-001-Q001 (SEQ ID NO:47); N11025-001-Q001 (SEQ ID NO:48); N09969-001-Q001 (SEQ ID NO:49); N09882-001-Q001 (SEQ ID NO:50); N10389-001-Q001 (SEQ ID NO:51); N09940-001-Q001 (SEQ ID NO:52); N23409-001-Q001 (SEQ ID NO:53); N23119-001-Q001 (SEQ ID NO:54); N09861-001-Q001 (SEQ ID NO:55); N04807-1-Q1 (SEQ ID NO:56); N06778-1-Q1 (SEQ ID NO:57); N09897-001-Q001 (SEQ ID NO:58); N10499-001-Q001 (SEQ ID NO:59); N23447-001-Q001 (SEQ ID NO:60); N19834-001-Q001 (SEQ ID NO:61); N23362-001-Q001 (SEQ ID NO:62); N23266-001-Q001 (SEQ ID NO:63); N19862-001-Q001 (SEQ ID NO:64); N22187-001-Q001 (SEQ ID NO:65); N08651-1-Q1 (SEQ ID NO:66); N23296-001-Q001 (SEQ ID NO:67); N17314-001-Q001 (SEQ ID NO:68); N20380-001-Q001 (SEQ ID NO:69); N05490-1-Q1 (SEQ ID NO:70); N18849-001-Q001 (SEQ ID NO:71); N08200-1-Q1 (SEQ ID NO:72); N19827-001-Q001 (SEQ ID NO:73); N001R9W-001-Q001 (SEQ ID NO:74); N08264-1-Q1 (SEQ ID NO:75); N23132-001-Q001 (SEQ ID NO:76); N03615-1-Q1 (SEQ ID NO:77); N001RWT-001-Q001 (SEQ ID NO:78); N08465-1-Q1 (SEQ ID NO:79); N10774-001-Q001 (SEQ ID NO:80); N17035-001-Q001 (SEQ ID NO:81); N20834-001-Q001 (SEQ ID NO:82); N22903-001-Q001 (SEQ ID NO:83); N09920-001-Q001 (SEQ ID NO:84); N22822-001-Q001 (SEQ ID NO:85); N22688-001-Q001 (SEQ ID NO:86); N10074-001-Q001 (SEQ ID NO:87); N10057-001-Q001 (SEQ ID NO:88); N10086-001-Q001 (SEQ ID NO:89); N11084-001-Q001 (SEQ ID NO:90); N22814-001-Q001 (SEQ ID NO:91); N01564-2-Q1 (SEQ ID NO:92); N12902-001-Q001 (SEQ ID NO:93); N21144-001-Q001 (SEQ ID NO:94); N07534-1-Q1 (SEQ ID NO:95); N22993-001-Q001 (SEQ ID NO:96); N09963-001-Q001 (SEQ ID NO:97); N11542-001-Q001 (SEQ ID NO:98); N14681-001-Q001 (SEQ ID NO:99); N11636-001-Q001 (SEQ ID NO:100); N13732-001-Q001 (SEQ ID NO: 101); N11255-001-Q001 (SEQ ID NO: 102); N15511-001-Q001 (SEQ ID NO: 103); N10536-001-Q001 (SEQ ID NO:104); N09862-001-Q001 (SEQ ID NO:105); N23033-001-Q001 (SEQ ID NO:106); N06039-1-Q1 (SEQ ID NO:107); N10016-001-Q001 (SEQ ID NO:108); N22743-001-Q001 (SEQ ID NO:109); N22953-001-Q001 (SEQ ID NO: 110); N09987-001-Q001 (SEQ ID NO:111); N10092-001-Q001 (SEQ ID NO:112); N10096-001-Q001 (SEQ ID NO:113); N22728-001-Q001 (SEQ ID NO:114); N22747-001-Q001 (SEQ ID NO:115); N22840-001-Q001 (SEQ ID NO:116); N23027-001-Q001 (SEQ ID NO: 117); N22777-001-Q001 (SEQ ID NO:118); N09636-001-Q001 (SEQ ID NO: 119); N09879-001-Q001 (SEQ ID NO: 120); N10123-001-Q001 (SEQ ID NO:121); N10316-001-Q001 (SEQ ID NO: 122); N10507-001-Q001 (SEQ ID NO: 123); N09834-001-Q001 (SEQ ID NO: 124); N22934-001-Q001 (SEQ ID NO:125); N22700-001-Q001 (SEQ ID NO: 126); N22725-001-Q001 (SEQ ID NO: 127); N22881-001-Q001 (SEQ ID NO: 128); N23032-001-Q001 (SEQ ID NO:129); N22786-001-Q001 (SEQ ID NO:130); N23014-001-Q001 (SEQ ID NO:131); N10471-001-Q001 (SEQ ID NO:132); N11419-001-Q001 (SEQ ID NO: 133); N22724-001-Q001 (SEQ ID NO: 134); N12785-001-Q001 (SEQ ID NO: 135); N09910-001-Q001 (SEQ ID NO:136); N21146-001-Q001 (SEQ ID NO:137); N17618-001-Q001 (SEQ ID NO:138); N09776-001-Q001 (SEQ ID NO:139); N19296-001-Q001 (SEQ ID NO:140); N05205-1-Q1 (SEQ ID NO:141); N10406-001-Q001 (SEQ ID NO:142); N22941-001-Q001 (SEQ ID NO:143); N22875-001-Q001 (SEQ ID NO:144); N13286-001-Q001 (SEQ ID NO:145); N04503-1-Q1 (SEQ ID NO:146); N22925-001-Q001 (SEQ ID NO: 147); N05656-1-Q1 (SEQ ID NO: 148); N17581-001-Q001 (SEQ ID NO: 149); N001NVH-001-Q001 (SEQ ID NO: 150); N22928-001-Q001 (SEQ ID NO: 151); N08219-1-Q001 (SEQ ID NO:152); N05710-1-Q1 (SEQ ID NO: 153); N15338-001-Q001 (SEQ ID NO: 154); N10424-001-Q001 (SEQ ID NO: 155); N16006-001-Q001 (SEQ ID NO: 156); N07278-1-Q1 (SEQ ID NO: 761); N16343-001-Q001 (SEQ ID NO: 762); N23417-001-Q001 (SEQ ID NO: 763); N22902-001-Q001 (SEQ ID NO: 764); N23063-001-Q001 (SEQ ID NO: 765); N22723-001-Q001 (SEQ ID NO: 766); N23049-001-Q001 (SEQ ID NO: 767); N10321-001-Q001 (SEQ ID NO: 768); N15374-001-Q001 (SEQ ID NO: 769); N22802-001-Q001 (SEQ ID NO: 770), N22803-001-Q001 (SEQ ID NO: 771), N18929-001-Q001 (SEQ ID NO: 772); N16041-001-Q001 (SEQ ID NO: 773); and N18401-001-Q001 (SEQ ID NO: 774).

More particularly, the favorable allele is associated with a marker selected from the group consisting of: N88514-001-K001 (SEQ ID NO:18); N88515-001-K001 (SEQ ID NO:19); N88516-001-K001 (SEQ ID NO:20); N88517-001-K001 (SEQ ID NO:21); N88518-001-K001 (SEQ ID NO:22); N88519-001-K001 (SEQ ID NO:23); N88520-001-K001 (SEQ ID NO:24); N88521-001-K001 (SEQ ID NO:25); N001KFE-001-Q001 (SEQ ID NO:26); N88522-001-K001 (SEQ ID NO:27); N88523-001-K001 (SEQ ID NO:28); N88524-001-K001 (SEQ ID NO:29); N88525-001-K001 (SEQ ID NO:30); N88529-001-K001 (SEQ ID NO:31); N88530-001-K001 (SEQ ID NO:32); N88531-001-K001 (SEQ ID NO:33); N88533-001-K001 (SEQ ID NO:34); N88535-001-K001 (SEQ ID NO:35); N88536-001-K001 (SEQ ID NO:36); and N88537-001-K001 (SEQ ID NO:37).

In certain embodiments, the Brassica plant contains a plurality of favorable alleles for resistance to shatter. For instance, the plant may contain 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 favorable alleles for resistance to shatter. In certain embodiments, the plurality of alleles are associated with two or more different linkage groups.

The aforementioned Brassica plant is selected from Brassica napus; Brassica juncea; Brassica rapa; Brassica oleracea; and Brassica carina. In one embodiment, the plant is Brassica napus (canola), which can be spring canola, winter canola or semi-winter canola.

Other features and advantages of the invention will be understood from the detailed description and examples that follow.

DETAILED DESCRIPTION

Overview

The present invention relates to the identification of genetic markers, e.g., marker loci and nucleic acids corresponding to (or derived from) these marker loci, such as probes and amplification products useful for genotyping plants, correlated with resistance or improved resistance to shatter. The markers of the invention are used to identify plants, particularly plants of the species Brassica napus (B. napus) (canola), that are resistant or exhibit improved resistance to shatter (sometimes referred to herein simply as “shatter resistance”). Accordingly, these markers are useful for marker-assisted selection (MAS) and breeding of shatter resistant plants, and for identification of susceptible plants. The markers of the invention are also used to identify and define nucleic acids that are proximal to and/or chromosome intervals corresponding to, or including, quantitative trait loci associated with shatter resistance. Quantitative Trait Loci (QTLs) associated with shatter resistance are isolated by positional cloning, e.g., nucleic acids proximal to or of genetic intervals defined by a pair of markers described herein, or subsequences of an interval defined by and including such markers. Such isolated QTL nucleic acids can be used for the production of transgenic cells and plants exhibiting shatter resistance. In addition, QTL nucleic acids isolated from one organism, e.g., canola, can, in turn, serve to isolate homologs of QTLs for shatter resistance from other plants, including a variety of commercially and/or scientifically important dicots, such as soybean, alfalfa, sunflower, flax, beans, (for example, white beans), potatoes, peas, peanuts and Arabidopsis.

Definitions

Units, prefixes, and symbols are denoted in their International System of Units (SI) accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; and amino acid sequences are written left to right in amino to carboxy orientation. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Nucleotides may be referred to herein by their one-letter symbols recommended by the IUPAC-IUBMB Nomenclature Commission. The terms defined below are more fully defined by reference to the specification as a whole. Section headings provided throughout the specification are provided for convenience and are not limitations to the various objects and embodiments of the present invention.

“Resistance or improved resistance to shatter in Brassica”, or simply “shatter resistance,” refers to the resistance of a plant against pod shatter tendency, under field conditions and/or under extreme weather conditions such as a wind storm. Means of measuring the level of resistance to pod shatter tendency are known in the art and include, but are not limited to, the following: pendulum-based test (Kadkol et al., 1991; Liu et al. 1994), cantilever test (Kadkol et al., 1984), manual bending test (Roy 1982), microfracture test (MFT) (Child et al., 2003), siliqua twisting (Tys et al., 2007), ‘Ripping’ method (Tan et al., 2007), Random Impact Test (RIT) (Bruce et al., 2002; Morgan et al., 1998, 2003; Squires et al., 2003) and the device and method described in U.S. Pat. No. 7,412,880 B2. In one embodiment, a plant with field resistance to pod shatter has a rating of 5.0 or greater, based on the “shatter score” (SHTSC) rating scale. In other embodiments, a plant with resistance to pod shatter has a rating of 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 or 9.0, based on the SHTSC rating scale. Shatter scores are sometimes expressed in ranges; for instance in a range of 5-6, 6-7, 7-8, 8-9 or in a range of 5-7, 7-9 and so on, or by a number range within integers, such as 5.5-6.5, 5.5-7.5, 6-7.5, 7-8.5, for example. In those instances, a plant with resistance to shatter has a rating in the range of at least 5-6, or 6-7, or 7-8, or 8-9, based on the SHTSC rating scale.

It will be understood by the skilled artisan that the greater the number (or percentage) of favorable alleles for shatter resistance a plant possesses, the greater will be the level of resistance exhibited. In certain embodiments, a plant with shatter resistance has a genome containing at least about 50% favorable alleles. In more particular embodiments, a plant with shatter resistance has a genome containing at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or more favorable alleles. The percentage of favorable alleles can also be expressed as a number value. For instance, if a total number of 15 favorable alleles are possible in a certain mapping population, a plant having 12 of those alleles would have 80% favorable alleles. In certain embodiments, the number or percent of favorable alleles in a plant can serve as a rough predictor of the expected level of shatter resistance a plant will exhibit.

It will also be understood by the skilled artisan that the QTLs described herein represent regions of the genome comprising genes that contribute to the shatter resistance of a plant. Further, each QTL can contribute differently to that resistance level. Thus, breeding efforts are directed to increasing the number of those QTLs, particularly quantitatively significant QTLs, present in the germplasm. Early in a breeding program, fewer QTLs may be present in a particular germplasm, but that number will increase as the breeding program progresses. Thus, in certain embodiments, a plant exhibiting shatter resistance may contain at least 2 of the QTLs described herein. More particularly, the plant may contain at least 3, 4, 5 or 6 of the QTLs described herein. Yet more particularly, the plant may contain all of the QTLs described herein.

As mentioned above, the term “shatter” refers to a process by which the silique or pod, on maturation late in fruit development, releases and disperses the seeds contained within it. The siliques or pods are formed by two carpels that are separated by a thin replum. The dehiscence zone (DZ) is where the valve (fruit wall) margin connects to the replum, and extends throughout the entire length of the fruit between the valve and replum. As the pod matures late in fruit development, the valve margin detaches from the replum, leading to seed dispersal. The DZ demarcates the precise location where the valves detach.

The term “quantitative trait locus” or “QTL” refers to a polymorphic genetic locus with at least two alleles that differentially affect the expression of a continuously distributed phenotypic trait, for example, resistance to shatter. For example, the QTL may have a favorable allele that confers, or contributes to, shatter resistance.

The term “favorable allele” is an allele at a particular locus that confers, or contributes to, a desirable phenotype, e.g., resistance to shatter, or alternatively is an allele that allows the identification of plants with decreased resistance that can be removed from a breeding program or planting (“counterselection”). A favorable allele of a marker is a marker allele that segregates with the favorable phenotype, or alternatively, segregates with the unfavorable plant phenotype, therefore providing the benefit of identifying plants. Alleles that are favorable for resistance to shatter are provided, for example, in Table 6.

The term “associated with” or “associated” in the context of this invention refers to, e.g., a nucleic acid and a phenotypic trait or a second nucleic acid, that are in linkage disequilibrium, i.e., the nucleic acid and the trait/second nucleic acid are found together in progeny plants more often than if the nucleic acid and phenotype/second nucleic acid segregated separately.

The term “linkage” is used to describe the degree with which one marker locus is associated with another marker locus or some other locus (for example, a QTL). The linkage relationship between a molecular marker and a phenotype is given as a “probability” or “adjusted probability”. Linkage can be expressed as a desired limit or range. For example, in some embodiments, any marker is linked (genetically and/or physically) to any other marker when the markers are separated by less than 50, 40, 30, 25, 20, or 15 map units (or cM). In some aspects, it is advantageous to define a bracketed range of linkage, for example, between 10 and 20 cM, between 10 and 30 cM, or between 10 and 40 cM. The more closely a marker is linked to a second locus, the better an indicator for the second locus that marker becomes. Thus, “closely linked loci” such as a marker locus and a second locus display an inter-locus recombination frequency of 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less. In highly preferred embodiments, the relevant loci display a recombination frequency of about 1% or less, e.g., about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. Two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are also said to be “proximal to” or “in proximity of each other. Since one cM is the distance between two markers that show a 1% recombination frequency, any marker is closely linked (genetically and/or physically) to any other marker that is in close proximity, e.g., at or less than 10 cM distance. Two closely linked markers on the same chromosome can be positioned 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5 or 0.25 cM or less from each other.

The term “linkage disequilibrium” refers to a non-random segregation of genetic loci. This implies that such loci are in sufficient physical proximity along a length of a chromosome that they tend to segregate together with greater than random frequency.

The term “genetically linked” refers to genetic loci that are in linkage disequilibrium and statistically determined not to assort independently. Genetically linked loci assort dependently from 51% to 99% of the time or any whole number value there between, preferably at least 60%, 70%, 80%, 90%, 95% or 99%. Loci or alleles that are inherited in this way are said to be linked, and are referred to as “linkage groups”.

The “probability value” or “p-value” is the statistical likelihood that the particular combination of a phenotype and the presence or absence of a particular marker is random. The lower the probability value, the greater the likelihood that a phenotype and a particular marker will co-segregate. In some aspects, the probability value is considered “significant” or “non-significant”. In some embodiments, a probability value of 0.05 (p=0.05, or a 5% probability) of random assortment is considered a significant indication of co-segregation. However, an acceptable probability can be any probability of less than 50% (p=0.5). For example, a significant probability can be less than 0.25, less than 0.2, less than 0.15, less than 0.1, less than 0.05, less than 0.01 or less than 0.001.

The term “marker locus” is a specific chromosome location in the genome of a species where a specific marker can be found. A marker locus can be used to track the presence of a second linked locus, e.g., a linked locus that encodes or contributes to expression of a phenotypic trait. For example, a marker locus can be used to monitor segregation of alleles at a locus, such as a QTL or single gene, that are genetically or physically linked to the marker locus.

The term “marker” is a nucleotide sequence or encoded product thereof (e.g., a protein) used as a point of reference. For markers to be useful at detecting recombinations, they need to detect differences, or polymorphisms, within the population being monitored.

For molecular markers, this means differences at the DNA level due to polynucleotide sequence differences (e.g., SSRs, RFLPs, FLPs, SNPs). The genomic variability can be of any origin, for example, insertions, deletions, duplications, repetitive elements, point mutations, recombination events, or the presence and sequence of transposable elements. Molecular markers can be derived from genomic or expressed nucleic acids (e.g., ESTs) and can refer also to nucleic acids used as probes or primer pairs capable of amplifying sequence fragments via the use of PCR-based methods. A large number of Brassica molecular markers are known in the art, and are published or available from various sources.

Examples of markers associated with shatter-resistance are provided, in SEQ ID NOS: 1-156 and SEQ ID NOS: 761-774. It will be understood by one skilled in the art that a marker of the present invention may comprise the entire sequence of any one of the sequences set out in SEQ ID NOS: 1-156 and SEQ ID NOS: 761-774, or a fragment of such a sequence. The fragment can be, for example, the SNPs (as highlighted, for example, in Table 7, or sequences that flank and includes the SNPs. It will also be understood by one skilled in the art that the sequences of markers such as those set out in any of SEQ ID NOS: 1-156 and SEQ ID NOS: 761-774 or a fragment of such a sequence will have some variation. Therefore, the markers of the present invention include sequences that have 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence as provided in any of SEQ ID NOS: 1-156 and SEQ ID NOS: 761-774 or a fragment thereof.

Markers corresponding to genetic polymorphisms between members of a population can be detected by methods well-established in the art. These include, e.g., DNA sequencing, PCR-based sequence specific amplification methods, detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, detection of polynucleotide polymorphisms by allele specific hybridization (ASH), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of simple sequence repeats (SSRs), detection of single nucleotide polymorphisms (SNPs), or detection of amplified fragment length polymorphisms (AFLPs). Well established methods are also known for the detection of expressed sequence tags (ESTs) and SSR markers derived from EST sequences and randomly amplified polymorphic DNA (RAPD).

The term “molecular marker” may be used to refer to any type of nucleic acid based marker, or an encoded product thereof (e.g., a protein) used as a point of reference when identifying a linked locus. A marker can be derived from genomic nucleotide sequences or from expressed nucleotide sequences (e.g., from a spliced RNA, a cDNA, etc.), or from an encoded polypeptide. The term also refers to nucleic acid sequences complementary to or flanking the marker sequences, such as nucleic acids used as probes or primer pairs capable of amplifying the marker sequence. A “molecular marker probe” is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence. Alternatively, in some aspects, a molecular marker probe refers to a probe of any type that is able to distinguish (i.e., genotype) the particular allele that is present at a marker locus. Nucleic acids are “complementary” when they specifically hybridize in solution, e.g., according to Watson-Crick base pairing rules. Any suitable marker detection technology may be used to identify such a hybridization marker, e.g., SNP technology is predominantly used in the examples provided herein. A “marker allele”, alternatively an “allele of a marker locus”, can refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus in a population that is polymorphic for the marker locus.

The term “interval” refers to a continuous linear span of chromosomal DNA with termini that are typically defined by and including molecular markers.

The terms “nucleic acid,” “nucleotide”, “polynucleotide,” “polynucleotide sequence” and “nucleic acid sequence” refer to single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymers, or chimeras thereof. As used herein, the term can additionally or alternatively include analogs of naturally occurring nucleotides having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). Unless otherwise indicated, a particular nucleic acid sequence of this invention optionally encompasses complementary sequences, in addition to the sequence explicitly indicated.

The term “gene” is used to refer to, e.g., a cDNA and an mRNA encoded by the genomic sequence, as well as to that genomic sequence.

The term “homologous” refers to nucleic acid sequences that are derived from a common ancestral gene through natural or artificial processes (e.g., are members of the same gene family), and thus, typically, share sequence similarity. Typically, homologous nucleic acids have sufficient sequence identity that one of the sequences or its complement is able to selectively hybridize to the other under selective hybridization conditions. The term “selectively hybridizes” includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences have about at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with each other. A nucleic acid that exhibits at least some degree of homology to a reference nucleic acid can be unique or identical to the reference nucleic acid or its complementary sequence.

The term “isolated” refers to material, such as a nucleic acid or a protein, which is substantially free from components that normally accompany or interact with it in its naturally occurring environment. The isolated material optionally comprises material not found with the material in its natural environment, e.g., a cell. In addition, if the material is in its natural environment, such as a cell, the material has been placed at a location in the cell (e.g., genome or subcellular organelle) not native to a material found in that environment. For example, a naturally occurring nucleic acid (e.g., a promoter) is considered to be isolated if it is introduced by non-naturally occurring means to a locus of the genome not native to that nucleic acid. Nucleic acids that are “isolated” as defined herein, are also referred to as “heterologous” nucleic acids. In certain embodiments, the isolated nucleic acids described herein are operably linked to or inserted within a heterologous sequence. Such a heterologous sequence may be a sequence within a different plant genome, or it may be a sequence within a vector, as explained below.

The term “recombinant” indicates that the material (e.g., a nucleic acid or protein) has been synthetically (non-naturally) altered by human intervention. The alteration to yield the synthetic material can be performed on the material within or removed from its natural environment or state. For example, a naturally occurring nucleic acid is considered a recombinant nucleic acid if it is altered, or if it is transcribed from DNA that has been altered, by means of human intervention performed within the cell from which it originates. See, e.g., Compounds and Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In Vivo Homologous Sequence Targeting in Eukaryotic Cells; Zarling et al., PCT/US93/03868.

The term “introduced” when referring to a heterologous or isolated nucleic acid refers to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid can be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA). The term includes such nucleic acid introduction means as “transfection,” “transformation” and “transduction.

The terms “SSR” or “simple sequence repeat” refers to a polymorphic locus present in nuclear and organellar DNA that consist of repeating units of 1-6 base pairs in length. Different alleles can have different numbers of the repeating SSR, resulting in different lengths of the alleles, as detectable, for example, by gel electrophoresis after amplification of the allele. For example, a di-nucleotide repeat would be GAGAGAGA. It is believed that when DNA is being replicated, errors occur in the process and extra sets of these repeated sequences are added to the strand. Over time, these repeated sequences vary in length between one cultivar and another. An example of an allelic variation in SSRs would be: Allele A: 4 repeats of the GA sequence and Allele B: 6 repeats of the GA sequence. These variations in length are easy to trace in the lab and allow tracking of genotypic variation in breeding programs.

The term “microsatellite” is an alternative term for SSR.

The term “single nucleotide polymorphism” or “SNP” is a DNA sequence variation occurring when a single nucleotide—A, T, C, or G—in the genome (or other shared sequence) differs between members of a species (or between paired chromosomes in an individual). For example, two sequenced DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide. In this case we say that there are two alleles: C and T. Almost all common SNPs have only two alleles.

The term “host cell” means a cell that contains a heterologous nucleic acid, such as a vector, and supports the replication and/or expression of the nucleic acid. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells. The host cells can be monocotyledonous or dicotyledonous plant cells. The dicotyledonous host cell can be, for example, a canola host cell.

The term “transgenic plant” refers to a plant that comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. “Transgenic” is used herein to refer to any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenic organisms or cells initially so altered, as well as those created by crosses or asexual propagation from the initial transgenic organism or cell. The term “transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods (i.e., crosses) or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

The term “dicot” refers to the subclass of angiosperm plants also knows as “dicotyledoneae” and includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of the same. Plant cell, as used herein includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.

The term “crossed” or “cross” in the context of this invention means the fusion of gametes via pollination to produce progeny (i.e., cells, seeds, or plants). The term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, i.e., when the pollen and ovule are from the same plant).

The term “introgression” refers to the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny plant via a sexual cross between two parent plants, where at least one of the parent plants has the desired allele within its genome. Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be, e.g., a transgene or a selected allele of a marker or QTL.

Markers

The present invention provides molecular markers genetically linked to quantitative trait loci (“QTLs”) associated with resistance to shatter in Brassica. Such molecular markers are useful for identifying and producing dicotyledonous plants, in particular, such commercially important dicot crops as sunflower, canola, alfalfa, and soybean, displaying resistance to shatter.

Genetic mapping of several hundred molecular markers has developed a genetic linkage map covering approximately 1700 cM (centiMorgans) corresponding to 19 canola chromosomes. Additional details regarding the nature and use of molecular markers are provided below in the section entitled “Marker Assisted Selection and Breeding of Plants,” and in the Examples.

Exemplary marker loci associated with resistance to shatter are localized to the following linkage groups in Brassica napus: N1, N3, N4, N6, N7, N9, N13, N14, N15, N18 and N19. These exemplary marker loci delineate chromosomal intervals including quantitative trait loci (QTL's) associated with phenotypic measures of shatter resistance. For example, Tables 4 and 5 list markers that localize to those linkage groups and set out the intervals on the linkage groups that define the QTLs associated with shatter resistance; for instance: (a) an interval flanked by and including markers N20003-001-Q001 and N23426-001-Q001 on linkage group N1; (b) an interval flanked by and including markers N05671-1-Q1 and N12643-001-Q001 on linkage group N3; (c) an interval flanked by and including markers N05943-1-Q1 and N88537-001-K001 on linkage group N4; (d) an interval flanked by and including markers N07541-1-Q1 and N14649-001-Q001 on linkage group N6; (e) one or more intervals flanked by and including: (i) markers N23310-001-Q001 and N23409-001-Q001 on linkage group N7, or (ii) markers N07278-1-Q1 and N23417-001-Q001 on linkage group N7; (f) one or more intervals flanked by and including: (i) markers N23119-001-Q001 and N20380-001-Q001 on linkage group N9, or (ii) markers NO5490-1-Q1 and N20834-001-Q001 on linkage group N9; (g) one or more intervals flanked by and including: (i) markers N21144-001-Q001 and N09862-001-Q001 on linkage group N13, or (ii) markers N22903-001-Q001 and N12902-001-Q001 on linkage group N13; (h) one or more intervals flanked by and including: (i) markers N23033-001-Q001 and N22724-001-Q001 on linkage group N14, or (ii) markers N23033-001-Q001 and N22802-001-Q001 on linkage group N14; (i) an interval flanked by and including markers N12785-001-Q001 and N19296-001-Q001 on linkage group N15; (j) one or more intervals flanked by and including: (i) markers N05205-1-Q1 and N22925-001-Q001 on linkage group N18, or (ii) markers N22803-001-Q001 and N18401-001-Q001 on linkage group N18; and (k) an interval flanked by and including markers N05656-1-Q1 and N16006-001-Q001 on linkage group N19. As described in detail herein, primers and probes corresponding to these markers or fragments of these markers can be designed based on the sequence information provided herein.

The following markers (sometimes referred to as “the markers exemplified by SEQ ID NOs: 1-156 and SEQ ID NOS: 761-774”) contain single nucleotide polymorphisms (SNPs) or simple sequence repeats (SSRs) that identify QTLs contributing to shatter resistance and can be used as markers thereof: N20003-001-Q001 (SEQ ID NO: 1); N03491-1-Q1 (SEQ ID NO:2); N0017NR-001-Q001 (SEQ ID NO:3); N10336-001-Q001 (SEQ ID NO:4); N23133-001-Q001 (SEQ ID NO:5); N16487-001-Q001 (SEQ ID NO:6); N23426-001-Q001 (SEQ ID NO:7); N05671-1-Q1 (SEQ ID NO:8); N12643-001-Q001 (SEQ ID NO:9); N05943-1-Q1 (SEQ ID NO:10); N06007-1-Q1 (SEQ ID NO: 11); N10105-001-Q001 (SEQ ID NO:12); N08181-1-Q1 (SEQ ID NO:13); N06675-1-Q1 (SEQ ID NO:14); N001KH2-001-Q001 (SEQ ID NO:15); N29313-001-Q001 (SEQ ID NO:16); N88512-001-K001 (SEQ ID NO:17); N88514-001-K001 (SEQ ID NO:18); N88515-001-K001 (SEQ ID NO:19); N88516-001-K001 (SEQ ID NO:20); N88517-001-K001 (SEQ ID NO:21); N88518-001-K001 (SEQ ID NO:22); N88519-001-K001 (SEQ ID NO:23); N88520-001-K001 (SEQ ID NO:24); N88521-001-K001 (SEQ ID NO:25); N001KFE-001-Q001 (SEQ ID NO:26); N88522-001-K001 (SEQ ID NO:27); N88523-001-K001 (SEQ ID NO:28); N88524-001-K001 (SEQ ID NO:29); N88525-001-K001 (SEQ ID NO:30); N88529-001-K001 (SEQ ID NO:31); N88530-001-K001 (SEQ ID NO:32); N88531-001-K001 (SEQ ID NO:33); N88533-001-K001 (SEQ ID NO:34); N88535-001-K001 (SEQ ID NO:35); N88536-001-K001 (SEQ ID NO:36); N88537-001-K001 (SEQ ID NO:37); N07541-1-Q1 (SEQ ID NO:38); N23413-001-Q001 (SEQ ID NO:39); N08344-1-Q1 (SEQ ID NO:40); N23533-001-Q011 (SEQ ID NO:41); N14649-001-Q001 (SEQ ID NO:42); N23310-001-Q001 (SEQ ID NO:43); N10526-001-Q001 (SEQ ID NO:44); N23373-001-Q001 (SEQ ID NO:45); N23353-001-Q001 (SEQ ID NO:46); N23206-001-Q001 (SEQ ID NO:47); N11025-001-Q001 (SEQ ID NO:48); N09969-001-Q001 (SEQ ID NO:49); N09882-001-Q001 (SEQ ID NO:50); N10389-001-Q001 (SEQ ID NO:51); N09940-001-Q001 (SEQ ID NO:52); N23409-001-Q001 (SEQ ID NO:53); N23119-001-Q001 (SEQ ID NO:54); N09861-001-Q001 (SEQ ID NO:55); N04807-1-Q1 (SEQ ID NO:56); N06778-1-Q1 (SEQ ID NO:57); N09897-001-Q001 (SEQ ID NO:58); N10499-001-Q001 (SEQ ID NO:59); N23447-001-Q001 (SEQ ID NO:60); N19834-001-Q001 (SEQ ID NO:61); N23362-001-Q001 (SEQ ID NO:62); N23266-001-Q001 (SEQ ID NO:63); N19862-001-Q001 (SEQ ID NO:64); N22187-001-Q001 (SEQ ID NO:65); N08651-1-Q1 (SEQ ID NO:66); N23296-001-Q001 (SEQ ID NO:67); N17314-001-Q001 (SEQ ID NO:68); N20380-001-Q001 (SEQ ID NO:69); N05490-1-Q1 (SEQ ID NO:70); N18849-001-Q001 (SEQ ID NO:71); N08200-1-Q1 (SEQ ID NO:72); N19827-001-Q001 (SEQ ID NO:73); N001R9W-001-Q001 (SEQ ID NO:74); N08264-1-Q1 (SEQ ID NO:75); N23132-001-Q001 (SEQ ID NO:76); N03615-1-Q1 (SEQ ID NO:77); N001RWT-001-Q001 (SEQ ID NO:78); N08465-1-Q1 (SEQ ID NO:79); N10774-001-Q001 (SEQ ID NO:80); N17035-001-Q001 (SEQ ID NO:81); N20834-001-Q001 (SEQ ID NO:82); N22903-001-Q001 (SEQ ID NO:83); N09920-001-Q001 (SEQ ID NO:84); N22822-001-Q001 (SEQ ID NO:85); N22688-001-Q001 (SEQ ID NO:86); N10074-001-Q001 (SEQ ID NO:87); N10057-001-Q001 (SEQ ID NO:88); N10086-001-Q001 (SEQ ID NO:89); N11084-001-Q001 (SEQ ID NO:90); N22814-001-Q001 (SEQ ID NO:91); N01564-2-Q1 (SEQ ID NO:92); N12902-001-Q001 (SEQ ID NO:93); N21144-001-Q001 (SEQ ID NO:94); N07534-1-Q1 (SEQ ID NO:95); N22993-001-Q001 (SEQ ID NO:96); N09963-001-Q001 (SEQ ID NO:97); N11542-001-Q001 (SEQ ID NO:98); N14681-001-Q001 (SEQ ID NO:99); N11636-001-Q001 (SEQ ID NO:100); N13732-001-Q001 (SEQ ID NO: 101); N11255-001-Q001 (SEQ ID NO: 102); N15511-001-Q001 (SEQ ID NO: 103); N10536-001-Q001 (SEQ ID NO:104); N09862-001-Q001 (SEQ ID NO:105); N23033-001-Q001 (SEQ ID NO:106); N06039-1-Q1 (SEQ ID NO:107); N10016-001-Q001 (SEQ ID NO: 108); N22743-001-Q001 (SEQ ID NO: 109); N22953-001-Q001 (SEQ ID NO: 110); N09987-001-Q001 (SEQ ID NO:111); N10092-001-Q001 (SEQ ID NO: 112); N10096-001-Q001 (SEQ ID NO: 113); N22728-001-Q001 (SEQ ID NO: 114); N22747-001-Q001 (SEQ ID NO:115); N22840-001-Q001 (SEQ ID NO:116); N23027-001-Q001 (SEQ ID NO: 117); N22777-001-Q001 (SEQ ID NO: 118); N09636-001-Q001 (SEQ ID NO: 119); N09879-001-Q001 (SEQ ID NO: 120); N10123-001-Q001 (SEQ ID NO:121); N10316-001-Q001 (SEQ ID NO: 122); N10507-001-Q001 (SEQ ID NO: 123); N09834-001-Q001 (SEQ ID NO:124); N22934-001-Q001 (SEQ ID NO:125); N22700-001-Q001 (SEQ ID NO: 126); N22725-001-Q001 (SEQ ID NO: 127); N22881-001-Q001 (SEQ ID NO: 128); N23032-001-Q001 (SEQ ID NO:129); N22786-001-Q001 (SEQ ID NO:130); N23014-001-Q001 (SEQ ID NO:131); N10471-001-Q001 (SEQ ID NO:132); N11419-001-Q001 (SEQ ID NO: 133); N22724-001-Q001 (SEQ ID NO: 134); N12785-001-Q001 (SEQ ID NO: 135); N09910-001-Q001 (SEQ ID NO:136); N21146-001-Q001 (SEQ ID NO:137); N17618-001-Q001 (SEQ ID NO:138); N09776-001-Q001 (SEQ ID NO:139); N19296-001-Q001 (SEQ ID NO:140); N05205-1-Q1 (SEQ ID NO:141); N10406-001-Q001 (SEQ ID NO:142); N22941-001-Q001 (SEQ ID NO:143); N22875-001-Q001 (SEQ ID NO:144); N13286-001-Q001 (SEQ ID NO:145); N04503-1-Q1 (SEQ ID NO:146); N22925-001-Q001 (SEQ ID NO: 147); N05656-1-Q1 (SEQ ID NO: 148); N17581-001-Q001 (SEQ ID NO: 149); N001NVH-001-Q001 (SEQ ID NO: 150); N22928-001-Q001 (SEQ ID NO: 151); N08219-1-Q001 (SEQ ID NO:152); N05710-1-Q1 (SEQ ID NO:153); N15338-001-Q001 (SEQ ID NO:154); N10424-001-Q001 (SEQ ID NO:155); N16006-001-Q001 (SEQ ID NO:156); N07278-1-Q1 (SEQ ID NO: 761); N16343-001-Q001 (SEQ ID NO: 762); N23417-001-Q001 (SEQ ID NO: 763); N22902-001-Q001 (SEQ ID NO: 764); N23063-001-Q001 (SEQ ID NO: 765); N22723-001-Q001 (SEQ ID NO: 766); N23049-001-Q001 (SEQ ID NO: 767); N10321-001-Q001 (SEQ ID NO: 768); N15374-001-Q001 (SEQ ID NO: 769); N22802-001-Q001 (SEQ ID NO: 770), N22803-001-Q001 (SEQ ID NO: 771), N18929-001-Q001 (SEQ ID NO: 772); N16041-001-Q001 (SEQ ID NO: 773); and N18401-001-Q001 (SEQ ID NO: 774). It will be appreciated that the number of repeats in any SSR can vary. Favorable alleles that contribute to shatter resistance are provided, for example, in Table 6.

It will be noted that, regardless of their molecular nature, e.g., whether the marker is a SNP, SSR, AFLP, RFLP, etc., markers are typically strain specific. That is, a particular polymorphic marker, such as the exemplary markers of the invention described above, is defined relative to the parental lines of interest. For each marker locus, resistance-associated, and conversely, susceptibility-associated alleles are identified for each pair of parental lines. Following correlation of specific alleles with susceptibility and resistance in parents of a cross, the marker can be utilized to identify progeny with genotypes that correspond to the desired resistance phenotype. In some circumstance, i.e., in some crosses of parental lines, the exemplary markers described herein will not be optimally informative.

In such cases, additional informative markers, e.g., certain linked markers and/or homologous markers are evaluated and substituted for genotyping, e.g., for marker-assisted selection, etc. In the case where a marker corresponds to a QTL, following identification of resistance- and susceptibility-associated alleles, it is possible to directly screen a population of samples, e.g., samples obtained from a seed bank, without first correlating the parental phenotype with an allele.

Linked Markers

Those of skill in the art will recognize that additional molecular markers can be identified within the intervals defined by the above-described pairs of markers. Such markers are also genetically linked to the QTLs identified herein as associated with shatter resistance, and are within the scope of the present invention. Markers can be identified by any of a variety of genetic or physical mapping techniques. Methods of determining whether markers are genetically linked to a QTL (or to a specified marker) associated with shatter resistance are known to those of skill in the art and include, e.g., interval mapping (Lander and Botstein (1989) Genetics 121:185), regression mapping (Haley and Knott (1992) Heredity 69:315) or MQM mapping (Jansen (1994) Genetics 138:871). In addition, such physical mapping techniques as chromosome walking, contig mapping and assembly, and the like, can be employed to identify and isolate additional sequences useful as markers in the context of the present invention.

Homologous Nucleotide Sequences

In addition, the markers exemplified by SEQ ID NOs: 1-156 and SEQ ID NOS: 761-774 are useful for the identification of homologous nucleotide sequences with utility in identifying QTLs associated with shatter resistance in different lines, varieties, or species of dicots. Such homologous markers are a feature of the invention.

Such homologous sequences can be identified by selective hybridization to a reference sequence. The reference sequence is typically a unique sequence, such as a unique oligonucleotide primer sequence, EST, amplified fragment (e.g., corresponding to AFLP markers) and the like, derived from any of the marker loci listed herein or its complement.

Two single-stranded nucleic acids “hybridize” when they form a double-stranded duplex. The double stranded region can include the full-length of one or both of the single-stranded nucleic acids, or all of one single stranded nucleic acid and a subsequence of the other single-stranded nucleic acid, or the double stranded region can include a subsequence of each nucleic acid. Selective hybridization conditions distinguish between nucleic acids that are related, e.g., share significant sequence identity with the reference sequence (or its complement) and those that associate with the reference sequence in a non-specific manner. Generally, selective hybridization conditions are those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Selective hybridization conditions may also be achieved with the addition of destabilizing agents such as formamide. Selectivity can be achieved by varying the stringency of the hybridization and/or wash conditions. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically a function of post-hybridization washes, with the critical factors being ionic strength and temperature of the final wash solution. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (T_m) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (T_m); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (T_m); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (T_m).

The T_m, is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. For DNA-DNA hybrids, the T_m, can be approximated from the equation of Meinkoth and Wahl ((1984) Anal. Biochem. 138:267-284): T_m, =81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form) 500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. T_m, is reduced by about 1° C. for each 1% of mismatching; thus, T_m, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T_m, can be decreased 10° C.

Using the equation, hybridization and wash compositions, and desired T_m, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T_m, of less than 45° C. (aqueous solution) or 32° C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. Hybridization and/or wash conditions can be applied for at least 10, 30, 60, 90, 120, or 240 minutes. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York. General Texts that discuss considerations relevant to nucleic acid hybridization, the selection of probes, and buffer and incubation conditions, and the like, as well as numerous other topics of interest in the context of the present invention (e.g., cloning of nucleic acids that correspond to markers and QTLs, sequencing of cloned markers/QTLs, the use of promoters, vectors, etc.) can be found in Berger and Kimmel (1987) Guide to Molecular Cloning Techniques, Methods in Enzymology vol. 152, Academic Press, Inc., San Diego (“Berger”); Sambrook et al., (2001) Molecular Cloning—A Laboratory Manual, 3^rded. Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor (“Sambrook”); and Ausubel et al., (eds) (supplemented through 2001) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., (“Ausubel”).

In addition to hybridization methods described above, homologs of the markers of the invention can be identified in silico using any of a variety of sequence alignment and comparison protocols. For the purposes of the ensuing discussion, the following terms are used to describe the sequence relationships between a marker nucleotide sequence and a reference polynucleotide sequence.

A “reference sequence” is a defined sequence used as a basis for sequence comparison with a test sequence, e.g., a candidate marker homolog, of the present invention. A reference sequence may be a subsequence or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

As used herein, a “comparison window” is a contiguous and specified segment, (e.g., a subsequence) of a polynucleotide/polypeptide sequence to be compared to a reference sequence. The segment of the polynucleotide/polypeptide sequence in the comparison window can include one or more additions or deletions (i.e., gaps) with respect to the reference sequence, which (by definition) does not comprise addition(s) or deletion(s), for optimal alignment of the two sequences. An optimal alignment of two sequences yields the fewest number of unlike nucleotide/amino acid residues in a comparison window. Generally, the comparison window is at least 20 contiguous nucleotide/amino acid residues in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a falsely high similarity between two sequences, due to inclusion of gaps in the polynucleotide/polypeptide sequence, a gap penalty is typically assessed and is subtracted from the number of matches.

“Sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences refers to residues that are the same in both sequences when aligned for maximum correspondence over a specified comparison window.

“Percentage sequence identity” refers to the value determined by comparing two optimally aligned sequences over a comparison window. The percentage is calculated by determining the number of positions at which both sequences have the same nucleotide or amino acid residue, determining the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity.

When percentage of sequence identity is used in reference to proteins it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ by conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller (1988) Computer Applic. Biol. Sci. 4:11-17, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).

Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman ((1981) Adv. Appl. Math. 2:482); by the homology alignment algorithm of Needleman and Wunsch ((1970) J. Mol. Biol. 48:443); by the search for similarity method of Pearson and Lipman ((1988) Proc. Natl. Acad. Sci. USA 85:2444); by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), Madison, Wis., USA; the CLUSTAL program is well described by Higgins and Sharp ((1988) Gene 73:237-244); Higgins and Sharp ((1989) CABIOS 5:151-153); Corpet et al. ((1988) Nucleic Acids Research 16:10881-90); Huang et al. ((1992) Computer Applications in the Biosciences 8: 155-65), and Pearson et al. ((1994) Methods in Molecular Biology 24:307-331).

The BLAST family of programs that can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, e.g., Current Protocols in Molecular Biology, Chapter 19, Ausubel et al., Eds., (1995) Greene Publishing and Wiley-Interscience, New York; Altschul et al. (1990) J. Mol. Biol. 215:403-410; and, Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.

Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad. Sci. USA 90:5873-5877). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.

BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences that may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen (1993) Comput. Chem. 17:149-163) and XNU (Claverie and States (1993) Comput. Chem. 17:191-201) low-complexity filters can be employed alone or in combination.

Unless otherwise stated, nucleotide and protein identity/similarity values provided herein are calculated using GAP (CGC Version 10) under default values.

GAP (Global Alignment Program) can also be used to compare a polynucleotide or polypeptide of the present invention with a reference sequence. GAP uses the algorithm of Needleman and Wunsch ((1970) J. Mol. Biol. 48: 443-453), to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100. Thus, for example, the gap creation and gap extension penalties can each independently be: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60 or greater.

GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see, e.g., Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

Multiple alignment of the sequences can be performed using the CLUSTAL method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the CLUSTAL method are KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The percentage sequence identity of a homologous marker to its reference marker (e.g., any one of the markers described herein) is typically at least 70% and, rounded upwards to the nearest integer, can be expressed as an integer selected from the group of integers between 70 and 99. Thus, for example, the percentage sequence identity to a reference sequence can be at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99%. Sequence identity can be calculated using, for example, the BLAST, CLUSTALW, or GAP algorithms under default conditions.

Detection of Marker Loci

Markers corresponding to genetic polymorphisms between members of a population can be detected by numerous methods, well-established in the art (e.g., restriction fragment length polymorphisms, isozyme markers, allele specific hybridization (ASH), amplified variable sequences of the plant genome, self-sustained sequence replication, simple sequence repeat (SSR), single nucleotide polymorphism (SNP), or amplified fragment length polymorphisms (AFLP)).

The majority of genetic markers rely on one or more properties of nucleic acids for their detection. For example, some techniques for detecting genetic markers utilize hybridization of a probe nucleic acid to nucleic acids corresponding to the genetic marker. Hybridization formats include but are not limited to, solution phase, solid phase, mixed phase, or in situ hybridization assays. Markers that are restriction fragment length polymorphisms (RFLP), are detected by hybridizing a probe, which is typically a sub-fragment (or a synthetic oligonucleotide corresponding to a sub-fragment) of the nucleic acid to be detected to restriction digested genomic DNA. The restriction enzyme is selected to provide restriction fragments of at least two alternative (or polymorphic) lengths in different individuals, and will often vary from line to line. Determining a (one or more) restriction enzyme that produces informative fragments for each cross is a simple procedure, well known in the art. After separation by length in an appropriate matrix (e.g., agarose) and transfer to a membrane (e.g., nitrocellulose, nylon), the labeled probe is hybridized under conditions that result in equilibrium binding of the probe to the target followed by removal of excess probe by washing.

Nucleic acid probes to the marker loci can be cloned and/or synthesized. Detectable labels suitable for use with nucleic acid probes include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels include biotin for staining with labeled streptavidin conjugate, magnetic beads, fluorescent dyes, radiolabels, enzymes, and colorimetric labels. Other labels include ligands that bind to antibodies labeled with fluorophores, chemiluminescent agents, and enzymes. Labeling markers is readily achieved such as by the use of labeled PCR primers to marker loci.

The hybridized probe is then detected using, most typically by autoradiography or other similar detection technique (e.g., fluorography, liquid scintillation counter, etc.). Examples of specific hybridization protocols are widely available in the art, see, e.g., Berger, Sambrook, Ausubel, all supra.

Amplified variable sequences refer to amplified sequences of the plant genome that exhibit high nucleic acid residue variability between members of the same species. All organisms have variable genomic sequences and each organism (with the exception of a clone) has a different set of variable sequences. Once identified, the presence of specific variable sequence can be used to predict phenotypic traits. Preferably, DNA from the plant serves as a template for amplification with primers that flank a variable sequence of DNA. The variable sequence is amplified and then sequenced.

In vitro amplification techniques are well known in the art. Examples of techniques sufficient to direct persons of skill through such in vitro methods, including the polymerase chain reaction (PCR), the ligase chain reaction (LCR), Q13-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA), are found in Berger, Sambrook and Ausubel (all supra) as well as Mullis et al. ((1987) U.S. Pat. No. 4,683,202); PCR Protocols, A Guide to Methods and Applications ((Innis et al., eds.) Academic Press Inc., San Diego Academic Press Inc. San Diego, Calif. (1990) (Innis)); Arnheim & Levinson ((Oct. 1, 1990) C&EN 36-47); The Journal Of NIH Research (1991) 3, 81-94; Kwoh et al. ((1989) Proc. Natl. Acad. Sci. USA 86, 1173); Guatelli et al. ((1990) Proc. Natl. Acad. Sci. USA 87, 1874); Lomell et al. ((1989) J. Clin. Chem. 35, 1826); Landegren et al. ((1988) Science 241, 1077-1080); Van Brunt ((1990) Biotechnology 8, 291-294); Wu and Wallace ((1989) Gene 4, 560); Barringer et al. ((1990) Gene 89, 117), and Sooknanan and Malek ((1995) Biotechnology 13: 563-564). Improved methods of cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improved methods of amplifying large nucleic acids by PCR are summarized in Cheng et al. (1994) Nature 369: 684, and the references therein, in which PCR amplicons of up to 40 kb are generated. One of skill will appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See, Ausubel, Sambrook and Berger, all supra. Oligonucleotides for use as primers, e.g., in amplification reactions and for use as nucleic acid sequence probes are typically synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers ((1981) Tetrahedron Lett. 22:1859), or can simply be ordered commercially.

Alternatively, self-sustained sequence replication can be used to identify genetic markers. Self-sustained sequence replication refers to a method of nucleic acid amplification using target nucleic acid sequences that are replicated exponentially in vitro under substantially isothermal conditions by using three enzymatic activities involved in retroviral replication: (1) reverse transcriptase, (2) Rnase H, and (3) a DNA-dependent RNA polymerase (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874). By mimicking the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target.

Amplified fragment length polymorphisms (AFLP) can also be used as genetic markers (Vos et al. (1995) Nucl. Acids Res. 23:4407. The phrase “amplified fragment length polymorphism” refers to selected restriction fragments that are amplified before or after cleavage by a restriction endonuclease. The amplification step allows easier detection of specific restriction fragments. AFLP allows the detection large numbers of polymorphic markers and has been used for genetic mapping of plants (Becker et al. (1995) Mol. Gen. Genet. 249:65; and Meksem et al. (1995) Mol. Gen. Genet. 249:74.

Allele-specific hybridization (ASH) can be used to identify the genetic markers of the invention. ASH technology is based on the stable annealing of a short, single-stranded, oligonucleotide probe to a completely complementary single-strand target nucleic acid. Detection is via an isotopic or non-isotopic label attached to the probe.

For each polymorphism, two or more different ASH probes are designed to have identical DNA sequences except at the polymorphic nucleotides. Each probe will have exact homology with one allele sequence so that the range of probes can distinguish all the known alternative allele sequences. Each probe is hybridized to the target DNA. With appropriate probe design and hybridization conditions, a single-base mismatch between the probe and target DNA will prevent hybridization. In this manner, only one of the alternative probes will hybridize to a target sample that is homozygous or homogenous for an allele. Samples that are heterozygous or heterogeneous for two alleles will hybridize to both of two alternative probes.

ASH markers are used as dominant markers where the presence or absence of only one allele is determined from hybridization or lack of hybridization by only one probe. The alternative allele may be inferred from the lack of hybridization. ASH probe and target molecules are optionally RNA or DNA; the target molecules are any length of nucleotides beyond the sequence that is complementary to the probe; the probe is designed to hybridize with either strand of a DNA target; the probe ranges in size to conform to variously stringent hybridization conditions, etc.

PCR allows the target sequence for ASH to be amplified from low concentrations of nucleic acid in relatively small volumes. Otherwise, the target sequence from genomic DNA is digested with a restriction endonuclease and size separated by gel electrophoresis. Hybridizations typically occur with the target sequence bound to the surface of a membrane or, as described in U.S. Pat. No. 5,468,613, the ASH probe sequence may be bound to a membrane.

In one embodiment, ASH data are obtained by amplifying nucleic acid fragments (amplicons) from genomic DNA using PCR, transferring the amplicon target DNA to a membrane in a dot-blot format, hybridizing a labeled oligonucleotide probe to the amplicon target, and observing the hybridization dots by autoradiography.

Single nucleotide polymorphisms (SNP) are markers that consist of a shared sequence differentiated on the basis of a single nucleotide. Typically, this distinction is detected by differential migration patterns of an amplicon comprising the SNP on e.g., an acrylamide gel. However, alternative modes of detection, such as hybridization, e.g., ASH, or RFLP analysis are not excluded.

In yet another basis for providing a genetic linkage map, Simple sequence repeats (SSR), take advantage of high levels of di-, tri-, tetra-, penta- or hexa-nucleotide tandem repeats within a genome. Dinucleotide repeats have been reported to occur in the human genome as many as 50,000 times with n varying from 10 to 60 or more (Jacob et al. (1991) Cell 67:213. Dinucleotide repeats have also been found in higher plants (Condit and Hubbell (1991) Genome 34:66).

Briefly, SSR data are generated by hybridizing primers to conserved regions of the plant genome that flank the SSR sequence. PCR is then used to amplify the nucleotide repeats between the primers. The amplified sequences are then electrophoresed to determine the size and therefore the number of di-, tri-, and tetra-nucleotide repeats. The number of repeats distinguishes the favorable allele from an unfavorable allele.

Alternatively, isozyme markers are employed as genetic markers. Isozymes are multiple forms of enzymes that differ from one another in their amino acid, and therefore their nucleic acid sequences. Some isozymes are multimeric enzymes containing slightly different subunits. Other isozymes are either multimeric or monomeric but have been cleaved from the proenzyme at different sites in the amino acid sequence. Isozymes can be characterized and analyzed at the protein level, or alternatively, isozymes that differ at the nucleic acid level can be determined. In such cases, any of the nucleic acid based methods described herein can be used to analyze isozyme markers.

In alternative embodiments, in silico methods can be used to detect the marker loci. For example, the sequence of a nucleic acid comprising the marker can be stored in a computer. The desired marker locus sequence or its homolog can be identified using an appropriate nucleic acid search algorithm as provided by, for example, in such readily available programs as BLAST.

QTL Mapping

Multiple experimental paradigms have been developed to identify and analyze QTLs. In general, these paradigms involve crossing one or more parental pairs, which can be, for example, a single pair derived from two inbred strains, or multiple related or unrelated parents of different inbred strains or lines, which each exhibit different characteristics relative to the phenotypic trait of interest. The parents and a population of progeny are genotyped, typically for multiple marker loci, and evaluated for the trait of interest. In the context of the present invention, the parental and progeny plants are genotyped for any one or more of the molecular markers exemplified herein, or homologs, or alternative markers linked to any one or more of the markers exemplified herein, and evaluated for shatter resistance. QTLs associated with shatter resistance are identified based on the significant statistical correlations between the marker genotype(s) and the resistance phenotype of the evaluated progeny plants. Numerous methods for determining whether markers are genetically linked to a QTL (or to another marker) associated with shatter resistance are known to those of skill in the art and include, e.g., interval mapping (Lander and Botstein (1989) Genetics 121:185), regression mapping (Haley and Knott (1992) Heredity 69:315) or MQM mapping (Jansen (1994) Genetics 138:871). In addition, the following patent publications provide additional details regarding alternative statistical methods applicable to complex breeding populations that can be used to identify and localize QTLs associated with shatter resistance: U.S. Ser. No. 09/216,089 by Beavis et al. “QTL MAPPING IN PLANT BREEDING POPULATIONS” and PCT/US00/34971 by Jansen et al. “MQM MAPPING USING HAPLOTYPED PUTATIVE QTLS ALLELES: A SIMPLE APPROACH FOR MAPPING QTLS IN PLANT BREEDING POPULATIONS.”

Marker Assisted Selection and Breeding of Plants

A primary motivation for development of molecular markers in crop species is the potential for increased efficiency in plant breeding through marker assisted selection (MAS). Genetic marker alleles, or alternatively, identified QTL alleles, are used to identify plants that contain a desired genotype at one or more loci, and that are expected to transfer the desired genotype, along with a desired phenotype to their progeny. Genetic marker alleles (or QTL alleles) can be used to identify plants that contain a desired genotype at one locus, or at several unlinked or linked loci (e.g., a haplotype), and that would be expected to transfer the desired genotype, along with a desired phenotype to their progeny. The present invention provides the means to identify plants, particularly dicots, e.g., Brassica, that have resistance to shatter by identifying plants having a specified allele, e.g., at one or more of the markers exemplified herein, or other markers within the intervals set forth herein. Similarly, by identifying plants lacking a desired allele of the marker, susceptible plants can be identified, and eliminated from subsequent crosses, if desired. It will be appreciated that, for the purposes of MAS, the term marker can encompass both marker and QTL loci as both can be used to identify plants that display shatter resistance.

After a desired phenotype, e.g., shatter resistance, and a polymorphic chromosomal locus, e.g., a marker locus or QTL, are determined to segregate together, it is possible to use those polymorphic loci to select for alleles corresponding to the desired phenotype—a process called marker-assisted selection (MAS). In brief, a nucleic acid corresponding to the marker nucleic acid is detected in a biological sample from a plant to be selected. This detection can take the form of hybridization of a probe nucleic acid to a marker, e.g., using allele-specific hybridization, southern blot analysis, northern blot analysis, in situ hybridization, hybridization of primers followed by PCR amplification of a region of the marker or the like. A variety of procedures for detecting markers are described herein, e.g., in the section entitled “DETECTION OF MARKER LOCI.” After the presence (or absence) of a particular marker in the biological sample is verified, the plant is selected, i.e., used to make progeny plants by selective breeding.

Plant breeders need to combine stress tolerant loci with genes for high yield and other desirable traits to develop improved plant varieties. Screening for large numbers of samples can be expensive, time consuming, and unreliable. Use of the polymorphic loci described herein, and genetically-linked nucleic acids, as genetic markers for shatter resistance loci is an effective method for selecting tolerant varieties in breeding programs. For example, one advantage of marker-assisted selection over field evaluations for shatter resistance is that MAS can be done at any time of year regardless of the growing season. Moreover, environmental effects are irrelevant to marker-assisted selection. When a population is segregating for multiple loci affecting one or multiple traits, e.g., multiple loci involved in resistance to a single stress, or multiple loci each involved in resistance to different stresses, the efficiency of MAS compared to phenotypic screening becomes even greater because all the loci can be processed in the lab together from a single sample of DNA. In the present instance, this means that multiple markers selected from among the markers exemplified by SEQ ID NOs: 1-156 and SEQ ID NOS: 761-774 or markers homologous or linked thereto can be assayed simultaneously or sequentially in a single sample or population of samples. Thus, any one or more of these markers, e.g., two or more, up to and including all of the established markers, can be assayed simultaneously. In some instances, it is desirable to evaluate a marker corresponding to each of the linkage groups associated with shatter resistance.

Another use of MAS in plant breeding is to assist the recovery of the recurrent parent genotype by backcross breeding. Backcross breeding is the process of crossing a progeny back to one of its parents. Backcrossing is usually done for the purpose of introgressing one or a few loci from a donor parent into an otherwise desirable genetic background from the recurrent parent. The more cycles of backcrossing that are done, the greater the genetic contribution of the recurrent parent to the resulting variety. This is often necessary, because tolerant plants may be otherwise undesirable, i.e., due to low yield, low fecundity, or the like. In contrast, strains that are the result of intensive breeding programs may have excellent yield, fecundity or the like, merely being deficient in one desired trait such as resistance to a particular stress (e.g., resistance to shatter).

The presence and/or absence of a particular genetic marker allele, or a homolog thereof, in the genome of a plant exhibiting a preferred phenotypic trait is determined by any method listed above, e.g., RFLP, AFLP, SSR, etc. If the nucleic acids from the plant are positive for a desired genetic marker, the plant can be selfed to create a true breeding line with the same genotype, or it can be crossed with a plant with the same marker or with other desired characteristics to create a sexually crossed hybrid generation.

As mentioned above, the skilled artisan will understand that the QTLs described herein represent regions of the genome comprising genes that contribute to the shatter resistance of a plant. Further, each QTL can contribute differently to that resistance level. Thus, breeding efforts are directed to increasing the number of those QTLs, particularly quantitatively significant QTLs, present in the germplasm. Early in a breeding program, fewer QTLs may be present in a particular germplasm, but that number will increase as the breeding program progresses. Thus, in certain embodiments, a plant exhibiting shatter resistance may contain at least 6 of the QTLs described herein. More particularly, the plant may contain at least 2 or 3 of the QTLs described herein. Yet more particularly, the plant may contain 4, 5, 6 or all of the QTLs described herein.

Positional Cloning

The molecular markers of the present invention and nucleic acids homologous thereto, can be used, as indicated previously, to identify additional linked marker loci, which can be cloned by well established procedures, e.g., as described in detail in Ausubel, Berger and Sambrook, supra. Similarly, the exemplified markers, as well as any additionally identified linked molecular markers can be used to physically isolate, e.g., by cloning, nucleic acids associated with QTLs contributing to shatter resistance. Such nucleic acids, i.e., linked to QTLs, have a variety of uses, including as genetic markers for identification of additional QTLs in subsequent applications of marker assisted selection (MAS).

These nucleic acids are first identified by their genetic linkage to markers of the present invention. Isolation of the nucleic acid of interest is achieved by any number of methods as discussed in detail in such references as Ausubel, Berger and Sambrook, supra, and Clark, Ed. (1997) Plant Molecular Biology: A Laboratory Manual Springer-Verlag, Berlin.

For example, positional gene cloning uses the proximity of a genetic marker to physically define an isolated chromosomal fragment that is linked to a QTL. The isolated chromosomal fragment can be produced by such well known methods as digesting chromosomal DNA with one or more restriction enzymes, or by amplifying a chromosomal region in a polymerase chain reaction (PCR), or alternative amplification reaction. The digested or amplified fragment is typically ligated into a vector suitable for replication, e.g., a plasmid, a cosmid, a phage, an artificial chromosome, or the like, and, optionally, expression of the inserted fragment. Markers that are adjacent to an open reading frame (ORF) associated with a phenotypic trait can hybridize to a DNA clone, thereby identifying a clone on which an ORF is located. If the marker is more distant, a fragment containing the open reading frame is identified by successive rounds of screening and isolation of clones, which together comprise a contiguous sequence of DNA, a “contig.” Protocols sufficient to guide one of skill through the isolation of clones associated with linked markers are found in, e.g., Berger, Sambrook and Ausubel, all supra.

Nucleic Acids in Proximity to Markers/Isolated Chromosome Intervals

The present invention provides isolated nucleic acids comprising a QTL associated with resistance to shatter. The QTL is in proximity to a marker described herein and/or is localized within an interval defined by two markers of the present invention wherein each marker flanks the QTL. Such nucleic acids and/or intervals can be utilized to identify homologous nucleic acids and/or can be used in the production of transgenic plants displaying improved shatter resistance conferred by the introduced QTL. The nucleic acid and/or chromosome interval comprising a QTL is isolated, e.g., cloned via positional cloning methods outlined above. A chromosome interval can contain one or more ORFs associated with resistance, and can be cloned on one or more individual vectors, e.g., depending on the size of the chromosome interval.

It will be appreciated that numerous vectors are available in the art for the isolation and replication of the nucleic acids of the invention. For example, plasmids, cosmids and phage vectors are well known in the art, and are sufficient for many applications (e.g., in applications involving insertion of nucleic acids ranging from less than 1 to about 20 kilobases (kb). In certain applications, it is advantageous to make or clone large nucleic acids to identify nucleic acids more distantly linked to a given marker, or to isolate nucleic acids in excess of 10-20 kb, e.g., up to several hundred kilobases or more, such as the entire interval between two linked markers, i.e., up to and including one or more centiMorgans (cM), linked to QTLs as identified herein. In such cases, a number of vectors capable of accommodating large nucleic acids are available in the art, these include, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), plant artificial chromosomes (PACs) and the like. For a general introduction to YACs, BACs, PACs and MACs as artificial chromosomes, see, e.g., Monaco and Larin (1994) Trends Biotechnol. 12:280. In addition, methods for the in vitro amplification of large nucleic acids linked to genetic markers are widely available (e.g., Cheng et al. (1994) Nature 369:684, and references therein). Cloning systems can be created or obtained from commercially; see, for example, Stratagene Cloning Systems, Catalogs 2000 (La Jolla, Calif.).

Generation of Transgenic Plants and Cells

The present invention also relates to host cells and organisms that are transformed with nucleic acids corresponding to QTLs and other genes identified according to the invention. For example, such nucleic acids include chromosome intervals, ORFs, and/or cDNAs or corresponding to a sequence or subsequence included within the identified chromosome interval or ORF. Additionally, the invention provides for the production of polypeptides corresponding to QTLs by recombinant techniques. Host cells are genetically engineered (i.e., transduced, transfected or transformed) with the vectors of this invention (i.e., vectors that comprise QTLs or other nucleic acids identified according to the methods of the invention and as described above) that include, for example, a cloning vector or an expression vector. Such vectors include, in addition to those described above, e.g., an Agrobacterium, a virus (such as a plant virus), a naked polynucleotide, or a conjugated polynucleotide. The vectors are introduced into plant tissues, cultured plant cells or plant protoplasts by a variety of standard methods including electroporation (From et al. (1985) Proc. Natl. Acad. Sci. USA 82; 5824), infection by viral vectors such as cauliflower mosaic virus (CaMV) (Hohn et al. (1982) Molecular Biology of Plant Tumors (Academic Press, New York, pp. 549-560); Howell U.S. Pat. No. 4,407,956), high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al. (1987) Nature 327; 70), use of pollen as vector (WO 85/01856), or use of Agrobacterium tumefaciens or A. rhizogenes carrying a T-DNA plasmid in which DNA fragments are cloned. The T-DNA plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens, and a portion is stably integrated into the plant genome (Horsch et al. (1984) Science 233; 496; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80; 4803). The method of introducing a nucleic acid of the present invention into a host cell is not critical to the instant invention. Thus, any method, e.g., including but not limited to the above examples, which provides for effective introduction of a nucleic acid into a cell or protoplast can be employed.

The engineered host cells can be cultured in conventional nutrient media modified as appropriate for such activities as, for example, activating promoters or selecting transformants. These cells can optionally be cultured into transgenic plants. Plant regeneration from cultured protoplasts is described in Evans et al. ((1983) “Protoplast Isolation and Culture,” Handbook of Plant Cell Cultures 1, 124-176 (MacMillan Publishing Co., New York); Davey ((1983) “Recent Developments in the Culture and Regeneration of Plant Protoplasts,” Protoplasts, pp. 12-29, (Birkhauser, Basel)); Dale ((1983) “Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops,” Protoplasts, pp. 31-41, (Birkhauser, Basel)); and Binding ((1985) “Regeneration of Plants,” Plant Protoplasts, pp. 21-73, (CRC Press, Boca Raton)).

The present invention also relates to the production of transgenic organisms, which may be bacteria, yeast, fungi, or plants, transduced with the nucleic acids, e.g., cloned QTLs of the invention. A thorough discussion of techniques relevant to bacteria, unicellular eukaryotes and cell culture may be found in references enumerated above and are briefly outlined as follows. Several well-known methods of introducing target nucleic acids into bacterial cells are available, any of which may be used in the present invention. These include: fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the cells with liposomes containing the DNA, electroporation, projectile bombardment (biolistics), carbon fiber delivery, and infection with viral vectors (discussed further, below), etc. Bacterial cells can be used to amplify the number of plasmids containing DNA constructs of this invention. The bacteria are grown to log phase and the plasmids within the bacteria can be isolated by a variety of methods known in the art (see, for instance, Sambrook). In addition, a plethora of kits are commercially available for the purification of plasmids from bacteria. For their proper use, follow the manufacturer's instructions (see, for example, EasyPrep™, FlexiPrep™, both from Pharmacia Biotech; StrataClean™, from Stratagene; and, QJAprep™ from Qiagen). The isolated and purified plasmids are then further manipulated to produce other plasmids, used to transfect plant cells or incorporated into Agrobacterium tumefaciens related vectors to infect plants. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. See, Giliman & Smith ((1979) Gene 8:81); Roberts et al. ((1987) Nature 328:731); (Schneider et al. (1995) Protein Expr. Purif 6435:10); Ausubel, Sambrook, Berger (all supra). A catalogue of Bacteria and Bacteriophages useful for cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria and Bateriophage (1992) Gherna et al. (eds) published by the ATCC. Additional basic procedures for sequencing, cloning and other aspects of molecular biology and underlying theoretical considerations are also found in Watson et al. (1992) Recombinant DNA Second Edition, Scientific American Books, NY.

Transforming Nucleic Acids into Plants

Embodiments of the present invention pertain to the production of transgenic plants comprising the cloned nucleic acids, e.g., chromosome intervals, isolated ORFs, and cDNAs associated with QTLs, of the invention. Techniques for transforming plant cells with nucleic acids are generally available and can be adapted to the invention by the use of nucleic acids encoding or corresponding to QTLs, QTL homologs, isolated chromosome intervals, and the like. In addition to Berger, Ausubel and Sambrook, useful general references for plant cell cloning, culture and regeneration include Jones (ed.) ((1995) Plant Gene Transfer and Expression Protocols—Methods in Molecular Biology, Volume 49 Humana Press Towata N.J.); Payne et al. ((1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y. (Payne)); and Gamborg and Phillips (eds) ((1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) (Gamborg)). A variety of cell culture media are described in Atlas and Parks (eds.) (The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla. (Atlas)). Additional information for plant cell culture is found in available commercial literature such as the Life Science Research Cell Culture Catalogue (1998) from Sigma-Aldrich, Inc. (St Louis, Mo.) (Sigma-LSRCCC) and, e.g., the Plant Culture Catalogue and supplement (1997) also from Sigma-Aldrich, Inc. (St Louis, Mo.) (Sigma-PCCS). Additional details regarding plant cell culture are found in Croy, (ed.) ((1993) Plant Molecular Biology Bios Scientific Publishers, Oxford, U.K.)

The nucleic acid constructs of the invention, e.g., plasmids, cosmids, artificial chromosomes, DNA and RNA polynucleotides, are introduced into plant cells, either in culture or in the organs of a plant by a variety of conventional techniques. Where the sequence is expressed, the sequence is optionally combined with transcriptional and translational initiation regulatory sequences that direct the transcription or translation of the sequence from the exogenous DNA in the intended tissues of the transformed plant.

Isolated nucleic acids of the present invention can be introduced into plants according to any of a variety of techniques known in the art. Techniques for transforming a wide variety of higher plant species are well known and described in the technical, scientific, and patent literature. See, for example, Weising et al. (1988) Ann. Rev. Genet. 22:421-477. The DNA constructs of the invention, for example, plasmids, cosmids, phage, naked or variously conjugated-DNA polynucleotides, (e.g., polylysine-conjugated DNA, peptide-conjugated DNA, liposome-conjugated DNA, etc.), or artificial chromosomes, can be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant cells using ballistic methods, such as DNA particle bombardment.

Microinjection techniques for injecting e.g., cells, embryos, callus and protoplasts, are known in the art and well described in the scientific and patent literature. For example, a number of methods are described in Jones (ed.) ((1995) Plant Gene Transfer and Expression Protocols—Methods in Molecular Biology, Volume 49 Humana Press Towata N.J.), as well as in the other references noted herein and available in the literature.

For example, the introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski, et al. (EMBO J. 3:2717 (1984)). Electroporation techniques are described in Fromm, et al. (Proc. Nat'l. Acad. Sci. USA 82:5824 (1985)). Ballistic transformation techniques are described in Klein, et al. (Nature 327:70-73 (1987)). Additional details are found in Jones (1995) and Gamborg and Phillips (1995), supra, and in U.S. Pat. No. 5,990,387.

Alternatively, Agrobacterium-mediated transformation is employed to generate transgenic plants. Agrobacterium-mediated transformation techniques, including disarming and use of binary vectors, are also well described in the scientific literature. See, for example, Horsch, et al. (1984) Science 233:496; and Fraley et al. (1984) Proc. Nat'l. Acad. Sci. USA 80:4803 and reviewed in Hansen and Chilton (1998) Current Topics in Microbiology 240:22 and Das (1998) Subcellular Biochemistry 29: Plant Microbe Interactions pp. 343-363.

The DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. See, U.S. Pat. No. 5,591,616. Although Agrobacterium is useful primarily in dicots, certain monocots can be transformed by Agrobacterium. For instance, Agrobacterium transformation of maize is described in U.S. Pat. No. 5,550,318. Other methods of transfection or transformation include (1) Agrobacterium rhizogenes-mediated transformation (see, e.g., Lichtenstein and Fuller (1987) In: Genetic Engineering, vol. 6, PWJ Rigby, Ed., London, Academic Press; and Lichtenstein; C. P., and Draper (1985) In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press); WO 88/02405, published Apr. 7, 1988, describes the use of A. rhizogenes strain A4 and its Ri plasmid along with A. tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNA uptake (see, e.g., Freeman et al. (1984) Plant Cell Physiol. 25:1353), (3) the vortexing method (see, e.g., Kindle (1990) Proc. Natl. Acad. Sci., (USA) 87:1228).

DNA can also be introduced into plants by direct DNA transfer into pollen as described by Zhou et al. ((1983) Methods in Enzymology, 101:433); Hess ((1987) Intern Rev. Cytol. 107:367); and Luo et al. ((1988) Plant Mol. Biol. Reporter 6:165). Expression of polypeptide coding genes can be obtained by injection of the DNA into reproductive organs of a plant as described by Pena et al. ((1987) Nature 325:274). DNA can also be injected directly into the cells of immature embryos and the desiccated embryos rehydrated as described by Neuhaus et al. ((1987) Theor. Appl. Genet. 75:30); and Benbrook et al. ((1986) in Proceedings Bio Expo Butterworth, Stoneham, Mass., pp. 27-54). A variety of plant viruses that can be employed as vectors are known in the art and include cauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.

Regeneration of Transgenic Plants

Transformed plant cells that are derived by any of the above transformation techniques can be cultured to regenerate a whole plant that possesses the transformed genotype and thus the desired phenotype. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al. ((1983) Protoplasts Isolation and Culture, Handbook of Plant Cell Culture pp. 124-176, Macmillian Publishing Company, New York); and Binding ((1985) Regeneration of Plants, Plant Protoplasts pp. 21-73, CRC Press, Boca Raton). Regeneration can also be obtained from plant callus, explants, somatic embryos (Dandekar et al. (1989) J. Tissue Cult. Meth. 12:145; McGranahan, et al. (1990) Plant Cell Rep. 8:512) organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. ((1987)., Ann. Rev. of Plant Phys. 38:467-486). Additional details are found in Payne (1992) and Jones (1995), both supra, and Weissbach and Weissbach, eds. ((1988) Methods for Plant Molecular Biology Academic Press, Inc., San Diego, Calif.). This regeneration and growth process includes the steps of selection of transformant cells and shoots, rooting the transformant shoots and growth of the plantlets in soil. These methods are adapted to the invention to produce transgenic plants bearing QTLs and other genes isolated according to the methods of the 10 invention.

In addition, the regeneration of plants containing the polynucleotide of the present invention and introduced by Agrobacterium into cells of leaf explants can be achieved as described by Horsch et al. ((1985) Science 227:1229-1231). In this procedure, transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species being transformed as described by Fraley et al. ((1983) Proc. Natl. Acad. Sci. (U.S.A.) 80:4803). This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Transgenic plants of the present invention may be fertile or sterile.

Plants for the transformation and expression of QTLs associated with shatter resistance and other nucleic acids identified and cloned according to the present invention include, but are not limited to, agronomically and horticulturally important species. Such species include primarily dicots, e.g., of the families: Brassicaceae, Leguminosae (including pea, beans, lentil, peanut, yam bean, cowpeas, velvet beans, soybean, clover, alfalfa, lupine, vetch, lotus, sweet clover, wisteria, and sweetpea); and, Compositae (the largest family of vascular plants, including at least 1,000 genera, including important commercial crops such as sunflower).

Additionally, targets for modification with the nucleic acids of the invention, as well as those specified above, plants from the genera: Allium, Apium, Arachis, Brassica, Capsicum, Cicer, Cucumis, Curcubita, Daucus, Fagopyrum, Glycine, Helianthus, Lactuca, Lens, Lycopersicon, Medicago, Pisum, Phaseolus, Solanurn, Trifolium, Vigna, and many others.

Common crop plants that are targets of the present invention include soybean, sunflower, canola, peas, beans, lentils, peanuts, yam beans, cowpeas, velvet beans, clover, alfalfa, lupine, vetch, sweet clover, sweetpea, field pea, fava bean, broccoli, brussel sprouts, cabbage, cauliflower, kale, kohlrabi, celery, lettuce, carrot, onion, pepper, potato, eggplant, and tomato.

In construction of recombinant expression cassettes of the invention, which include, for example, helper plasmids comprising virulence functions, and plasmids or viruses comprising exogenous DNA sequences such as structural genes, a plant promoter fragment is optionally employed to direct expression of a nucleic acid in any or all tissues of a regenerated plant. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, and other transcription initiation regions from various plant genes known to those of skill. Alternatively, the plant promoter may direct expression of the polynucleotide of the invention in a specific tissue (tissue-specific promoters) or may be otherwise under more precise environmental control (inducible promoters). Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues, such as fruit, seeds, or flowers.

Any of a number of promoters that direct transcription in plant cells can be suitable. The promoter can be either constitutive or inducible. In addition to the promoters noted above, promoters of bacterial origin that operate in plants include the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from native Ti plasmids. See, Herrara-Estrella et al. ((1983), Nature 303:209). Viral promoters include the 35S and 19S RNA promoters of cauliflower mosaic virus. See, Odell et al. ((1985) Nature 313:810). Other plant promoters include the ribulose-1,3-bisphosphate carboxylase small subunit promoter and the phaseolin promoter. The promoter sequence from the E8 gene and other genes may also be used. The isolation and sequence of the E8 promoter is described in detail in Deikman and Fischer ((1988) EMBO J. 7:3315). Many other promoters are in current use and can be coupled to an exogenous DNA sequence to direct expression of the nucleic acid.

If expression of a polypeptide, including those encoded by QTLs or other nucleic acids correlating with phenotypic traits of the present invention, is desired, a polyadenylation region at the 3′-end of the coding region is typically included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from, e.g., T-DNA.

The vector comprising the sequences (e.g., promoters or coding regions) from genes encoding expression products and transgenes of the invention will typically include a nucleic acid subsequence, a marker gene that confers a selectable, or alternatively, a screenable, phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosluforon, or phosphinothricin (the active ingredient in the herbicides bialaphos or Basta). See, e.g., Padgette et al. (1996) In: Herbicide-Resistant Crops (Duke, ed.), pp 53-84, CRC Lewis Publishers, Boca Raton (“Padgette, 1996”). For example, crop selectivity to specific herbicides can be conferred by engineering genes into crops that encode appropriate herbicide metabolizing enzymes from other organisms, such as microbes. See, Vasil (1996) In: Herbicide-Resistant Crops (Duke, ed.), pp 85-91, CRC Lewis Publishers, Boca Raton) (“Vasil”, 1996).

One of skill will recognize that after the recombinant expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. In vegetatively propagated crops, mature transgenic plants can be propagated by the taking of cuttings or by tissue culture techniques to produce multiple identical plants. Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use. In seed propagated crops, mature transgenic plants can be self-crossed to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced heterologous nucleic acid. These seeds can be grown to produce plants that would produce the selected phenotype.

Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are included in the invention, provided that these parts comprise cells comprising the isolated nucleic acid of the present invention. Progeny and variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced nucleic acid sequences.

Transgenic plants expressing a polynucleotide of the present invention can be screened for transmission of the nucleic acid of the present invention by, for example, standard immunoblot and DNA detection techniques. Expression at the RNA level can be determined initially to identify and quantitate expression-positive plants. Standard techniques for RNA analysis can be employed and include PCR amplification assays using oligonucleotide primers designed to amplify only the heterologous RNA templates and solution hybridization assays using heterologous nucleic acid-specific probes. The RNA-positive plants can then be analyzed for protein expression by Western immunoblot analysis using the specifically reactive antibodies of the present invention. In addition, in situ hybridization and immunocytochemistry according to standard protocols can be done using heterologous nucleic acid specific polynucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue. Generally, a number of transgenic lines are usually screened for the incorporated nucleic acid to identify and select plants with the most appropriate expression profiles.

One embodiment is a transgenic plant that is homozygous for the added heterologous nucleic acid; i.e., a transgenic plant that contains two added nucleic acid sequences, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced for altered expression of a polynucleotide of the present invention relative to a control plant (i.e., native, non-transgenic). Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated.

High Throughput Screening

In one aspect of the invention, the determination of genetic marker alleles is performed by high throughput screening. High throughput screening involves providing a library of genetic markers, e.g., RFLPs, AFLPs, isozymes, specific alleles and variable sequences, including SSR. Such libraries are then screened against plant genomes to generate a “fingerprint” for each plant under consideration. In some cases a partial fingerprint comprising a sub-portion of the markers is generated in an area of interest. Once the genetic marker alleles of a plant have been identified, the correspondence between one or several of the marker alleles and a desired phenotypic trait is determined through statistical associations based on the methods of this invention.

High throughput screening can be performed in many different formats. Hybridization can take place in a 96-, 324-, or a 1524-well format or in a matrix on a silicon chip or other format.

In one commonly used format, a dot blot apparatus is used to deposit samples of fragmented and denatured genomic DNA on a nylon or nitrocellulose membrane. After cross-linking the nucleic acid to the membrane, either through exposure to ultra-violet light or by heat, the membrane is incubated with a labeled hybridization probe. The labels are incorporated into the nucleic acid probes by any of a number of means well-known in the art. The membranes are washed to remove non-hybridized probes and the association of the label with the target nucleic acid sequence is determined.

A number of well-known robotic systems have been developed for high throughput screening, particularly in a 96 well format. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; ORCA™, Beckman Coulter, Fullerton Calif.). Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art.

In addition, high throughput screening systems themselves are commercially available (see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick, Mass., etc.). These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate or membrane in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for the use of their products in high throughput applications.

In one variation of the invention, solid phase arrays are adapted for the rapid and specific detection of multiple polymorphic nucleotides. Typically, a nucleic acid probe is linked to a solid support and a target nucleic acid is hybridized to the probe. Either the probe, or the target, or both, can be labeled, typically with a fluorophore. If the target is labeled, hybridization is evaluated by detecting bound fluorescence. If the probe is labeled, hybridization is typically detected by quenching of the label by the bound nucleic acid. If both the probe and the target are labeled, detection of hybridization is typically performed by monitoring a color shift resulting from proximity of the two bound labels.

In one embodiment, an array of probes is synthesized on a solid support. Using chip masking technologies and photoprotective chemistry, it is possible to generate ordered arrays of nucleic acid probes. These arrays, which are known, e.g., as “DNA chips” or as very large scale immobilized polymer arrays (VLSIPS™ arrays) can include millions of defined probe regions on a substrate having an area of about 1 cm²to several cm².

In another embodiment, capillary electrophoresis is used to analyze a polymorphism. This technique works best when the polymorphism is based on size, for example, AFLP and SSR. This technique is described in detail in U.S. Pat. Nos. 5,534,123 and 5,728,282. Briefly, capillary electrophoresis tubes are filled with the separation matrix. The separation matrix contains hydroxyethyl cellulose, urea and optionally formamide. The AFLP or SSR samples are loaded onto the capillary tube and electrophoresed. Because of the small amount of sample and separation matrix required by capillary electrophoresis, the run times are very short. The molecular sizes and therefore, the number of nucleotides present in the nucleic acid sample are determined by techniques described herein. In a high throughput format, many capillary tubes are placed in a capillary electrophoresis apparatus. The samples are loaded onto the tubes and electrophoresis of the samples is run simultaneously. See, Mathies and Huang (1992) Nature 359:167.

Integrated Systems

Because of the great number of possible combinations present in one array, in one aspect of the invention, an integrated system such as a computer, software corresponding to the statistical models of the invention, and data sets corresponding to genetic markers and phenotypic values, facilitates mapping of phenotypic traits, including QTLs. The phrase “integrated system” in the context of this invention refers to a system in which data entering a computer corresponds to physical objects or processes external to the computer, e.g., nucleic acid sequence hybridization, and a process that, within a computer, causes a physical transformation of the input signals to different output signals. In other words, the input data, e.g., hybridization on a specific region of an array is transformed to output data, e.g., the identification of the sequence hybridized. The process within the computer is a set of instructions, or “program,” by which positive hybridization signals are recognized by the integrated system and attributed to individual samples as a genotype. Additional programs correlate the genotype, and more particularly in the methods of the invention, the haplotype, of individual samples with phenotypic values, e.g., using the HAPLO-IM⁺, HAPLO-MQM, and/or HAPLO-MQM⁺ models of the invention. For example, the programs JoinMap® and MapQTL® are particularly suited to this type of analysis and can be extended to include the HAPLO-IM⁺, HAPLO-MQM, and/or HAPLO-MQM⁺ models of the invention. In addition there are numerous e.g., C/C++ programs for computing, Delphi and/or Java programs for GUI interfaces, and Active X applications (e.g., Olectra Chart and True WevChart) for charting tools. Other useful software tools in the context of the integrated systems of the invention include statistical packages such as SAS, Genstat, and S-Plus. Furthermore additional programming languages such as Fortran and the like are also suitably employed in the integrated systems of the invention.

In one aspect, the invention provides an integrated system comprising a computer or computer readable medium comprising a database with at least one data set that corresponds to genotypes for genetic markers. The system also includes a user interface allowing a user to selectively view one or more databases. In addition, standard text manipulation software such as word processing software (e.g., Microsoft Word™ or Corel Wordperfect™) and database or spreadsheet software (e.g., spreadsheet software such as Microsoft Excel™ Corel Quattro Pro™, or database programs such as Microsoft Access™ or Paradox™) can be used in conjunction with a user interface (e.g., a GUI in a standard operating system such as a Windows, Macintosh or Linux system) to manipulate strings of characters.

The invention also provides integrated systems for sample manipulation incorporating robotic devices as previously described. A robotic liquid control armature for transferring solutions (e.g., plant cell extracts) from a source to a destination, e.g., from a microtiter plate to an array substrate, is optionally operably linked to the digital computer (or to an additional computer in the integrated system). An input device for entering data to the digital computer to control high throughput liquid transfer by the robotic liquid control armature and, optionally, to control transfer by the armature to the solid support is commonly a feature of the integrated system.

Integrated systems for genetic marker analysis of the present invention typically include a digital computer with one or more of high-throughput liquid control software, image analysis software, data interpretation software, a robotic liquid control armature for transferring solutions from a source to a destination operably linked to the digital computed, an input device (e.g., a computer keyboard) for entering data to the digital computer to control high throughput liquid transfer by the robotic liquid control armature and, optionally, an image scanner for digitizing label signals from labeled probes hybridized, e.g., to expression products on a solid support operably linked to the digital computer. The image scanner interfaces with the image analysis software to provide a measurement of, e.g., differentiating nucleic acid probe label intensity upon hybridization to an arrayed sample nucleic acid population, where the probe label intensity measurement is interpreted by the data interpretation software to show whether, and to what degree, the labeled probe hybridizes to a label. The data so derived is then correlated with phenotypic values using the statistical models of the present invention, to determine the correspondence between phenotype and genotype(s) for genetic markers, thereby, assigning chromosomal locations.

Optical images, e.g., hybridization patterns viewed (and, optionally, recorded) by a camera or other recording device (e.g., a photodiode and data storage device) are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and/or storing and analyzing the image on a computer. A variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or optical image, e.g., using PC (Intel x86 or pentium chip-compatible DOS™, OS2™ WINDOWS™, WINDOWS NT™ or WINDOWS95™ based machines), MACINTOSH™ LINUX, or UNIX based (e.g., SUN™ work station) computers.

Kits

Kits are also provided to facilitate the screening of germplasm for the markers of the present invention. The kits comprise the polynucleotides of the present invention, fragments or complements thereof, for use as probes or primers to detect the markers for shatter resistance. Examples of suitable primers and probes for use in the present invention are set forth in Table 8. The skilled artisan will understand that such primers and probes typically are made by nucleotide synthesis, and can be chemically modified, for instance, to improve stability or to detectably label the probe or primer. Primers and probes also can be affixed to solid supports, such as the arrays described above, and/or other solid supports as is well known in the art. Instructions for using the polynucleotides, as well as buffers and/or other solutions may also be provided to facilitate the use of the polynucleotides. The kit is useful for high throughput screening and in particular, high throughput screening with integrated systems. In certain embodiments, the kits contain a plurality of polynucleotides, e.g., to detect a plurality of the markers and/or polymorphisms associated with shatter resistance.

EXAMPLES

The following experimental methods and results provide additional details regarding specific aspects of protocols and procedures relevant to the practice of the present invention. The examples, which are provided without limitation to illustrate the claimed invention, involve the application of protocols well known to those of skill in the art, and detailed in the references cited herein.

Example 1: Description of Mapping Populations

Three mapping populations were examined. Parental lines were susceptible or resistant to shatter, as determined by one of two tests. In one test (Stirks), mature plants were challenged manually by dragging a wooden bar over the crop at the approximate height of the pods. In another test, a wind machine was used to simulate natural wind conditions.

Shatter resistance or susceptibility was ranked by way of a shatter score (SHTSC) from 1 to 9 (1=highly susceptible, 9=highly resistant). Table 1 below shows details of the ranking, along with the SHTSC of five parental lines used in the mapping populations. Table 2 shows details pertaining to the three mapping populations.

TABLE 1

Rating scale under high shatter pressure in the field

Rating
Percent

Relative response of

scale
shatter
Category
mapping parents

1
80-100
Highly susceptible
NS5902

2
70
Susceptible
06DSB13911

3
60
Susceptible

4
50
Moderately susceptible

5
40
Moderately resistant

6
30
Moderately resistant
AV Jade, 09DSB12654

7
20
Resistant
NS6184

8
10
Resistant

9
0
Highly resistant

TABLE 2

Three shattering sources, population size, marker number

and phenotypic data in three mapping populations

Population
Shatter
QTL
Pop
Marker
Shatter

Population
number
source
nomenclature
size
No.
field data

06DSB13911/
SH-Pop 1
09DSB12564
SH-564
188
494
Year 1-3

09DSB12564

reps; Year 2-2

locations, 2

reps

06DSB13911/
SH-Pop 2
AV Jade
SH-Jade
180
415
Year 2-2

AV Jade

reps

NS5902/
SH-Pop 3
NS6184
SH-6184
180
389
Year 2-2

NS6184

reps

The parents used for the mapping population #1 (SH-Pop 1) are double haploid lines. 09DSB 12564 is a shatter resistant parent, whereas 06DSB 13911 is a susceptible parent. The lines were used to develop a double haploid mapping population consisting of 188 progeny. The progeny were phenotyped with three replicates in Year 1 and two replicates at each of two locations in Year 2. The progeny were mapped with 494 SNPs.

The parents used for the mapping population #2 (SH-Pop 2) were AV Jade (a shatter resistant variety) and 06DSB 13911 (a susceptible parent). The lines were used to develop a double haploid mapping population consisting of 180 progeny. The progeny were phenotyped with two replicates in Year 2 and mapped with 415 SNPs.

The parents used for the mapping population #3 (SH-Pop 3) were NS6184 contributing shatter resistance and NS5902 (a susceptible parent). The lines were used to develop a double haploid mapping population consisting of 180 progeny. The progeny were phenotyped with two replicates in Year 2 and mapped with 389 SNPs.

Example 2. Genetic Mapping and QTL Analysis

Genetic mapping and QTL analysis were performed using JoinMap v3.0 (Van Ooijen, J. W. and R. E. Voorrips, 2001 JoinMap® 3.0, Software for the calculation of genetic linkage maps. Plant Research International, Wageningen, the Netherlands). The Kosambi centiMorgan function was used. A QTL was declared if its LOD score exceeded the threshold of 2.0. LOD stands for logarithm of the odds (to the base 10).

Genetic Mapping

Genetic mapping of the three above-described mapping populations has placed 389-494 molecular markers in 19 linkage groups (LG) that correspond to 19 canola chromosomes and public linkage group nomenclature. The linkage map covers ˜1600-1700 cM.

TABLE 3

Breakdown of Genetic Mapping.

Pop1

Pop2

Pop3

(06DSB13911/

(06DSB13911/

(NS5902/

09DSB12564)

AV Jade)

NS6184)

Number of
cM on
Number of
cM on
Number of
cM on

markers
PHD
markers
PHD
markers
PHD

N01
26
106.3
17
106.3
13
86.8

N02
7
93.3
11
51.2
8
71.4

N03
19
135.8
27
131.9
26
135.8

N04
81
69.3
23
54.2
25
69.3

N05
10
94.5
5
94.5
9
88.5

N06
13
77.3
27
78.9
20
78.9

N07
27
55.1
31
66.5
28
60.2

N08
7
80.7
19
80.7
3
64.3

N09
31
126.0
16
126.0
20
73.7

N10
14
67.1
20
64.1
20
67.1

N11
11
106.7
28
82.0
33
106.4

N12
37
79.4
15
129.1
17
97.7

N13
37
113.0
51
108.4
33
113.0

N14
27
94.8
44
82.1
46
108.0

N15
13
109.6
16
90.3
12
109.6

N16
14
68.3
23
68.3
17
68.1

N17
27
93.7
25
93.1
25
87.9

N18
30
92.5
17
92.8
28
94.1

N19
63
49.0
0
0.0
6
49.0

Total
494
1712.5
415
1600.3
389
1629.9

QTL Analysis

QTL analysis using simple interval mapping and composite interval mapping (CIM) (Zeng, 1994, Genetics 136:1457) identified 11 linkage groups (N1, N3, N4, N6, N7, N9, N13, N14, N15, N18 and N19) contributing to shatter resistance. In addition, regions identified by interval mapping as being associated with shatter resistance were confirmed by single-factor analysis of variance (PROC GLM, SAS Enterprise Guide 4.2) on shatter parameters (using the above-described Shatter Score) at the P≤0.01 significance level. These QTLs and the markers associated therewith are identified in Tables 4-7 below. As shown by the “Phenotypic Variation Explained” values in Table 5, some QTLs had a larger effect on shatter resistance than others. With respect to marker designations, nomenclature is as follows: (1) the locus designation is indicated first; for instance, N20003-001; (2) the amplification chemistry is designated second; for instance, -Q001. Markers amplified using TaqMan chemistry (Life Technologies, Inc., Grand Island N.Y.) are designated with a Q, while markers amplified using KASPr chemistry (LGC Genomics, Boston Mass.) are designated with a K.

TABLE 4

Markers significantly associated with shatter resistance at P ≤ 0.01.

Linkage

Genetic

Group
SNP marker
Position
Year for Pop1
Year for Pop2
Year for Pop3

N1
N20003-001-Q001
34.7
Year 1 (rep1)

N03491-1-Q1
37.9
Year 1 (rep1)

N0017NR-001-
38.6
Year 1 (rep1)

Q001

N10336-001-Q001
43.3
Year 1 (rep1)
Year 2 (rep2)

N23133-001-Q001
43.3
Year 1 (rep1)
Year 2 (rep2)

N16487-001-Q001
46.2
Year 1 (rep1)
Year 2 (rep2)

N23426-001-Q001
48.8

Year 2 (rep2)

N3
N05671-1-Q1
76.3

Year 2 (rep1)

N12643-001-Q001
90.8

Year 2 (rep1)

N4
N05943-1-Q1
42.9

Year 2

N06007-1-Q1
45.2

Year 2

N10105-001-Q001
54.2

Year 2

N08181-1-Q1
55.3

Years 2 and 3

N06675-1-Q1
69.3
Years 1 and 2

Years 2 and 3

N001KH2-001-
64.6
Years 1 and 2

Q001

N29313-001-Q001
64.6
Years 1 and 2

N88512-001-K001
64.3
Years 1 and 2

N88514-001-K001
66.4
Years 1 and 2

N88515-001-K001
66.4
Years 1 and 2

N88516-001-K001
66.4
Years 1 and 2

N88517-001-K001
66.4
Years 1 and 2

N88518-001-K001
67.0
Years 1 and 2

N88519-001-K001
65.9
Years 1 and 2

N88520-001-K001
64.9
Years 1 and 2

N88521-001-K001
65.1
Years 1 and 2

N001KFE-001-
66.4
Years 1 and 2

Q001

N88522-001-K001
66.4
Years 1 and 2

N88523-001-K001
67.1
Years 1 and 2

N88524-001-K001
66.8
Years 1 and 2

N88525-001-K001
67.5
Years 1 and 2

N88529-001-K001
67.3
Years 1 and 2

N88530-001-K001
68.2
Years 1 and 2

N88531-001-K001
69.0
Years 1 and 2

N88533-001-K001
69.2
Years 1 and 2

N88535-001-K001
69.1
Years 1 and 2

N88536-001-K001
69.1
Years 1 and 2

N88537-001-K001
69.1
Years 1 and 2

N6
N07541-1-Q1
39.6

Years 2 and 3

N23413-001-Q001
56.6

Years 2 and 3

N08344-1-Q1
58.9

Years 2 and 3

N23533-001-Q011
59.7

Years 2 and 3

N14649-001-Q001
65.6

Years 2 and 3

N7
N23310-001-Q001
27.9
Year 2

N10526-001-Q001
28.6
Year 2

N23373-001-Q001
29.2
Year 2

N23353-001-Q001
30.9
Year 2

N23206-001-Q001
33.1
Year 2

N11025-001-Q001
36.1
Year 2

N09969-001-Q001
40.0
Year 2

N09882-001-Q001
41.6
Year 2

N10389-001-Q001
41.6
Year 2

N09940-001-Q001
41.8
Year 2

N23409-001-Q001
46.0
Year 2

N7
N07278-1-Q1
55.1

Year 3

N16343-001-Q001
58.6

Year 3

N23417-001-Q001
64.5

Year 3

N9
N23119-001-Q001
45.4

Years 2 and 3

N09861-001-Q001
53.3

Years 2 and 3

N04807-1-Q1
55.7

Years 2 and 3
Year 2

N06778-1-Q1
60.0

Years 2 and 3
Year 2

N09897-001-Q001
60.1

Years 2 and 3
Year 2

N10499-001-Q001
60.1

Years 2 and 3
Year 2

N23447-001-Q001
60.1

Years 2 and 3
Year 2

N19834-001-Q001
60.1

Years 2 and 3
Year 2

N23362-001-Q001
64.1

Years 2 and 3
Year 2

N23266-001-Q001
70.0

Years 2 and 3
Year 2

N19862-001-Q001
70.7

Years 2 and 3
Year 2

N22187-001-Q001
70.7

Years 2 and 3
Year 2

N08651-1-Q1
71.9

Years 2 and 3
Year 2

N23296-001-Q001
73.1

Years 2 and 3
Year 2

N17314-001-Q001
73.7

Years 2 and 3
Year 2

N20380-001-Q001
74.2

Years 2 and 3

N9
N05490-1-Q1
104.5
Year 1

N18849-001-Q001
104.8
Year 1

N08200-1-Q1
105.4
Year 1

N19827-001-Q001
105.4
Year 1

N001R9W-001-
105.4
Year 1

Q001

N08264-1-Q1
112.4
Year 1

N23132-001-Q001
113.6
Year 1

N03615-1-Q1
118.4
Year 1

N001RWT-001-
118.4
Year 1

Q001

N08465-1-Q1
119.0
Year 1

N10774-001-Q001
119.3
Year 1

N17035-001-Q001
122.1
Year 1

N20834-001-Q001
122.7
Year 1

N13
N22903-001-Q001
−22.6

Years 2 and 3

N09920-001-Q001
−15.5

Years 2 and 3

N22822-001-Q001
−13.0

Years 2 and 3

N22688-001-Q001
−9.8

Years 2 and 3

N10074-001-Q001
−9.7

Years 2 and 3

N10057-001-Q001
−8.5

Years 2 and 3

N10086-001-Q001
−8.5

Years 2 and 3

N11084-001-Q001
−8.4

Years 2 and 3

N22814-001-Q001
2.5

Years 2 and 3

N01564-2-Q1
3.2

Years 2 and 3

N12902-001-Q001
3.5

Years 2 and 3

N13
N21144-001-Q001
59.8

Years 2 and 3

N07534-1-Q1
62.5

Years 2 and 3

N22993-001-Q001
62.5

Years 2 and 3

N09963-001-Q001
62.8

Years 2 and 3

N11542-001-Q001
63.2

Years 2 and 3

N14681-001-Q001
63.9

Years 2 and 3

N11636-001-Q001
64.3

Years 2 and 3

N13732-001-Q001
65.1

Years 2 and 3

N11255-001-Q001
67.4

Years 2 and 3

N15511-001-Q001
67.6

Years 2 and 3

N10536-001-Q001
69.5

Years 2 and 3

N09862-001-Q001
71.8

Years 2 and 3

N14
N23033-001-Q001
14.1

Year 2

N06039-1-Q1
31.7

Year 2

N10016-001-Q001
31.7

Years 2 and 3

N22743-001-Q001
32.4

Years 2 and 3

N22953-001-Q001
32.5

Years 2 and 3

N09987-001-Q001
33.4

Years 2 and 3

N10092-001-Q001
33.4

Years 2 and 3

N10096-001-Q001
33.4

Years 2 and 3

N22728-001-Q001
33.4

Years 2 and 3

N22747-001-Q001
33.4

Years 2 and 3

N22840-001-Q001
33.4

Years 2 and 3

N23027-001-Q001
33.4

Years 2 and 3

N22777-001-Q001
33.9

Years 2 and 3

N09636-001-Q001
34.2

Years 2 and 3

N09879-001-Q001
35.6

Years 2 and 3

N10123-001-Q001
35.6

Years 2 and 3

N10316-001-Q001
35.6

Years 2 and 3

N10507-001-Q001
35.6

Years 2 and 3

N09834-001-Q001
36.8

Years 2 and 3

N22934-001-Q001
37.2

Years 2 and 3

N22700-001-Q001
37.8

Years 2 and 3

N22725-001-Q001
37.8

Years 2 and 3

N22881-001-Q001
37.8

Years 2 and 3

N23032-001-Q001
37.8

Years 2 and 3

N22786-001-Q001
37.9

Years 2 and 3

N23014-001-Q001
37.9

Years 2 and 3

N10471-001-Q001
38.2

Years 2 and 3

N11419-001-Q001
39.5

Years 2 and 3

N22724-001-Q001
43.4

Years 2 and 3

N22902-001-Q001
43.0

Years 2 and 3

N23063-001-Q001
43.3

Years 2 and 3

N22723-001-Q001
43.4

Years 2 and 3

N23049-001-Q001
43.5

Years 2 and 3

N10321-001-Q001
47.1

Years 2 and 3

N15374-001-Q001
48.3

Years 2 and 3

N22802-001-Q001
49.4

Years 2 and 3

N15
N12785-001-Q001
40.5

Year 2

N09910-001-Q001
56.5

Years 2 and 3

N21146-001-Q001
60.0

Years 2 and 3

N17618-001-Q001
63.1

Years 2 and 3

N09776-001-Q001
64.2

Years 2 and 3

N19296-001-Q001
64.2

Years 2 and 3

N18
N22803-001-Q001
43.8

Year 3

N05205-1-Q1
57.2

Years 2 and 3

N10406-001-Q001
58.0
Year3

Years 2 and 3

N22941-001-Q001
58.6
Year3

Years 2 and 3

N22875-001-Q001
63.9
Year3

Years 2 and 3

N13286-001-Q001
63.9
Year3

Years 2 and 3

N04503-1-Q1
64.6
Year3

Years 2 and 3

N22925-001-Q001
65.0
Year3

Years 2 and 3

N18929-001-Q001
74.8
Year3

N16041-001-Q001
76.9
Year3

N18401-001-Q001
84.6
Year3

N19
N05656-1-Q1
27.7
Year 1

N17581-001-Q001
30.7
Year 1

N001NVH-001-
38.4
Year 1

Q001

N22928-001-Q001
38.9
Year 1

N08219-1-Q001
40.1
Year 1

N05710-1-Q1
40.2
Year 1

N15338-001-Q001
41.6
Year 1

N10424-001-Q001
41.7
Year 1

N16006-001-Q001
44.4
Year 1

TABLE 5

QTLs associated with shatter resistance.

Phenotypic

QTL interval

variation

Flanking Markers
length (PHD

LOD
explained

QTL
LG
(cM)
v1.3)
Year
score
(%)

a. Six SH-QTLs identified from 09DSB12564 in 06DSB13911/09DSB12564 (Pop1)

SH-564-N1.1
N1
N20003-001-Q001-
13.3
(34.7-48.0)
1 (rep1)
3.4
4.7

N23426-001-Q001

SH-564-N4.1
N4
N88514-001-K001-
6
(64.0-70.0)
1
23.6
36.3

N88537-001-K001

N4
N88514-001-K001-
6
(64.0-70.0)
2
16.3
23.1

N88537-001-K001

N4
N88514-001-K001-
6
(64.0-70.0)
3
10.6
15.5

N88537-001-K001

SH-564-N7.1
N7
N23310-001-Q001 -
18.1
(27.9-46.0)
2
6.5
7.9

N23409-001-Q001

SH-564-N9.1
N9
N05490-1-Q1 -
18.2
(104.5-122.7)
1
4.4
5.9

N20834-001-Q001

N9
N001RWT-001-Q001 -
4.5
(118.4-122.7)
2 (one rep)
2.8
4.9

N20834-001-Q001

SH-564-N19.1
N18
N10406-001-Q001 -
26.6
(58.0-84.6)
3
8.2
12.8

N18401-001-Q001

SH-564-N19.1
N19
N05656-1-Q1 -
18
(28.0-46.0)
1
6.1
7.6

N87555-001-Q001

b. Four SH-QTLs identified from AV Jade in 06DSB13911/AV Jade (Pop2)

SH-Jade-N1.1
N1
N10336-001-Q001-
5.5
(43.3-48.8)
2 (Rep2)
3.6
10.4

N23426-001-Q001

SH-Jade-N7.1
N7
N07278-1-Q1-
9.4
(55.1-64.5)
3
6.2
10.4

N23417-001-Q001

SH-Jade-N9.1
N9
N23119-001-Q001 -
28.8
(45.4-74.2)
2
4.8
9.8

N20380-001-Q001

N9
N23119-001-Q001 -
28.8
(45.4-74.2)
3
9.9
20.2

N20380-001-Q001

SH-Jade-N13.1
N13
N21144-001-Q001 -
12
(59.8-71.8)
2
6.6
12.3

N09862-001-Q001

N13
N21144-001-Q001 -
12
(59.8-71.8)
3
5.3
8.5

N09862-001-Q001

c. Eight SH-QTLs identified from NS6184 in NS5902/NS6184 (Pop3)

SH-6184-N3.1
N3
N05671-1-Q1-
14.5
(76.3-90.8)
2 (Rep1)
4.2
9.0

N12643-001-Q001

SH-6184-N4.1
N4
N05943-1-Q1 -
26.5
(42.9-69.4)
2
6.0
12.1

N06675-1-Q1

N4
N08181-1-Q1-
14.1
(55.3-69.4)
3 (Rep2)
3.4
6.9

N06675-1-Q1

SH-6184-N6.1
N6
N07541-1-Q1 -
26.0
(39.6-65.6)
2
4.9
9.4

N14649-001-Q001

N6
N07541-1-Q1 -
26.0
(39.6-65.6)
3
3.9
7.9

N14649-001-Q001

SH-6184-N9.1
N9
N04807-1-Q1 -
18.0
(55.7-73.7)
2
4.2
7.5

N17314-001-Q001

SH-6184-N13.1
N13
N22903-001-Q001 -
26.1
(−22.6-3.5)
2
4.3
8.4

N12902-001-Q001

N13
N22903-001-Q001 -
26.1
(−22.6-3.5)
3 (Rep2)
2.3
4.2

N12902-001-Q001

SH-6184-N14.1
N14
N23033-001-Q001 -
29.3
(14.1-43.4)
2
6.4
12.8

N22724-001-Q001

N14
N10016-001-Q001 -
17.1
(31.7-49.4)
3 (Rep2)
2.7
5.1

N22802-001-Q001

SH-6184-N15.1
N15
N12785-001-Q001 -
23.7
(40.5-64.2)
2
5.6
9.6

N19296-001-Q001

N15
N09910-001-Q001 -
7.7
(56.5-64.2)
3
6.7
14.0

N19296-001-Q001

SH-6184-N18.1
N18
N05205-1-Q1 -
7.8
(57.2-65.0)
2
3.9
6.6

N22925-001-Q001

N18
N22803-001-Q001 -
21.2
(43.8-65.0)
3
2.6
7.9

N22925-001-Q001

Additional information about the alleles of each SNP marker flanking the QTLs associated with resistance to shatter is provided in Table 6.

TABLE 6

SNP marker alleles for Shatter QTLs:

Favorable

Favorable

allele

Favorable

allele

from

allele

from

SNP

09DSB -

from

NS61-

SNP marker
LG
Type
Pop1 -QTL
12564
Pop2-QTL
JADE
Pop3-QTL
84BR

N20003-001-Q001
N1
G/T
SH-564-N1.1
T

N03491-1-Q1
N1
C/G
SH-564-N1.1
G

N0017NR-001-Q001
N1
A/G
SH-564-N1.1
G

N10336-001-Q001
N1
A/G
SH-564-N1.1
A
SH-Jade-N1.1
A

N23133-001-Q001
N1
C/T
SH-564-N1.1
C
SH-Jade-N1.1
T

N16487-001-Q001
N1
G/T
SH-564-N1.1
G
SH-Jade-N1.1
T

N23426-001-Q001
N1
A/G

SH-Jade-N1.1
A

N05671-1-Q1
N3
C/T

SH-6184-N3.1
C

N12643-001-Q001
N3
A/C

SH-6184-N3.1
A

N05943-1-Q1
N4
A/G

SH-6184-N4.1
G

N06007-1-Q1
N4
C/T

SH-6184-N4.1
T

N10105-001-Q001
N4
A/T

SH-6184-N4.1
A

N08181-1-Q1
N4
G/T

SH-6184-N4.1
G

N06675-1-Q1
N4
C/T
SH-564-N4.1
T

SH-6184-N4.1
T

N001KH2-001-Q001
N4
A/G
SH-564-N4.1
G

N29313-001-Q001
N4
G/T
SH-564-N4.1
T

N88512-001-K001
N4
A/C
SH-564-N4.1
C

N88514-001-K001
N4
C/G
SH-564-N4.1
G

N88515-001-K001
N4
A/G
SH-564-N4.1
A

N88516-001-K001
N4
A/T
SH-564-N4.1
T

N88517-001-K001
N4
A/G
SH-564-N4.1
G

N88518-001-K001
N4
A/G
SH-564-N4.1
A

N88519-001-K001
N4
A/G
SH-564-N4.1
A

N88520-001-K001
N4
G/T
SH-564-N4.1
G

N88521-001-K001
N4
A/G
SH-564-N4.1
A

N001KFE-001-Q001
N4
A/G
SH-564-N4.1
A

N88522-001-K001
N4
A/C
SH-564-N4.1
A

N88523-001-K001
N4
A/G
SH-564-N4.1
A

N88524-001-K001
N4
G/T
SH-564-N4.1
G

N88525-001-K001
N4
A/T
SH-564-N4.1
A

N88529-001-K001
N4
C/T
SH-564-N4.1
C

N88530-001-K001
N4
C/T
SH-564-N4.1
C

N88531-001-K001
N4
A/G
SH-564-N4.1
A

N88533-001-K001
N4
A/G
SH-564-N4.1
A

N88535-001-K001
N4
A/C
SH-564-N4.1
C

N88536-001-K001
N4
C/G
SH-564-N4.1
G

N88537-001-K001
N4
C/G
SH-564-N4.1
G

N07541-1-Q1
N6
C/G

SH-6184-N6.1
C

N23413-001-Q001
N6
A/T

SH-6184-N6.1
T

N08344-1-Q1
N6
A/C

SH-6184-N6.1
C

N23533-001-Q011
N6
C/T

SH-6184-N6.1
T

N14649-001-Q001
N6
A/C

SH-6184-N6.1
A

N23310-001-Q001
N7
A/G
SH-564-N7.1
A

N10526-001-Q001
N7
G/T
SH-564-N7.1
T

N23373-001-Q001
N7
C/G
SH-564-N7.1
G

N23353-001-Q001
N7
C/G
SH-564-N7.1
C

N23206-001-Q001
N7
C/T
SH-564-N7.1
T

N11025-001-Q001
N7
A/G
SH-564-N7.1
A

N09969-001-Q001
N7
C/T
SH-564-N7.1
C

N09882-001-Q001
N7
A/C
SH-564-N7.1
C

N10389-001-Q001
N7
C/T
SH-564-N7.1
C

N09940-001-Q001
N7
A/G
SH-564-N7.1
A

N23409-001-Q001
N7
C/G
SH-564-N7.1
G

N07278-1-Q1
N7
A/G

SH-Jade-N7.1
G

N16343-001-Q001
N7
A/C

SH-Jade-N7.1
C

N23417-001-Q001
N7
C/G

SH-Jade-N7.1
C

N23119-001-Q001
N9
A/G

SH-Jade-N9.1
A

N09861-001-Q001
N9
A/G

SH-Jade-N9.1
A

N04807-1-Q1
N9
A/G

SH-Jade-N9.1
G
SH-6184-N9.1
G

N06778-1-Q1
N9
C/G

SH-Jade-N9.1
G
SH-6184-N9.1
C

N09897-001-Q001
N9
C/T

SH-Jade-N9.1
C
SH-6184-N9.1
T

N10499-001-Q001
N9
A/C

SH-Jade-N9.1
A
SH-6184-N9.1
C

N23447-001-Q001
N9
A/G

SH-Jade-N9.1
A
SH-6184-N9.1
G

N19834-001-Q001
N9
A/G

SH-Jade-N9.1
A
SH-6184-N9.1
G

N23362-001-Q001
N9
A/G

SH-Jade-N9.1
A
SH-6184-N9.1
G

N23266-001-Q001
N9
C/G

SH-Jade-N9.1
G
SH-6184-N9.1
G

N19862-001-Q001
N9
A/C

SH-Jade-N9.1
A
SH-6184-N9.1
A

N22187-001-Q001
N9
A/G

SH-Jade-N9.1
G
SH-6184-N9.1
G

N08651-1-Q1
N9
A/T

SH-Jade-N9.1
A
SH-6184-N9.1
T

N23296-001-Q001
N9
A/G

SH-Jade-N9.1
A
SH-6184-N9.1
A

N17314-001-Q001
N9
G/T

SH-Jade-N9.1
T
SH-6184-N9.1
G

N20380-001-Q001
N9
A/C

SH-Jade-N9.1
C

N05490-1-Q1
N9
C/G
SH-564-N9.1
G

N18849-001-Q001
N9
G/T
SH-564-N9.1
T

N08200-1-Q1
N9
C/G
SH-564-N9.1
G

N19827-001-Q001
N9
A/G
SH-564-N9.1
G

N001R9W-001-Q001
N9
A/C
SH-564-N9.1
C

N08264-1-Q1
N9
C/T
SH-564-N9.1
C

N23132-001-Q001
N9
A/G
SH-564-N9.1
G

N03615-1-Q1
N9
A/T
SH-564-N9.1
A

N001RWT-001-Q001
N9
A/G
SH-564-N9.1
A

N08465-1-Q1
N9
A/G
SH-564-N9.1
A

N10774-001-Q001
N9
A/C
SH-564-N9.1
A

N17035-001-Q001
N9
A/G
SH-564-N9.1
A

N20834-001-Q001
N9
C/T
SH-564-N9.1
T

N22903-001-Q001
N13
C/G

SH-6184-N13.1
C

N09920-001-Q001
N13
A/T

SH-6184-N13.1
T

N22822-001-Q001
N13
C/G

SH-6184-N13.1
G

N22688-001-Q001
N13
C/G

SH-6184-N13.1
C

N10074-001-Q001
N13
G/T

SH-6184-N13.1
G

N10057-001-Q001
N13
C/T

SH-6184-N13.1
T

N10086-001-Q001
N13
C/T

SH-6184-N13.1
C

N11084-001-Q001
N13
A/G

SH-6184-N13.1
A

N22814-001-Q001
N13
A/T

SH-6184-N13.1
T

N01564-2-Q1
N13
A/C

SH-6184-N13.1
C

N12902-001-Q001
N13
C/T

SH-6184-N13.1
C

N21144-001-Q001
N13
A/C

SH-Jade-N13.1
A

N07534-1-Q1
N13
G/T

SH-Jade-N13.1
G

N22993-001-Q001
N13
C/G

SH-Jade-N13.1
C

N09963-001-Q001
N13
G/T

SH-Jade-N13.1
G

N11542-001-Q001
N13
C/T

SH-Jade-N13.1
T

N14681-001-Q001
N13
A/C

SH-Jade-N13.1
C

N11636-001-Q001
N13
A/G

SH-Jade-N13.1
G

N13732-001-Q001
N13
C/T

SH-Jade-N13.1
T

N11255-001-Q001
N13
C/T

SH-Jade-N13.1
T

N15511-001-Q001
N13
A/G

SH-Jade-N13.1
A

N10536-001-Q001
N13
G/T

SH-Jade-N13.1
G

N09862-001-Q001
N13
A/G

SH-Jade-N13.1
A

N23033-001-Q001
N14
A/T

SH-6184-N14.1
T

N06039-1-Q1
N14
G/T

SH-6184-N14.1
G

N10016-001-Q001
N14
C/T

SH-6184-N14.1
T

N22743-001-Q001
N14
A/T

SH-6184-N14.1
T

N22953-001-Q001
N14
A/T

SH-6184-N14.1
A

N09987-001-Q001
N14
A/G

SH-6184-N14.1
G

N10092-001-Q001
N14
A/G

SH-6184-N14.1
A

N10096-001-Q001
N14
A/G

SH-6184-N14.1
A

N22728-001-Q001
N14
C/G

SH-6184-N14.1
G

N22747-001-Q001
N14
C/G

SH-6184-N14.1
C

N22840-001-Q001
N14
A/T

SH-6184-N14.1
T

N23027-001-Q001
N14
A/T

SH-6184-N14.1
T

N22777-001-Q001
N14
C/G

SH-6184-N14.1
C

N09636-001-Q001
N14
G/T

SH-6184-N14.1
T

N09879-001-Q001
N14
A/G

SH-6184-N14.1
G

N10123-001-Q001
N14
A/G

SH-6184-N14.1
A

N10316-001-Q001
N14
C/T

SH-6184-N14.1
T

N10507-001-Q001
N14
C/T

SH-6184-N14.1
T

N09834-001-Q001
N14
C/T

SH-6184-N14.1
C

N22934-001-Q001
N14
A/T

SH-6184-N14.1
A

N22700-001-Q001
N14
A/T

SH-6184-N14.1
A

N22725-001-Q001
N14
A/T

SH-6184-N14.1
A

N22881-001-Q001
N14
A/T

SH-6184-N14.1
A

N23032-001-Q001
N14
A/T

SH-6184-N14.1
T

N22786-001-Q001
N14
C/G

SH-6184-N14.1
C

N23014-001-Q001
N14
C/G

SH-6184-N14.1
G

N10471-001-Q001
N14
C/T

SH-6184-N14.1
T

N11419-001-Q001
N14
C/T

SH-6184-N14.1
T

N22724-001-Q001
N14
A/T

SH-6184-N14.1
T

N22902-001-Q001
N14
C/G

SH-6184-N14.1
G

N23063-001-Q001
N14
A/T

SH-6184-N14.1
A

N22723-001-Q001
N14
C/G

SH-6184-N14.1
C

N23049-001-Q001
N14
A/T

SH-6184-N14.1
A

N10321-001-Q001
N14
A/T

SH-6184-N14.1
A

N15374-001-Q001
N14
A/C

SH-6184-N14.1
A

N22802-001-Q001
N14
C/G

SH-6184-N14.1
C

N12785-001-Q001
N15
A/G

SH-6184-N15.1
G

N09910-001-Q001
N15
A/T

SH-6184-N15.1
T

N21146-001-Q001
N15
A/C

SH-6184-N15.1
A

N17618-001-Q001
N15
A/G

SH-6184-N15.1
G

N09776-001-Q001
N15
A/C

SH-6184-N15.1
A

N19296-001-Q001
N15
A/C

SH-6184-N15.1
C

N22803-001-Q001
N18
A/T

SH-6184-N18.1
A

N05205-1-Q1
N18
G/T

SH-6184-N18.1
T

N10406-001-Q001
N18
C/G
SH-564-N18.1
G

SH-6184-N18.1
G

N22941-001-Q001
N18
C/G
SH-564-N18.1
C

SH-6184-N18.1
G

N22875-001-Q001
N18
C/G
SH-564-N18.1
G

SH-6184-N18.1
C

N13286-001-Q001
N18
A/G
SH-564-N18.1
A

SH-6184-N18.1
G

N04503-1-Q1
N18
C/G
SH-564-N18.1
G

SH-6184-N18.1
G

N22925-001-Q001
N18
C/G
SH-564-N18.1
G

SH-6184-N18.1
C

N18929-001-Q001
N18
A/G
SH-564-N18.1
G

N16041-001-Q001
N18
C/T
SH-564-N18.1
C

N18401-001-Q001
N18
C/T
SH-564-N18.1
T

N05656-1-Q1
N19
G/T
SH-564-N19.1
G

N17581-001-Q001
N19
A/C
SH-564-N19.1
C

N001NVH-001-Q001
N19
A/G
SH-564-N19.1
A

N22928-001-Q001
N19
A/T
SH-564-N19.1
T

N08219-1-Q001
N19
G/T
SH-564-N19.1
T

N05710-1-Q1
N19
A/G
SH-564-N19.1
G

N15338-001-Q001
N19
C/T
SH-564-N19.1
T

N10424-001-Q001
N19
A/G
SH-564-N19.1
A

N16006-001-Q001
N19
C/T
SH-564-N19.1
T

Example 3: Marker Sequences Containing Polymorphisms, and Exemplary Primers

Set forth below in Table 7 is sequence information for markers of QTLs significantly associated with shatter resistance at P≤0.01, as set forth in the foregoing examples. In the sequences, n=an unknown nucleotide; underlined sequences indicate the primer sequences from Table 8 that follows, and sequences in brackets indicate polymorphic regions (SNPs). Publicly available markers are indicated with an asterisk (*).

TABLE 7

Markers of QTLs significantly associated

with shatter resistance (P ≤ 0.01)

SEQ

ID NO:
Marker Name and Sequence

1
N20003-001-Q001

AAGGAGAGACTAAGGAAGGAGCATATGCACTGACCTTTGC

ATCCGTCCGGTGTATATGCGTTTCCTGTGTGCAACCACAA

TGTGCATAACTAATATTAGAAATGGTGGTTTTGTCGCAGA

TGCACTTCATTTGGGATTTAGTCGAGGAAGTATTACGTCG

GTACTCTTTTCTGTTGTCGCAGGGCAAGGAACTCAGGAAT

[G/T]AATGATTCTTTGTTTGTATCACCCATCCTAGAGTA

AGGGTAGCATATCCCTTACCAAGTAACTGTGTTGGTCTTG

TCGCATTGGACAAAGTGTAGACTTCATCCGTCATGAAGGC

CACTCTACAATGTTCTTCTCTTGTTGTTGTTGTTGTTGTC

GAGTTTCCACCATTGCTCTCTCTATCATGATGCCAATAGT

TTGTT

2
N03491-1-Q1*

GCTTGATCTCTTCAATTCGGGGATTAGAGCTTTCCGGTAC

TCATGCGGCCCCAATCCAGAATCGATCTCTCACTA[C/G]

GGACTACTTCCCTTGGTACAGNCGTNCCCAAGGTCGCCAN

TTCGCTTCGAAATCAAACGGTACTGATGAAAGCAG

3
N0017NR-001-Q001

TTTTGTCCNTAGCTATTCATAATTAATCAAAAAGGTGGTC

CAATTTTACACCTTAGTGCT[A/G]TGANTATCTTTCATA

CATCTCTAGAGTGGAACATATGATACTGCNAATTGCAGTT

ATATT

4
N10336-001-Q001

TTATGATGGTGACAGGAGTTAAGTGTGCATGTGAATGTAG

ATGACTGAAAAAGATAGCCAACTACTTATAACCAACATAC

GACCTTTTGGTCTTTCCTCTTCTCTCTCTCAACTTATTTT

GATTATACAAAATGTTATGTTTGCAAACTGGCATTTAACT

GGGCCGCTTAGCTCTCTTCGGTTATTTTTTTCTTGTGATA

[A/G]ACCACATTCAGATATATATATGTCATCTCGTCATG

TGCTGTTGTTGTTTTCTATATCGTTTCGATTAATCAAGAA

GTTGGGAACGTCGGAACTCCAAACCAAATGTCCTACGATT

ATTAATTATACATGTATCCTGATCATATCTATCTAACATG

AACGAAAATTTGAATCTACTATAAAAGAAATATTAGAGAC

AATTC

5
N23133-001-Q001

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNATAACTCTTTTATTCAAGTGGAGTTTCAAC

TTTTTGTAAACCTCAGAATGCTTCTTTTGTTCCTTCAAAT

TCAAACAATAATGAATAATATAACATACTCTTTTTATACG

CGATATTACATTANATTCAAATTCGAATNTTCTTGGATCA

AATGCTTCAGGCCTCATGTATGTGGGCCAATTTTGAATGG

AATCGTTGGGTTATACTTAGAAGTAGATTCATCAGGTTTA

GTTATGTGGGGCTCATGACTCGCGTCCATTGATCAAACAA

CAAGCCCTCTCATGTACAATGTANGATTATGTTTTCTTCA

[C/T]AATCAACTAGTTAGATTTGATGCATAGCGGTGGTT

AACACATAACCGATTTGTTTTTCAATTAGTTGTGGACAAG

CAACTCTAATTTCTGAATATAGATTTTATTTAGTCATATG

ATTAGCGCCAAAGATTAACACAAAAGTTATTGACCATAGT

CTACNGAAATCACAACCAAACACGAGATACGGCATTGTCA

AACACAAAGTCTAAAAGAGAATATAAAGTTTGAGACNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNAGGTCAGGAGAAGGTTCTACAAGA

ACTGGGCAAAGTCCAAGAAGAAGGCTTTCACCGGGTACGC

CAAGC

6
N16487-001-Q001

ACAATAAAAATATNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCACTTTG

ATATCCATAAACCTAATCTTTTATAAACCCCCTCTTGAGG

TTCTATCCTCTGCGCTCGGCTGTACTTTAGGAGAAATCTA

CACCGTTTATTTTCCCTGAGATTAAAATTTAATCACCCTA

CGCTCTTATTGGGCCGGGCCGGGCCGAGTTTGTACTGAGA

AATTGGTTTGAGTGAACTAATCCTCTGTAGAAAGCTATAC

CGTACACAAAATCAGNGCNTTTTCTTCACCGTACGATGAA

TCAGACGAAAGTAGGATTTTTTTTTAGTGAACAGAGAGAT

[G/T]AATTGGGCCAGGCCCATTAGTAGCTTCGTTCTATA

ATTTATTCGTTTCTTAGAACAGAAGAGTTTTTGATTGTGC

AAAAATCAGAAAGAGACGATCACGAAGATGGCGACGGAGA

GCTCTTCAGCTAAGAGATGGCTTCCTCTTGAAGCTAACCC

AGAGGTTATGAATCAGGTATTCGTCTCACTTTCCCTTCTT

CTTCTTCTCCGATCATCNGCTTTTTTTTCGAAATTGGGAA

GATTGATTCTAGGTCAGTTCAGTGATTTTTTTTTGTATTG

GTGTGTATTGTGTACAGTTTCTTTGGGGNCTGGGTCTTGC

ACCAGATGCAGCGGAGTGCAATGATGTGTTTGGATTCGAC

GACGAACTTCTTGAGATGGTTCCNAAGCCTGTTCTTGCTG

TTCTC

7
N23426-001-Q001

AGCACCACCATCGACATGCGACCCCTTAGGTAACACCATA

TCCGAAACATTTTAGTAGCTGTTATTTTNTTTCTGATTAA

GATTTAGCCTTCATATTCTTCTTGGATCATAACTCTTTAT

TTGCTATATTCAATGCACAGTCATGAATTCATATTCCATC

CATTATTTTCGTTAATCACTCGTAAAATGCATATTATATT

GAGGAAAAATAACAACTCCACTTAATTAGACTTATATGAG

CCGTTTCAAATGTTTGAAAAATCAACACAACTAGATATAT

AATTTTCTTACTGATATTGTGGAAATTGGCTGGATGTTCA

AATGAAATAATTAATCCGCATGAATTGATGATGCTTCCCT

TCAAAGAAAGACATTTCTAATATGGATACCTTGTTTTTGT

[A/G]CAACTTCTAATATGGATACCANGCATTCAAAAATA

TGTAAATGTAATATAGGCTTTGATTGGTAACATGTAAATA

CTTTTGAGTTAGACATACAACTAACAAATGTTACCAACTT

TGAATTTTTGAAATTGTCTTTGAGTTGTGATGGATTATTG

TTGAGTTACAATTTTGTGTTATAACCTTTATAAAATTGAC

CACTCAAATGTTAAATCAAGATAAAAAAAATCTCATGTAT

TAAAATTTGAATTGGAAAAATGGGTTTCATAATAATTGCN

CACGTCCATATTTTATTTTAACAAATTAAACAATTTACAA

AACATGATCATATAGTTTAGTACGTTTAGGTNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNN

8
N05671-1-Q1

AATCTCCGAACAGAGAGACATCCTTGTNAAAAGTNNGAGA

CTTNGCGAACCCATCAAATGAAAAGGAGGAGTTCTCNTGT

GTAGTAGCCATGGTTCC[A/G]TAACTCACGAAGCACAGA

GCAAAGAAAAAGGATAGAGCTTTGAGCAACATCGCCATAG

CTTTGAACAGGGACAAGCTCTTNTTCTTCTTCTTCTTCTT

CTAGATGGAATCTTTTAGGAATCGAGAAAGAGTTTTACTT

TTTCAAGAAGACAAGTAAAGTTGTTTTCTTTGTTTTTGTG

TGGATTTACGTGAAGAAGAAGATTAAAATATAATCTTTGC

AGAAACCAGAGACCACGATTCGCTCTCATTCTCTTTCTTG

TAGTGCTTGCTGACTAAAGGTTGTGAATGCATTTAATGCT

GTTTCTGATGTTTTTTAATTCAATGTTATTATTACATTCT

CTTTTGTTTAGCCTTTGAGATTATGAAACCTATGCGCATT

TTCTTCAAAACGTTTATAACATACATTAAAATGGTTGAAG

AGGCTTTGCTTCACACTTTTTGTTTTACTTACGATCAGAT

GGTTACATACATATATGTAAGGACAGCTTGAGTGAATAGT

AC

9
N12643-001-Q001

TCGAGCTCGTTGCGCGCATAGGGAAACACGTGTACAGCAG

AAATGGAGTACTCACGGAGGTCAAATCCTTCGGCAAGGTC

GAATTGGGTTACGGCATTAGAAAGCTCGACGGCAGACACT

ATCAGGTACCAATGAGAATTGATTCAATTTTTGCCTTGTT

GAATCGGTGATTAGTCGAGTCCGTAGANTTTGTTTTGATT

AGGTTGGCCTTTTGGTAAAAGCTTTGCTTTAGCATCTTTC

TGATCGATTCCTATTGTTAAAGAAACTAGCTTTTGAGCCT

TTGAGTTGAATTATCGAAGCAGCANAGTTTTGAATTTTGA

ATTAGTTTATTCAAATTAGTCACAAGGATTCTTGATTCAC

CTCTTTGAGCTAACACTAGTCACAAAGATATACAGAACCA

[A/C]CATGGGTTTGCTTGGTAGCATTTTGTCCCTTGGAT

CTTTCAGTGTNTATATATAATAACATAGTTATGTGTGGGT

GAATGGCGCAGGGACANTTGATGCAGATAACAATGATGGC

AACACCAAACATGAACAAGGAGCTTCACTACCTCAACAAG

GAAGACAAACTCCTGCGCTGGCTCCTCGTTAAACACCGCG

ACATCAAGATTGGAGCTTNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAC

AATCAAAGCTAATCGAGAATGGTTAACCAAATCGAATAAA

GAGTAGTCGGGATCGTTACCCATAACGACGGAACTGATCC

CTAAC

10
N05943-1-Q1

AAGAGCAAGGGTACTAGCATTCACTGTGTGTTGCTGCAGA

ACNGGTCCAAAGACCATGGTAGGGCAAATGCTGACGAGAC

TAATCCCGGTTCGTTTTGCAAACTCGAAAGCTTCACTTTC

TGCTTGTGTCTTTGACGCACAATACCANTTCTCAGTTCTT

TTGCAAAACTCCAGGTCAGACCAACAAGACTCATCTATNA

CTCGATCCTTTGACCAATTAGGGTTCATCATAAGCGCAGC

TGCAGANGAAACGTACA[C/T]AACACGCTTAACATTTGC

TTCGACACAAGCTTTAAGCACATTCAACGTGCCATCCACA

GCTGGTGCCATCACTTCCACCTCAGGGTTTGGGACTGAAG

ATGATGGAACAGGGCTAGCGACATGGAAGACGCCGATGCA

ACCTGCGATCGCAGATTGAAGAGAGGCGTANTCAAGCAAA

TCAGCCTTGACGAGCTTGAGTTTNTCACCNGCTTTCTCCA

GCTTCTTCAAATGAGAATACTTTTCGTTATCAGGGTCTCT

GACAGTGCCATGGACNAAGTAATCCTTGGAGAGGAGGAGA

TCGACGACCCACGAAGCNAGAAANCCTCCGGCACCNGTAA

CGCAAACCTTTCCTTTCGCCGCCACCGACATTTTTTTATT

TTTTTTATCTCCGAGATGCTTCTTCAATATTCTTCTACCT

TGTCTGAATGCCAGGTGTGTC

11
N06007-1-Q1

TCCCGTGTTAATGATTTTACACGTTATTATCACAATAAAA

CCAGAGCTTCCAAATTCTTGGTTCTGTGTAATCAT[C/T]

TGGAAAAGAAAAAAGAGAAAAAAANTGAATATGTAAATGA

TGTGATTTTTGGTTCGTTGCGGTTTGCATGCTCTA

12
N10105-001-Q001

CCCTAAAAACAGTCATATTAATCGGGGGCCTGAGGCGAAT

ACCTTTTTCAATACACTGTAGGCACGCCTCTGATGAAAAC

TACAGAAAACTTTTCATTTTGCCGTGTTCATATCGGCTTA

CATATAGAGAACAATTAACACATCACCTGACCGAAAGAAA

CACTAGTTAAGAAAATATAGTACTAAAGATAAAATTACTG

[A/T]TATTAGCAATTTAGCATAGCTAAATGAAGAATAAT

TCTTACAGAAAGAATTGTAAACCTTAATTTTCTCAAAAAA

AAAAGAACTAGGATAATAAATTATGGTTTAGTATAGCTTA

TAAGGTTTTAAATATAATGTGTATATCATATAATTCTTTC

AGAGAAAAATCATTTTTTCTACGGTAGGAATGTAGATAAG

AAAAA

13
N08181-1-Q1

ATGAGCAGACCCNTTTTTTATCTTTTGTTGAATGGGGATT

TTTNNGACAGTGGAGGAGCTTCTCCAACTTCCAAA[G/T]

TATCAAATTTAGCTGTCTTTTTCTATTTTCTTTTGACGTA

TGTAACTCTTACATGCTCAATTCTAAGTTGGTAAC

14
N06675-1-Q1

AGAAGCTTGCTAATATTCCGACGCCGGAAGCCACCGTGGA

CGACGTAGACTTCAAAGGTGTGACTCGTCAAGGAGTTGAT

TATCACGCCAAGGTCTCCGTCAAGAATCCTTACCCTCAGG

CCATCCCTATTTGCCAGATCTCTTACATCCTCAAGAGTGA

CACAAGGATGATAGCGTCTGGNACAATACCNGATCCGGGT

TCGTTGATCGCGAACGGGTCGACGGTTCTTGACGTACCGG

TCAAGGTGCCTTATAGCATAGCGGTGAGTTTGATGAAGGA

CATGTGTTTGGACTGGGACATTGACTATCAACTCGATATT

GGACTGACCATCGATATTCCTATTGTTGGTGACATTACTA

TTCCTGTCTCNACTCAGGGTGAGATGAAGCTCCCTTCCCT

TCGCGACTTCTTTTAATCATCTNTATAAGTTATAATCTGA

TTTTTNAATAAGTACGATCCGTAAACGACATAGACGATCG

TTGGATGTTTCAGTTGTGGA[C/T]TCTTGTGTTTGTTGG

TTATATGTATTTGTTGCTTTGAATATTTTGTTGGTGAGAG

TTAAAAACTACAAGATGTCATAGTTCGAATACTAACGAAG

TGATGGAGANNNAAAAAAAAAAAAAAAAAA

15
N001KH2-001-Q001*

AGCTGGATTTTGTTTCTTCTACAAAAATGTTGGATTTAAA

GAGTTTTTTACTGTGCTTGATAATGGGATTACTGTGGCTT

TTTTGGTAGCAGGAAGCAGAGAGAAAGAGTGGGAAAGAAA

AGAGTAAGAAGAAAGGCTTTGGGAAGCTAA[A/G]GCGTG

GAGAGAGCAGTTCATTTCTTCCCATCTTCAGAGAGCCTAG

TAGTATTGAAAAGATTTTGGCAGAAGCTGAGAGAGATCAT

AATCTTGTTTTCAGGCCTCCTACTCCTCCTGATCAATCAA

ATCCACCCTCAGCCTCTCCTCCACC

16
N29313-001-Q001

CTACTTGTATTTCAATTCACATTCATTGTTTTGCTAAAAA

CAGACAAAGGCTTTTGATATACACCTACTTTTCGGCAATG

AATACAGTTGCTTGTGACCAGTCAAAGCGATGAGTCAAAG

CAATGAACAATCTGGCCACACAGACGGGGTGAATATCTGT

TTCTATTCACATATGGGAATGGAACAAGAGAAGAATCAAC

[G/T]AGAAGTANCAGAGAAAGTCATGAGTTTTTCAAGNT

TAAAGAAGTGAAAACCTTGGCTTTATGAATAACTTTAACA

GCAACTTCTTCTTGACCCTTCAGGCTGCCTTTTTTACCCT

TGGCNGAGCAAGTGTCCTCGACCCACCTNGCCGTCCATCT

CATACTCCTTGTCTTCAGGAATAGAGACCTCGTTGGGAGC

CTAAA

17
N88512-001-K001

AATAGCTATACTACCAGTCTCTGACTACNACAACAAATNA

CAATTTTACTCAAAAAGGACCATTTCTTCACCTATTAACA

ACAAATGACTAGTAGAAAAATGGATTGATAACAAAGGCNA

ATACTGAAACAAACAANATTCGTTTGAAAAAAGCAATCGA

ATTTCAGATGTTGATCAACAACTAGCAAATCAAAAGTGAC

[A/C]AAGTTTTAAACTTGTAATNATAAGTTCGTCAAAGA

ACACTTTATTCCACCATTATCAACAAAACCAAAAGACACT

GCTTCAAAGTTCCAAAGCATCAAACATCTGATCCTCTCAA

TCGGAAGAATCTCCGGCGAGCCTCTCCGCCTCGAGCCGCT

CCAGGGCCTTCTGATGAGCCCAAGTCTCGATCGAGAGATC

AGGCT

18
N88514-001-K001

ACACGAGAAGCGCTCTTCACCGAGGNGATGATGCCCTTTA

CAGGTTAATGGGCTTTCTCTTGTTTTTTGCTTAATGGGCT

TTCTCTTGTTTTTTGCTTAATGGGCTTTCTCTTGNTAACT

CTTTAACTAACTGTGAACAGTGTTGTTTGTTCTTGTTGTG

NTAGCAAGAAACAAGAATGTAAAGGAGAAGAAGAAGAGCA

[C/G]TCGAGGAGTTACTGGCGACCAATTGGTTGGGACAG

GCTCTCTGAGCTTGTTCAGACAGTGAAGGTTGATGGTGAG

TGGTCAGTGCAGAACGTTGATGTAGATCATGAGGATGNTG

ATACAACGGTTGCTGAGCTGGCTGCTCCTTACTGGGACCG

ACCACTCGCGGGTCCCACGTGGTGGTGCCACGTGGATGCT

AGCCA

19
N88515-001-K001

ATCAGGAGAACAATAAGAAACTTGTTTNAAAGGCAAAACA

AAATGATAAAAGCCGTTTTTGCTAACTCTTATGGTAATGC

AATTAGAACAATGATAATAAAAAGGTTCCATCTAAGGCCT

AACAACATACTTTGAATATGTTCCTCTAGAAATATGATAA

CATGTTGTATAGAAGTAACAGATCATTCTAACTCATTGCC

[A/G]CCAAGTTTAACNCAATGAACTAAACACTAATATAT

ATAAGGGGCGGTGTAGATTACCTCTGAGCTGAGGAAGTTA

TGAAGCACAATAATTCGAGGGGACCAACTAACTACTTCAG

GCTTGACCTGCACTAAACCAAATCAACATTAAGAACAGAC

CTTGAATTTACAGAATGGATATGATAAGACTTACATAGCC

AAGCC

20
N88516-001-K001

TAAACTGAAACCCATATTAACGACAGANGAATATACGTAA

CATGTGGTGTTTACTTAGTTTTATTTTATTTAACAATTCT

TTTGTCTTGTAGGGTTCTGATTGTAGTTTCGTTTTGAAGC

AAGCATAATATTGTATTCATATTTTTGTTATANAAGTATT

TCATGTTTCTGTCGTGAAAAAATAATTTTCTATATTTCCA

[A/T]TTTTTTTGGTAATGTGAAATTTATTGATTAGTAAA

AGATAGTTCATTACAATAAAATGGTACTATAAATGATAAA

AGAAAATATGATAAGCGAATAAATTTTGAATATCGAAGTC

GCTATAATACTTTAAAATGAATATAGCCAGTAAGTACGGA

ATCCTTCTTTGTTATATAGCGACTTGTATAATCGTTTTAT

TCCTT

21
N88517-001-K001

ACGGATTCACCTTCTCCCTCTCTCTCTGTATATTCTTCGC

ATCTTCTCAAGAAGCTCNAGCTTGAGGAGAGAGAGAGAGA

GAGAGCTTTAGATTCTTGAGTGTCTGTAAAATTAGATCTC

ATTGAGANAGAGAGAGTATCCAATTCTACAAGGTTTTGGG

CTTGTGAATGCACTNGTTGAGCTGATCTTACAGGTCCATT

[A/G]AGGTTAGTTTGATCGATCTGNTCTCTGTTTTCTTC

ACCGGAGCTGATAAAAATGTNANCTTTACAATGTGGCCAT

GCTTGATTCTGCTCCCAAGTTTACATTTTTATCTTATGGG

TTTTGAGATCTATCACGGCTACTGAGATCTGATGGCTCTC

GTGACTTGTCATTTTGAATGCTTATGTTGTTTTCATTGTG

NAGCG

22
N88518-001-K001

GACAAAAAAGAGACCCAACAACTCANGTGTTTCTTTTTCT

GCTTCTTCGGCTCANCAAATCAGCACCACATTTCACACTA

GTCTTCACGACATTCATGTTCTCNTTGNTCCNTCATTTCC

ATCACTATCATTTATCCATTTACCACAANCNTACTATTCN

TTTATCATTTTAAAGTTTACTTTTATACATCACGAGATTA

[A/G]TACTAAAATTATAACTATATACTAGTTTTTTAAAG

ATTTTTATANNTATATATCATTTTGTTTCAACAGAAATTA

AAAGAAACTAGTTGAGGAAAAAATGAAACAATGGCTAACA

TAACATCAAAAAACTTATTAATATTTTCTGTAATCAGAAA

ACTTTAGACCAAACCTCAAACTTATTTATGAGAACATATA

TTCAC

23
N88519-001-K001

GNGATGTGTTTCATCAGAATGATTTATGGTTTCTTNTAGA

GGCATATATTTGTGAGATGAGGGATGTGGAGAGTTACAGG

GCCTCATAATTTGATGGTTTATTGAAGAGCATAGAAACTC

CATCAGAAGCATTANGCNGGAGGAGTTNGAGAGACAAACA

AATTCNGCTATGAGATAATTCAAGGTGATTAAGTGATATT

[A/G]GATAATATTAGAATTAGNGGTGGGCCAAAAAACCC

AAAAACTTTTGGAAATGGGCTGGCTTTTTTATGGATCCGG

ATGGTTCAAGGTCCATCAGCTTAATATACGCTATGACGTG

GCTAAATAATAGACTTAGAATATTTCAGGTGATGTGGCAT

CNANATAAAACCAGAGATTTACTTTCTTTTATATAGTTAA

ATGAT

24
N88520-001-K001

TAGTGTNGATATAAAGCCCTAATTTACGGCAGCTACCGAA

TAAATTTNCTCCGGCCGGTGGGGTGTTCGAGAGCAGATCA

TCAGCTTCTCTAGTCTCCGGTAAGTTAATTTTTTTTAATT

CTTATATCTCGTAGGTTTTTTTTTTTTTGTAATTTGATCN

NTTTAATTAGCTTCGAATCGATCAGAATCTAAACGGTTAT

[G/T]AGNAATTNTGTTTAAATTGTGCTTTTAATATTTCA

AATTGATTAAGTGCAAAGTCAATTTTGATCAGATCTCACT

TTCTGGTAGAGGAGAAGAAGAAGAAGAAGTTTTTTTTCAG

TGCTTGAGGAGGATCTGCCATATCCATCAGAGGTTTTCAA

ACAATGGTAAATTTTGATCCTTAGCTATATGAATACTCTA

TATAA

25
N88521-001-K001

TTTTCGAAAAAAAAAATAAAAAAAAAAATGGATGGCATTT

TCGTAAATTATATGAACTTGTGGGGTGAATAGGGCAAAAN

CAATTTTCAAAAAAAAAGGAGGTTAGTTTTGTGTTTGACT

TTAAGTTATAGGTCAATTTTGCAAAAATCCCATTTTTTAT

ACATGCCAAAACCAATATAGAAAAAAACAAAACACTCTTC

[A/G]TATACNAGACAAGCACTATAAATACATTCCATAAC

CGTTAAAGCTTCCAACACCACCACCACCACCACTACAGCA

CTACTCCACTCTCTCTCCCTCTTTAATTATCTCTGAACAA

GTGTAAGTTAGCGACATACAATGGCTTCACTTCTTTTCCT

CTTCGTCTTCTTCTTCTCCATCTCCTCTTGCTTTGCTCAT

TCCTA

26
N001KFE-001-Q001*

ATGTTCATTGTAGTTAATTAGTANAGAACTATCGGGGCAG

AAAAAAATAAGTCAACGCGTTGCCCGAAAAATTGTATACG

AATGTACCTGATTTAAAGAA[A/G]GAAAATTCCATAAAA

ATACTTCAACTAAATTTTTCTTAAGCTTTTTAAGANTCAT

TTTTCCGCTATCCACTTTAGTACTTCAACTAATTATTTTG

CCCAG

27
N88522-001-K001

TTGTATGAACTGGTTTTAATTAAGTAATGCAAGTAGTTGG

TTGACAAAAAAAAAGTACTACACCGTGGATCTTCAATCGC

CTGATCAGCATAATTAGTATATTCAATCATATGCANAATT

AATCTAGGAACTAATTGATAAACTAATTCTTTTACAGTGT

AGCTAAAGCTTTTATTTTCTTCTGGATACAGATAAGAATA

[A/C]ATACTATATGGGGACANATNCTTTTGTCGATTTTT

CTATTCAGCTTTGCAATGAAGTCTGAGCAAAGATATGACC

AGACTGAAAAGGNAATTAAAGGATGATAACATGGAAAAAA

TTAAAAACAAAATTCATGCATCTGCTAGTGAGGTTATTTG

GATTCATTGCATGTATGTTGATGATTCGTTTTCCTTTCAC

CTCCC

28
N88523-001-K001

AATTTTTACACTTTGGCCAGAGATATAANGAAAGGTTGTA

AACTCATAGACTTAAGAGATTTTTTGGTTTAGTCNGTANG

TGATTTTGTAGAAATAATAGCGCATATTGATAAATATGCA

ACACTTGCTTTAGCCTTTGTTTGTGAATTGTGGTGACATA

AGTCCATCCCTACAAGTCATGTATTTGTAACGACTTGAAA

[A/G]TAAAATATTTTCGGAAGACATNTCGATNTATAATC

TGCATTTCAGTCCGGACTCCGAATATCCATATATATGATC

TAAATTGGGTTTCTGAAATAAAAANAAANCACAACTTACG

TCAAGTCAACGGAGGTTAAATCCACCCTGTCGCTCTTGTT

TGCTCCTTTGGCAAAACAAAAGTCAACTACTATTTTAGGT

GCTTG

29
N88524-001-K001

TTGAATTGGTCTGAAAGTTTGTTAGCTGTTACTTTGAATA

GATGCCTCGGAGACCATCAGGAGGAAGAAGGTTCATAAAG

CACCAACCTTTAGCATTTTCACCGTTTATGCGGTCACTTG

CTTTAGCTTCAAGGCGTAAACTGCATCGTCATCAACAAGA

AGANGACTCTCNNCGTTCTGAAGAGCTGATGTCTTTTGGT

[G/T]AGTAGTAGTAGTAATCATTAAAACTGAGAAGTTTC

TGTTCAGAAACATTGTAANAACGAATCATTCTTGTTTTGT

TAGATCAAAAGCTCCCAACTTTGTCNAAAAAGGAGCAAAA

AGAACAGCTTTCTGACTCCTCTGATGAAGAAGACTCTCAG

GTAATAAGATTATTCAGACTCCTCTGTTTTGATCCTTGAN

TCTCT

30
N88525-001-K001

AAGTGTAAAAGTTATAAAACNGAGACGTTGTGTTTGGNCA

TGTCATGTAATTGTTGAATGTTGTGNATATGACACAGACG

GTGATGAANTCAACAAGGAGACAGCTGGAGAGAGAGCTGA

TGCTGGAAGACATAATGCATCTTGAAGACTTGCCTTCCTA

CGCCCTCCTCTCTCAATAGCCTTTTAAGAGTTTTCTAACC

[A/T]CAACACTCCCGCTTTTAATAACACAAAAGGTTGCT

CGCTGCTGCCTCTTCTTTGATCTTTTTCTTCTACTGTTTG

TTGGTTGGCTATATGAAAAACAGCTCTAAAACTGAGTTGT

TGTTGTTATATTAANACAAGNAAGAGGGAGATAGAGAAGG

AGGTTGTGGGATAAAATCTCAATTTGGTTGTGGGTTTGGA

AAGTG

31
N88529-001-K001

TATTNGAAATAATATTGAGGGCTGTGACGAAGGATGCACG

AGAAATNTCAACAGCTTCTTCTCTATCACTGCATTCACTC

ACGAAGNTGATTAGTTCTTGCACCTTCTNCATCCGTAGAG

CTTGGGTGGCCTCGATACGCTGTGGTGAGAACAGATGAGT

AACTGATATTTTCCTCAACAGCCTGAAAAATATAACATAA

[C/T]TATTTTTTTAATNAAATACACAAATCTATTGGTTC

NGTTAAGGTAACTATATGTTATGTATACAGCAGTTATATA

TCCGGGACAGTCNTCTAGTATAATGAGGATAATATTTTGG

TAATGACCGGGATAGCTCAGTTGGTAGAGCAGATGACAAA

TAAAAAGACTACCAATAGGAAAAAAAAACTTAGCAACGGT

TACCT

32
N88530-001-K001

GGCAACATATATCACAGTATAAGGAAGTCAATACAAAAAC

ATTATAACATTTCCAAATATTGAAAGATTTCTTTCTCATA

ATACNAATGCACTGAATCAAAATAAAAAATCAATAATTTT

CCAAAAACAAAATCTAATAATTCTAAGGATGATATTAACT

AGAAAATATTAATCCTCCATTGTTGCGTATGACAAGCTCG

[C/T]ACGTTTTAGCCAATCTCTCACGAACGATACCGGCG

CGTTTCNTACACTCGCTCTTCTNTCCTCCTCCCTTATATT

ACTTACCCTCGAACTACTTCGTATCAACCTTTAACCTTTA

GACAAACTCACTCGTCACACATTCTACTCATCCTTGACCT

TGTGTTGTGTGTTATATATACACTACTACACACGCTTATT

ATATA

33
N88531-001-K001

CTGCTGCTGTTGCTGTTGCACGGATGAAGANGAAGAAGTC

GGAGACGCAGCGGATGATGTGGAGTTGGTCCGACCCGCCG

GACCCATGCCCGGATTCCGNGCGGGGCCTCCGCCACTGCC

TCCAGATTGCATTCAATTCTTCGATCAATCAAACCTAATT

CAAATGCACAATAAGACAAAAATTCAAAACAAGAAAAAAT

[A/G]AAGCTGGGANTTAGCAATAAACGTTTGAAAACGGA

AATTTACAAAGCCACACACNCACTCACTCACTCTCTGCCA

CTTTCCTTCACTTGATTTGAATCGATTTTCTGAGGGAGGG

AGGGAGAGAGAAACGAAAGTCGCGGGATGCAACGGAATAA

ACCAGATCCTNTGNGGGTGGCTTCTCGAGAAACGNAATCC

ACGGA

34
N88533-001-K001

GTATCCTTCCGGCTATCAATTCCCACCTATGAACCACATA

CCATAGATTCTAAATCGTCAAATACTATATTTTATGTAAA

TAGCTTTTTAAAATAATTTAACTTAAACAATCTGNTGAAT

AAATAACTTTCAAGTAAAATCAANAAATAGAAGATTAGCA

TGGCCGTTGCGCAAGGATTACACGCACAAATTCGAGAAAT

[A/G]GTCCAATTTTTTTTCCAGTCAAAAANGTAAAATCA

AGAACAAACTGGATCAGCGAGATCAGGCTGAACATAGTCA

TTGACAGCTNGGTTCCAATAATAAGCAATAAAATACATAA

AAACGACCGACTTATGAGATAAATCAAAAGCCATAGTATA

TTCTTTTAAAAAAATCAGANTAAACTAAAGATGGAAACAG

ACAAA

35
N88535-001-K001

TGAGTATCATCTTTGCCTTTGTTGTACCTTTTCTGAGGAT

TTAGAACGAGATTGGATTGAGGATTAGTCCTCTNTATCCT

AAGAAATACATGCAATTTAGTGTNTTCCTTACTAANATCT

GATTTTCACAAATGGATTGCTGCTTCTCATGACTNATCTT

GAATCTCAAGACTTGGTTCATTTTATTTAATGGACCTTTG

[A/C]AACGTTGTTGTTTTGTTAACACAGTGGTTCGTTTA

CAAGAACATGATCTTCAAACCAGACAGGTCAAGCANTCTC

CCCTCAATTACATAATTTGGTTTCGTCTAGTGTTTTCTTT

NAGAAATATGAAACTCATATTTTTCTATCGTAACGTGTTT

CAGGGAGGTGCTGTTTCAGAAGGTCAAGGATACATACAGA

GACAT

36
N88536-001-K001

TTATTATNGTTTTTATTTATTTTTTGCCGGCCAATCATAT

CAATTAGACCAAGTTCTAATTTGTCACAAAGAGTTGTTCC

GAATAAAATATTTGTCTGACCATGTCTGATCTAGATGGAA

AAATACAATGCCTCTAGTCCTTCCATATGGTTTACAANGA

GTTAGATTAAAACTTCCCATTATCATTGTGCAATTTCCGA

[C/G]TGAGGAAATAACAAATTGTATCTGGAGAAGCAAGA

AAGGNGGTACAAAATCTTAGCTTATCAAAATGTTCACTTG

TCTTTGGTCTATTGGGAGTGTCACTTTTTGTCTTAGTTGA

AGCCCATAGAAAAGCCCAAATTATTAGTCGGTATCTGNCC

CATTTTTTAAAATTTGTGAACCGGTGTCTGTCTTCTTCGC

TTGCT

37
N88537-001-K001

TCCGAGTCAACTCAATCCGACTCGAGATCCACCACCAGTG

TGCCACGGACTCTCATCGGAGGGCTGTTGCTTGTCGAGAT

TGGACCTGAAGCATCGAGACAGGGCTTTGGTGGTCTCGTT

TAACGAAGAAGAAGGAGCGATGAACTTGTTGGAAGAATAA

CACAGGGAACCGCTTAAGGTTTGATCCTCAGTTCGCTCGT

[C/G]TCCTAAGATGATGAGGATCGGAAGGGGATGATTTA

AAGAGTTCGTATAATTATTCATCTGATGGGAGCGTTAATA

TATATAGATTTTGAATTTCAAATGAAACAAAAATATTACC

GTTATTCACTCAAGTCGGTNAAAAAAAAGTAACTTGTGCA

TCAAGCAAGTAACTTTTGTGGGCTGGCCTCTCTGTTTCTG

ATGTG

38
N07541-1-Q1

CTTGTTGCGAAGCTTCTCTTATTGGTTCTTCCATCGTCTC

TCCTGAATCAGAGTTTTGATATTCNAAGTCTCCCA[C/G]

CTATCAAAACGATCAGAGAACTTTCTTTCGAATAGATTAA

AACGATGATATCTAAAATCGAAACACTGAATTGCA

39
N23413-001-Q001

TCCTTCTTCTCTCCTTCTGATATTTTGGCTTTTTCTTAGA

ATCTTCCTTTTTTATTTTCACCAAAAAAAAGAAAAATCAA

AAAGTATTAATCATTTACCGGTATCAGTTACACCTACACT

TTGCGCCGCTGAACAAATCAATAAGGATAATAAGAAGAAG

CTCGTTTGCTTCCATTTTCAGACATTCTTTGCCTAGAGCA

AAAAAACAAAACAAAAAGATTGAGACTTGATCTTAGCAAA

ATGGGTAATTGTTTGGATTCATCAGCTAAAGTGGATAGTA

GCAGCCGCCATGCTAACTCTGGTTCGTCTCCTCCTCTCCT

TGCTTTCTCTTCCTTTACCCAGTTTCGTTGCTTCCTTAAG

ACTTAAAGACCTCTCCTTACTCTCCAATTCCAAGCCAAAG

[A/T]CTTAACCTTTAAGCTGTTTGGATCTTCAAAGATCA

ATCCTTTTCAATTACCAGCTGTCTGATTTCANAGATTGTA

GATATCTCTTTGGTACTCAATGTTGGATCAAGTTGATTTA

AGATGGTTGATATGCTTAACGTTGAGATTAANTTTATGAG

CCAAAGCTAAAAGTCTTAACCTTTTTAAGCTTCTGTTTTG

TCTGTGATCTTCAAAGATCAATCCTTTTGAATTACCCATC

TCTGCAGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNTCTATAAGAACAG

CGAGGAGAAATTAAACAAAACAACATCACTTCATATTTAC

AGTGT

40
N08344-1-Q1

ACNTCGGANTTGTCCTTCTCCTCGGATGCAGGCTCGTCTC

CGTTGGTGGTGGTNGTGGTTTCACCGAGACAGGTC[G/T]

CCTCCACGTGGACGGAGACTTCTTCCTCTGTTTTGAAAGG

GTTGCCGCAGATCGGACACTCGAACATGTTCAGAG

41
N23533-001-Q011

GTCCGGTGTTTCTCCTGTGCTAAGAAACTCCANTANAGAC

CAAACCGTTTTATGGACCANGGTGGTTGTTGAACNACCAT

TGAGTTCNTNTNTAACCGACGATAATCTATGGTTCACTTT

GAAATCCNCAGGAAAGTTGAATTTGATTCGCCNTGTGGTT

GACGCAAACCCTTTGATCAANAAGGTAACTTGATCATCAC

[A/G]AAACTCGACTGGNCATAGTGAAACNTTTTAAGTTA

GGGGTTGTTGTTGAGGAATGANTTTTCTTTCTTTCTTTCT

CAGNTAATTGTGNNGGGATGCACGNNGTTAGTTCCAGAAG

GAATCATTGCATGTGNTGAAANNTTGNNGAAGAACAATCA

TAAGTTGGAGACACTTCACATCAATGGCGTCCCTGGCTTC

ACTAA

42
N14649-001-Q001

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTC

TGTAGTAGCTGCTGGGTGATGGTCCTGAAACGTTCTTGGC

CTGCTGTGTCCCACTGTATACACGGTCATTGCCACGAGTT

ATAGTAGTATACACGCTTGCCAAGTTTTAAANGAATATAT

GTTATCAAGAACACTTACAATCTGGAGTTTGATTGTCTTT

CCGTCTTGTTCAACTGTGCGGATTTTCTGAATCACATCTA

ACATTAGCCAACACTAATATTTTTTTGTTTTCCGAGTACT

AACAAGTGATGGGAAGAGTAAACTTACAAAGTCAACACCA

ATGGTACTGATGTAGCTATCCAGGTAAGAATCATCCTGTA

CACACAAATTCAAGACATCAAACTATAAGCACACAAAGAG

[A/C]AACAACGATACTATACTGTAACCTGAGAACTAAAC

AAGTATTAAACTTCAAAGACCCATAGATTTGTTACTCGTG

TTCAATGTTAAAACGCAATTACCGAAGATAATACACTCAT

AGTAACAATGATAGCATAGTGTAAACTATTTTGGAGAGAC

AAGTAATTGAACTTGAAGGACGCACAGATTGTTATTAAAC

GGCAATTAACACATGCAAGTAAGAAATCTAAAACAATTAT

TCAGCTGGAGAAAGAGTTACTTACAGCAAACCTTAGAAGC

AAGCAAGATTTTCCAACACCAGAGTCACCGATAAGCAAAA

GCTTGAACAGGTAGTCACTGCAAATTAAAANNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNN

43
N23310-001-Q001

TTTCCCATACCGTGTAAGAATATGGACAATCAAAGAACAA

GTGATCTCGAGTCTCATCNCTTTCTCCACACAGTCCACAG

CTTTGTGTGNTTCCCCAAGCCCTATGTCTCCTGTAGCAAG

CCTGTTGAGCACTGCTAGCCACGTAATGAATGAATACCTA

GGAATGCTTTGCGTGAACCAAACCACCTTACTCCAANCAA

CCTTTGCTTTCTTGGGTCTAATCTGGTTCCAAGTGTTAGC

CGTTGGNAACATATCCCTGAAACCATCCTCCTTATGCCTC

CAAAGTATCAGATCAGTCCCCCTACTAGTATNTGGAAGTG

GCTCCGCGAGAATTTGAGTGTTTAAACTCTGAAACCTTCT

GCTGCGCTTGTTTCTAAGACTCCAACCGTCCCCTGAAACC

[A/G]CATTGCTCACTAGTGCACTCCTAGGAAGACCCAAA

TAGGTCGTACCGATGGCTCCAGTAACATCAATCAATCTTC

CACTGCCCATCCAATTATCGAACCAAAAATATGTGGTTTC

TCCATTTCGAANCTCTACTTTCATGAACTCATAAGCCAAA

TCNCTTAGCTTTAGTAGCTTCCTCCAAATCCATGAACNTT

TGGAATCATCTCTCACGTCCCAAAAAGAGCTTTGTCTGAG

TAGATAGTGCTTTATTCACTTCAGTTGAGAATTGTCAACT

ANTTGAAAGTTAAAAATGTGAAGCCAGAAATGATACATGT

TAACAGCTGAAGAAATTAATATATAACCAAAAAAAAAATT

CATTCTATAAGGAAACTTTTAAAAAAATTATACATACCAA

AGTTC

44
N10526-001-Q001

TGACCTCTCATTACTGTTACATCCCGATAAACATAGACAC

CATTTTTGACTTCACCGGCTCCAATCAGAGTCCTCGTAAA

ATGCTCCTGCAAAATACACAAAGTGTCTGTGAATACTGTC

AAGCAGCCAGTTTTTCGAAGCAATTTCGCTACAGACAATA

GAGTACAATTCAGATTAGGAACAAATAGAACATTTGCCAA

CAANAAACTCGCAGACAATCTCAAACTGCCACTCTTGGTA

GCCATCACGT[G/T]ACTACCGTCTGCAAAGCTCACATGA

CAGGAATGATACTACGTACATCAACCAGTAATAGAACATC

CCCCTTCCTGTNANGTGATATCTGGTGTCTATAATAACCT

CACCAGTTTGTACCGTACCGTTTAACCGATNAGGAGTTGG

AGAAGGNTTTTGACGCTCCAGCAGTGATGTCAAGGACGCC

CATTGCTCTANAGTGAACTGTGGCATAGAATTCCCTTGGT

TTGNAGACGAACTTCCAGTC

45
N23373-001-Q001

TGACCGATGTCTAAAACGGTTCGGGGGTGTTACNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNATTTTACATAGAAAACTAAAAACGTCA

TATAATTTGGAACATACAAATTTCTCTAAAACGACTTATA

TAAGAAAACGGTGGGAGTATATATTTTCATATATTAATTA

ATACAAATTAACAATAATGGACTCTTAAACCTAAAGTAAA

AGTGTAAAAGAAATTANGGATCTTGTGCATTTGCACTCTT

GGCNCATGGCCAGAGCCGATGCTGCTTGTAATGCTTCTGG

CCATTCACAGTTAATACGTTTTCTATGCCCGCATTCACTC

[C/G]GTTCAATTCGTTGGTTCAACATTGTAAGATAGTTG

GTGCTTTAATTTTCAAAAGTTTNGAATACTTTTTTACTAA

NTGACATAAAGGATANAATACATTTGGAAGAGTCAAATAA

TTTTCTTGAAGAAAATCTCAACATATATCAGAAATTGAGA

TATTGTAACTTANTTNGCCTAAAATGCAAAAANTGTCGTA

CAACTGATGGTTATAAGAAATTTAAGTTTTCAAGTTGNTG

AATTCAGTAAGCACTTCTGGAATCTAACCGGTAAGATTGT

TGTCAGGGAGAGTGGTAAAGAGATTTAAAATATAAACATC

AGTTAGGAATTTTCCACTNAACGGAAATCAAGAATTAACG

AAAATATAACCGGTAATTGTGNACGTATCATCACTGCCCA

TCTAC

46
N23353-001-Q001

TTGGTTAGATCGTAATTTTATGTATAAGAGGTCCACACAG

AATAGAAGAGACTGTTTGAGATTTTTTTGTATGCTTAGAA

GACTAAAAGAGATTAAATCCTGATTTGGCCTAGATTTTTT

TTTTAACTCACAAAAGGTTCATTAACTAAACNAACACAAA

TCCATTACTTCTTACACAAAAGAACAGACCCACTAGTTTT

TAGACCATCAAACTAATCTATTAAAATTCACAAATCATCA

ATTATCAAGTTGAACCTCCCAAAAAGCATTTCCTTCTTCC

ATCTCTTCTCAAATAAATGTTTTGTATGTATAGGTACCTT

CCACAAACCAAGTTTGACACAAGCCATGCAACATGGAAGC

CTATCTGGAAGTTTGAGCTTGCTCATTGAAGTTGAGCTAA

[C/G]AATATAGTTTTTCATTTGTTTCATAGATCTATGAC

GGTGACATCTTGTTGAGAACATTTTGACATTTGTCACTAT

AACTAGTGTAAGAGCAGGAGTCTCATCTTTTTTCTTCAAA

CGATGTACATAAATTTTCTTTAAAATATATCGAATAGAAT

CAGCTAAAATAGAACAATTTTCGCACAATCAATATCTTTT

GNGTGCATATCTGAAGGCAACTTTAGGTTTATCATATAAC

GGATAACTTTCACCACATAAATACATGTGATTGAGAAGAT

ACTTGGGATTAACATGTCTGAGTGCATTGGTGATGACTCT

TGGCAATAGATCATATTCGTTTTGTACAAGATAAACAATG

TTTTGTTGTAGATCCCATTTGATGTTGATTTATTAGTATA

AACAA

47
N23206-001-Q001

ACTGGGTAACATCAGATAAAATTTCANTTCCACTTCAGTT

NTGGTTACGTTCTAGTTTTAGGATAATTTCGGATAATTCA

CGTGAAAATCAGATTTTTTATTTTTTAGTTTTTCAAATCA

AATATCAGGTAATTTTGATAAATTTAGATAGTCCAGATAA

AAAAATATTTGGATAATTCAATTTTTTTATAGTTCATATA

ATATTAAATATTTTGGACAAAATATTAAAATAATTCAGTT

TATAAGAAGCATTTTAGACTCTTTGGTAATTTTAGAACTA

AAAATTGTTTTTAATTATATAAACGGAATTTTAGACGAAT

ACCGATTCAGTTTTTTGTTCGGTTTCAGTTTTTCAGTTTA

AGAAATATATAAACCGCTTAGATATTTGTGCATATCGGTT

[C/T]AGTTTACTTTTCGGTTCTNGGTTTATATGCTGAGG

TCTACCATGAACTATCGAGGTTCTTATCAAATTTTTGATT

ATCGTCATTGCCAATATCCTCTAGTCAAAAATCATGGATC

GTAAATACATGAATGCTGATTGCTAGTATAAGAAAGTAAA

TCATTGACTTCAATATTAACATTGACTTTGAACAGTACAC

AAAATAGTTACATGACTGGGATAGATATGCACTTTGCAGA

GNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNGATTTAAAGTTTTTTTTAC

TATTGACTACCCGTGGGCTTCCAGTTCGGTCCAATTTGGT

TTTGG

48
N11025-001-Q001

CGTCCAGATATCCAACTACTCGGCAANCTCACCTGTAATG

GGAAGAAAGAGGGGTGAGTAACATGGGAGTCACTCATCAA

GGTATGGGATGCTAAAAACGCAAACCACTGAATCAGTAAA

TAGAACTCTCACTATGGGAGTTTAGCTCTAACATGAACAA

ACACGATGTCTAAAGGATCACACAAAACAAACAATGCGAT

GATAACACATAGCTATTACACACACCCTGAATTCTCCTCA

TAAGGATATAAAATATCAATTGCGCCGGTAGTACAAAAGT

CTGTCTCTGTACCCCAGCAATGTCCGCTNTGTGTCTACAT

GCAAACNCCTTTGCACCCCGCAATCTCTGCTCTGTGTCAC

TATGCAAACCACTGCACCCCGTAATCTCTTGAACATCTGC

[A/G]TCGCTAGCTCAGACGTCTCTGGTCCCCGCGATCTC

CACATAGTACTTATATTTATAACTATATATCAGTTCAATC

AACAGTTGAATCCTCTAGACCCCTAATTCTGATTTACAAT

GAATGTACAAACTACCAAGCAAGACTCGAACAGACTCAGT

TCAAACAGACAGATCATGCTTCAAAACTAAATTACCATAG

AAAGAGTTCTACACTAGTTTGGGATTTAACGGATTAACGG

AATATACCCTCACCTTAGCCAATAGTAGAAACGGAAATGA

ATCTGACAACAACCAAGATAACTTGATTACCAAGAAACAT

AGGGTTCACCTTGGATCATGCAAGCAATGGGTTTGCCAAC

TTCATTGGAGATTTAACACGATCCAAAGCACGATTGCATA

AGGGC

49
N09969-001-Q001

TTCTCTGATAGTGCTGTGTCTCTCCTTTGCCCTCCGGTGA

AAGAAAGGAATTCTTACCTTCACACATATTTCATTGATTG

TCGGGCAATCATCCGCGTGGTGGATGATTAGGTTTTGGTT

TCTTCATCAATGGGTTTTCTTGGTGCTTACGGCTCCAAGT

TGCTTTTAGTTTGGGTCTTTCTTGTTTGGGCCATTTACTC

[C/T]GTTTGATAATTTATCTTTCTCAAGGCTTTTTTTTA

CGTAAGTATAGCCTTTGTTTAATAAACTTTCAGTGAAAAA

AAAATTATGATAGCTCATACAAAATTGAAACATGGTTGCC

TTGGCAGTATTATCTATATTATCTTATTGAAAGCTGGGCC

AACTAACGAGAGAGTTCGTGAAGCTCTCCATGTTCAAAAG

GTATT

50
N09882-001-Q001

GCACCCAAATTTGATAACGATACACAAGCAGCTAATGGCT

TATAAACACTGCTAGAGGAACTTGAAGGAAAAAAAATATT

GATGGTGTTGGATGATGTTTGGTCTGGAGCTGAGTCCTCC

TTACTTGAGAATTTACCCACTAACATACCGAATCTCAAGA

TCTTGTTGACTTCTCGGTTTAACTCGCTTGATTTCGGTGA

[A/C]ACTTTTAAATTGGAACCTTTGAAAAAGGAACATGC

CAAGACCCTTCTCATTCAATATGCATCGCGTCCTGATCAC

GCATCTGATGCCGAGTATGAACGTCTTTTCCAGAAGGTAT

TCTCTATTGAGCCTTTCCATTGGTGATCTAACATTTTGTA

AATGTTGTGGGTATAATGTGTGATGTGCCTCAGTCCGAGG

AACTC

51
N10389-001-Q001

CTTTGTTGCCGCGAGCGACAGGTTTATTGCTATCCCATCC

TCAAAAACCTCTCCTACAGCTTTATCGCTATCGATTCCCA

ACGTTGCTATCATGCAGAACAAAACAAACGACCACAAAAG

CCATTTGGGACCTCCATGGAGGTAAAACTACTCGGAGCGG

CAAAGTTGAAGCTAAAAATGAGAAACTTAAAAAGAGTTTT

AAAATAAACCTTTTTATATAGAGTAGTCGAGATGGTTTGA

TTTGCTTATG[C/T]TATTCTCTCATTAAAATTCAGATGA

GTGATAANGTGTAATTAGGCTTTAGAATTTAGATCCATCT

AACTTCACAGAGTCAACGACTGCAAAAGATTGATTTGCGA

GTAAGCTAAACCCTCGTGATATAGTCAATCATTCAATTAT

ACTATTATCGGACGACGGGATGGTGGGTTTACGTAGAAAC

GCCAAGGCAAGACGCAAACTCACAGTTTCCATGTAATTTT

GCAGGAAGTCAACGGCTACA

52
N09940-001-Q001

CGCAGATCAAGTTCTAGTTTCAGTCTAGTTGTATACAGCT

TTAATTCATGTTTATAGTCCTAGTTTAATTGTATCTCAGT

TTTGGTATATGTCGTGAGCCGCTAAACATGTCAAGCATAA

CGTACAAGATTTTCTCTCCTTAAATCTCATTCCCTCTTAA

AAATGTTATATTTTCGTTTAATTGTCTGCTGGTTTTGACC

[A/G]TCGCAAGTCACCCTGTGAAAAAGCCCACGTTTTTT

TTGTTTTCTTTCTTTCACCTTGCGCCGTGCTGGTCTCTTG

GCCTTGTCTCCTTTAGCGGCATTTGTAGGGTTGAGTATTT

CTTAGCGGTGGTGGTTGCGTGGTGGTCCGATGGTTTTGGC

TGAGGCTATGATCTCACGTATTGGAACTGAATGGTTGGAT

GTGGT

53
N23409-001-Q001

CTGGTGGAGTCCAGGGAGAAGCACAAGAGGAAATCATGGG

ACAAAGTATNTGTTAATAAAGAGAAAAGAGGATTGGGGTT

CAAAGATATTACTGATTTCAACACAGCGATGATTGGTAAA

CAGTTATGGCGTTTGATAGGGAAGACGAACACTTTATTTT

NTCGAGTTTTCAAANGTCGGTATTACAANAACGCATCACC

CTTGGAACTGATTTGTTCATATTCTCCGTCATATGGCTGG

CGGAGTATCNNATATGCTAGATCTTTGGTAAGCAAAACAC

TAATCAAAAAGGTNGAATCAGGATCATCCATATCTGTATG

GAACGATNTCTGGCTCTCAACCACTCGCCCGAGACTAGCT

AATAAAACCAACACAACTATTACCCAAACCTCAAAGTGGA

[C/G]ACTCTCATTGATTCTACTCCATGTTCTAAAACGCG

GTCACCGCGGCCGCAAACGCGGCGGTTAAGCGCTCCACGA

CTCTTAAGCGTCTTGATTTTGCTATACTCGGCTAATTATA

CAGAACAATTAGAAAAGTTAATTNTTTTGTTCTTTTTGAG

TTCAAAATCAATTGCTATATNATAGATCTGTGAGTTTAAT

GTGTAAACAACAAAAAGTAAGCACAAGATTTAAGGACTAT

GTATTTTTTAATGGCGGTCGCAGAAGGTTTACAGTAAACG

TAAACCCTTAAAAGAGGAAGACGAAGCTAAAAATTATGAT

TATGCCCTTCACATTAAAAAAAGAGAGCAAAAAGCGTTGT

TGCTGCGTTTCGAACACGAGTCCCGACAGTACTTAGGGAA

AGCCG

54
N23119-001-Q001

TCAATTGCTTGATAAAATAATTCAAAATACAACAGTATAA

CCCCGCACGTACGCCTCCGTAGTACCCTGTGAACATCTTT

AGANGCATTAACTAGTACTAATTACTTTAAACATTATATA

TACAAAAATACATTATCATGAATTAATCATATACGGCCAA

CNCTTAACTTCNAGAAAAAAATAGTTGTGGATATACTGCC

GATGCATGTATAGGCCATTAGGGATCCAACACATACATAT

TATGACCTTTCTAAACGTTATATATCAAATATAAGTTTGA

CATCCTCACAGTTTTTTTTTAATTTAACCACAGTGTCTGG

CGACCGAGATTAACCGACTATTCTGTGNAATCCAGAAGTA

CAATGTTAAATTCTGGTGGCCAACAAAAATCGAACTGCAG

[A/G]TTCACCGTATCAGGGTTATTCCTCAAGTGCTACTA

GCCTAAGGCCCGTTGGTTACATCCTCACAGTTACCAACAT

ATTTAACATGTTTACCAAAAAACACATAATTATAAAAAAA

ATTAAATATCACTAGATGCATATATTCCTAAATTATGACA

TCTTTGCAGCAATATATTAGTTTTATATTTNTAATGTTTA

GATGATATTCTAAATATTTTTATATCATCAAATATTATTT

TAATATGTAGTCAGTAAAGTGACGACAAAAAATGTAGTCA

GTAAAGTAAATGATCAAAATTAATGAAATAGAACGTCTCC

TTCGCGTTGACGATTGCACNAGTGAGGCTTGTGTGTTGTG

ACCAATAGGCAATAGCCCATATGAAGTGGTAGATTCCTTG

GGGTG

55
N09861-001-Q001

ATCGGGGAGGGAAAGCCAGTGGGAGAAGAGTTCCTCAGCG

ATAAGAGGGTTTGATTTAACGGACAAACCGGGAAGCTGCA

AGAGCTCTGGATCCAGTATATGAACTTCCTCTATATCCAT

CTTATCCCTAAACTCTACCTACCGAGTCAATTTTCGAAAA

CTGGATGATCGTTTACCACTGAAACCCTAATTTAGGATAT

[A/G]TCCCACCACGGAAAATTCAAAAGGCAGCTATGAAC

AATCGAGGAGGTCAACTAAGTACGAGAGGTTTGTATTATA

CTCGCGAGAAAAAGAGAAGAAAAAACGAAACCTAGATTCG

AATTTCTATAAACAAAGAGACTATACAACTTCTTCTTCTT

CTACGGTACGATATGTGCGATCGAACGAAGAGAGAGTCGG

AGAAA

56
N04807-1-Q1

GCCAAATTACCATTCAAACTTTGACGGCTATCGCTATGCA

CAAGATTGGGCCTAAGCTAGAGAAAGCTCATAACTAACTA

ATTTNCCTTGCCGGTGAGTATGATGACNCCTACAGAGCTA

TCGTCTCTGGCGTCNTTAAACGCACGCATAAGCTCCTTCA

CAGTCTGAGNCCGGAACGCGTTTCTTNTCTCCGGACGGTT

TATAGTGATCTNNATAACCAAAAACTATTCAACTGAAGAA

GAAAGAAACACACACACCAGAGAGAGATCATCGAACACTT

TACATTAACAAANCTTGGCAATATCTTCACCGACGGCTTT

CTCATATATAATGTCAACNAACTCTTT[C/T]GTTTCTCC

GTCGTCACCGACGAGATCAGCTTTTCTCCAAACGACTTGG

TGAGTCGGCACTTCACCGTGAACCTTATGGTANTTGTCAT

TCATCGACGANGCAGTAGAGAGTTCCACTGAGTGAACGCG

AGTTGGAATTGTTTCCANGGGGATGAGATGATTGGTGANG

ACGGAGATACGGCGGCTCGCGGTGCCGAGTTCCNTGGAGT

CNGCCATTGGAGAGGAAGACGCGAGAGACAGAGAAAGATG

AGACTGATTGATGATGAAGATGACAAGAGATTCTATCGCA

TGGACTTTGACAATATTTGGATAAAATATTTT

57
N06778-1-Q1

GTATGACAGTGATGATGAGTTTATTGCTCCTCCTAGGCAA

ATCAGGCAGCCATTCATCAACCGNCAACCCGCCCCTGTTA

CGGGTGTCCCTGTTGCTCCNACTTTGGACCAACGCCCGAG

CCGCAGTGACCCTTGGAGTGCACGTATGAGGGAGAAGTAT

GGACTNGACACGTCTGAGTTCACATACAATCCCTCACAGT

CACACCGGTTNNCAGCAAATGCCGACGCAACCAAATGAAG

AAAAAGGACGATGCACCATCATGTGAGTCCATGTCTTTAA

AAAAGTTCAGTCAGTTCTC[C/G]TTTCCTATTTTTATTT

CTGCTTGCGTATAACAATCAAGAGTGTCAGTGAAATTAGA

GGTGTGTTTCNGGGTNGTTNTTGATGATGATTACTCTAGC

ACTGGAGGTATCTCAAATCAACCTANNCATTTTGTTGAAA

CTCNTTTTATTGTGTCTGTGTAGTCTTTTTGTAAGTACTT

NANCATCATGAATGTTCATCTTGGTTATCTGATTCGGTCC

TATCATTCTTATTATATATAAAATTCATCTGTTTTA

58
N09897-001-Q001

GAGAAGAACTGTATAGAAGCTTCACTTGATTCCCTAAAAC

TGTTGTTCGAAACAACATAGAGTAGTTGAAAATGACGTTG

AAATACCCAAATGAATTTGGTTTGAGAGCGATTAGAATAA

ACTGTCTCTGGTGGTGAGATGATCTCCTTTGTGGTAGAGC

TGAAAGATGATGTTCTCTTCATTAAAGAGAGAAGTTATCT

[C/T]GTCATACCGAAGGACCATAGTGGTAATAAGCGGTT

GGCTTCTAAAACATAGCAGTTTCGTGGAGATGGAGCAGAG

CCTAGGACCGTGAAGTAGCTTATTTGGTGGTGACGGAGGA

TGCTGGCCGGAAATATCTGCTGTGAACCTATTGAAAACAT

CTAGAAAAAGGGATTTTGACGCTGAAGATCAGATTGGATG

AACAA

59
N10499-001-Q001

GTAATGAAGGCAAAACGAGGCGACCCTTGGACGAACCATA

CCACTTTACTCCGATTCTGCNTCTCTCTATGCGGTTGTAT

CTNCTCCCATGTTTGGAAAGANGAGAACCTGCACTTATAC

TCATCCGACCCTGATTTCCATAGAACCATGTCCTCCTCNT

TACCAACAACTAGTGCTTTGAAACTTCTAACACTCGTGAT

TATTTGCTGTAGAGTTTGGTCTCTGCATCTTCGAATCTGC

CAACCAGAAG[A/C]ATTTGCAGCATCAGCAACCTTGGTA

TATCGCCCAATATCAAGCCGTTGTGGACCACTATCCCCCN

CTATGTCNATAAGTCGGCCCCCAGGAAGCCAGGGGTCCGA

CCAAAATAGAGTTCTCTGCCCATTTTTAATTTGCATTCTG

ATGAATTGAGCANCCAGCGGCCTCAGCCTCAGGAGCTTCC

TCCACACCCACGATCCCTTAGCTGACTCCCTAATGTCCCA

NAATGAATCATTGCTCCATA

60
N23447-001-Q001

AGCGTTCACCCAATGTTCACCGTACCACTCCGTTAACGAG

GAGTGGTTGCTATATCTCACCGGGGCCGGGATTGAACCGG

NAATATACTATGCCTTCAACGGATGAACTCACAANCCGAG

CCACCGTCTTTACAACTCGATCACCTAGCTTCTCTCTCAC

TCTACAATACATCTGAGTNCTGATCTAACAAGATCCTAAT

CAGCTCAGCTCATNCTTATCCTAGTCTAACCGTCACACGT

GCGCACACTTCACACGTGAGATCAACTAACTCGAGAGAGA

GAGCCGGTTCTAATTGATTACAAGCTTAACCGACTAAACC

AACTTTAATCCCNCTATACAATTAGAACCTAAACCAATGG

ATAGAAACTTGACTATATCCAACAAAAGCCTTAGGGTGTA

[A/G]GTTAATTATCTTGTTCANCAAAACCTTAGCTTTTT

ATTTACTGCTTTAATCATAATCATCTAGTTTTAGTTCGAA

AACTACAAATTTATTGTGTAAATCCTAAAGTCTATGTCGA

TTCGATTCTTAAATATTGCAATTGAAACTTTTAATTAAAA

GAATAAAATCACTGTTTAGGGCAATTTAAATGATATCACA

CCTACTCTCCGGTAACAAAAATAAATGTAATCATAATGTT

GATGGATGGCACTACAAGATCTAAAANTCAATCAAATAGA

TTTTAAAACTTCCTTTCTATATGGTAATCAAGCAGTAGAG

ATTTATATGAAAAAATCTAAAAGGTTGGTTGTCCCATAAC

AAGAAAAGTCTACAGACTTGTTAAGTCATTATACAAACTA

AAACA

61
N19834-001-Q001

GAGGTAAAAAATCAGTAACTGTCATGTTCTTCGTTAGCAA

CGGAAGCCCTAAGTATCTGACAGGTATATTACCAGAAGCA

AAAGAAAATGATTCATGATCTCTTCTTTCTTTTGATCTAA

AAAACCAGCCATAAACAAGGTAGACTTTTCCAAGCTAATC

TTCAGACAAGACATTCTATTAAACTCCTCAAATACTTTCA

[A/G]TATCCCTTCAACTGATCTTCTAGTGCCTTCAATAA

ACACCATTAAGTCATCTGCAAAGCATAGGTGAGTGATACC

TATGTTTTTGCATTTTGGGTGGAATCCAATTAGATTTCTT

GAGGCTGCTTCATCCAGCATCTTTGATAGCACATTCATGC

AGATAACAAAGAAGTAGGGAGATAAAGAGAATCCTTGACG

CAGAC

62
N23362-001-Q001

AAATTGATCTGATACAGTTCTCAACTGGCATGCCTATGGG

TACCCTGCCTGTCTGTTATCTTGGTGTNCCATTGTGTACT

AAAAAGCTCACGCTCTTAAACTGTGAAGGTTTACTTCAGC

AGATTATCCTCNTGGAGTGCCAAATCACTCTCTTTCGCTG

GCAGGCTGCTGCTTATCAAAACANTCATCACAGGCATAAC

CACTTTCTGGTGTACAANGTTCATCCTTCCGCAAGCATGT

GTAAAACNCATAAATTCNCTATGTGGTGTCTTCCTCTGGA

AAGGTGATATTGAGGAGCACTACGCAGCACGAGCCTCTTG

GNAGGTTGTCACAAAGCCGAAGCAAGAAGGAGGCCTTGGG

ATTAAGAATCTTTCGATATGGAACAAAGCATGCTGCCTTA

[A/G]GTTGATCTGGCTACTTTTCTTTCAGGCAGGCTCGG

TTTGGGTTGCTTGGTTCAAGGAGGAAGTTCTGGATGGATA

TGTCTCAACAATCTCTGGACTATGGTTCCACATAGACGCT

ACTCTTGGCAGGTTAATAAGCTTCTTAAACTGAGCTCCTC

TATCTTCAATTGAGTTAAGCTTCGTGTCCAAAATAGTCTC

TCTTGTCGGTTCTGGAGTGACAACTGGTCCCCTTACGGCT

NTATGAGGTCTTATCTTAGCATCNGCTCCAACTCAACTAT

GGGGATCGCAGCACAAGCAACTTTAGCATCCCTTCATCAT

AACAACAACTGGTGGATTCCTCCTGCAAGATCAGAAGCTC

TTGTCAATGTCCATGCCCTATTGACCACCATTGAACTAAA

CAATA

63
N23266-001-Q001

CTTCTTAGACTAAAAATAGTAATTAAAAATTCTAAGATGT

TTGCTAAGAGTTTTCATTGATAATGTTGCTCTTAGGATGA

TGCACAAAACTGTGACTGTGGCATATAATATATCAAAGTT

ATCTGCAGCTTCACCACACAACAAAAAATACCTATAATTT

CTCCATTCTTCTACAAGGTAGCAGACTAGCAATCAATAAT

AATACACTACTAAATCTCTCTTCAAGAATAATCACGTGCA

CGAACGATTGTTCTNTATCGATATCTGCTTAACATTACAT

GCTCTCCCTGAAGTTAATAGATCTTGACATATCTGCAGGG

TTTGTTTGAAGGTTTAGATACGTCTTAGGGACAAATCTAT

TGTTGCAGCTCTTTGTTTCAAACCCATTAGAGGAAAAACT

[C/G]AATTGATGTGNCTTCTCGCGTTTCATCGGTACCGG

ATACAACACACGATTATTCGTAGNTACGTCAATACTTCGA

GATTGTGAGTCCAATCTTGCTCTTACAAAAGTGTCTTTGT

TTTCTCTAACACTTTCATGTGTTTTAGCACAAAAAACACT

TATCATGTAAAGCTTCGTCTCTTGTCCTTTTCCCTAGACA

CGTGGAAGTCTCTAGAGTATACCAAAATGGTACCATGCAG

CCCCAGTAGACAAGGATGTGGTAGAATGTAGGGTAAAGAG

GACACTGGTGAGGGCCGTGAGGCCATGGAGGCGAAAAAGG

GGATGTTACTTGCGAAGGAAGAAATCCTTGGGAGTTGTTG

AAATCTTTGAAAACAGAGTCAGAAGTTATCTTCACTTTGC

TTGAT

64
N19862-001-Q001

TGTACACTTTCCCTTGCTTGGCCATCCATGAGTTGAAGAT

CAGCATGGCCTCAGCGTCGAAAACGCTGTCGCTGTGGCTG

GGACCAGTGGCCACACGGTGGCCAGAACTGCCAACAACAT

GGTGGTTATAGTCGTATGAAACGACGGACATGTCCATGGC

CGTGGCACAAGATGCGATGAGCATTGCTAGTAAGAAGATC

[A/C]GCATGGCTGATTTAGCACTACCCATCAAAACTAAT

GAAACTTTAATTTATGTTTGTTTCAACAAGTTTAAAAAAG

CTCATAGAAAAAGAGAACAAGATTTTGTGCTGATTAATTT

TGCCTACTATATGAGAGTATTTATAGTATTCAGAGCTCAG

ATAGGTAACACTTGTGTTAAGAATCTCACATCAGAAGAGG

TGATA

65
N22187-001-Q001

GCCTCTGTTATTCCCTCCGGCTCTCTGGTTGGTGTTATTC

CTGCTACTGTTCAGAGCTCCGCCTCTGTTATTCCCTCCGG

TTCTCCGGTTGGTAGTCTTTCCTCCGGTCCTCCGATTGGT

ATTCTTCCTGCTGCAATTCAGAGCTCTGCCTCTGTCTCTC

CATCTGGATCACCGGTTGGTGCTCTCCCCTCTGGCTCTCT

[A/G]GCGGGCGTTGTTCCTCCTGAGACAGTGACGGTGAT

AAGTCCTCGCTCTGCTCATGCTCCTCTAGCTTCCATAGCT

CCATCACAAAACTATGCTTCTCTGTTGAAGAAATCTTCTC

AGTTGAAAGAGCTGGGAACGCCGGTGGAACACGTCTCTGG

TGTACCGTTTGTCATGATCCCTGATAAAAACATTGAGTCA

GCAAA

66
N08651-1-Q1

CNAACGACTCCCAAACGATCACACTTTGTTTTCAATTATT

CATCGATAACTATATTCATCGACTCCCAAACAACA[A/T]

AAGTTACCACCAGTTCACAACAAAAGTANAACATAAAGAT

CTACCACGCAGATGGTCCAAATAGCATNCGGACAA

67
N23296-001-Q001

GTTTGCGAATTATANNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTGTAAA

AGAAGCTTATGTACCCGTATTTTGTGGAAGCACGCAAGGG

CGAATCCAAAGATTATTTTCAGTGGGTGCACAAATTAAAG

TGTATNCTTACGTATGATCGTAGGTGTATACATCAAATTT

GTTAAGAAATATACTAAAAAATTTAAAATTATTATGGGGG

CAAGTGCATCCATAATCCTNTACCTAGNTTCGTCAGAACA

TCGTAGTCTATTGATTAAGGTTCAAAAGCTTATACACTTA

AGTCTTTGAATTTCAAACTATGCATTTTTTTTGCCGATTA

[A/G]GATGCGAAGCTCATCAGAAGTTTTAGAGTATTGTA

AGTAGAGCTGTTTGTTGTGGTGATGGAAAGAGTTTGTCTA

TCGCGGATGTCGTACATGCTGGACCGTCTGTCGATCCAAA

TGTTTCGGAAAGAACTTTATCNGAAAATTCTTATCCATCA

TCAATATTGCATATATTGNAGGTAGTTGAGCAGCACATTT

AAAAAGTTAAGTAGAATTTTGATCGCTGTTTTACCTACAG

AAGCTGGCGAGACACTAGTGTAAGAGAGATTGTATTGAGT

GAACATTTGATATTGATANTGGATTTTCCAAAACTAACCA

CCTCGTGAGTATATGCTGCAATCATTTTGATTAGACACTG

AAACAAGTAATCTTGTATGTCTTTTCGATTTCAACTTATG

CTTAT

68
N17314-001-Q001

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNCAATATTTTAAATTTTTTTAGAGGAAGACACC

TAGTATAATTTTTAATTAACTAAAANGGGGATTACATAAT

TGGATCCTACGTTACCGAAGCCGGACTCCTTCCGCCTCTG

TCGATCTGCAGTCTTGCCGCTCCTCCAAAACACCGCCACG

TAAAGCCATTCCGGTGAGAGACAACCAGGTTCAAAGTAAG

ATGCATATCGAACAGAAAAATCAACGATGGAGAGATAGGC

AAAAAGAAAAAAGAGTAACCTCCGGCACCGCTTATTGCTG

GACCGGAAAAAAGATCTTTTGCGCTTGAGAGCTTTTAAAG

AGAGATAGTTCTGTTAGAGAGAGGGTCCACTGCATGCAAT

[G/T]ATATATTCTTTCTCATCAGACTTTGATTGGTATGG

TGCTTAAATGGGCTGAAAGCCTTATATAGTATTTATCGTT

CATGATAATCAATTATATAAGTTTCTTAACAAATAAATTA

AAATAAAGATTTTTTGAATGATTGAATCTGAAAAATATAT

CGCAAAAGCGGCAGCCTAGGGTTAGATCAACTTCTCCATG

GACACGTACACTAGCCAGATTAGGTCCTCAAGGGGCCGAT

AAGGTTCGAAAGTGAGGCTGCTCGGGAAGCCTTCCTCGAG

AGGAACTTAAGGCCCCNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTTG

GTTTG

69
N20380-001-Q001

ATCACAGAGCTGCATATAACAGATACAGAAATACTAGATA

TTGGTTCATGGGTCAAGGAACTCTCTCATCTAGGCCGACT

TGTACTCTACGGATGCAAGAACCTGGTATCTCTCCCACAG

CTTCCAGGTTCCCTACTAGACCTTGATGCATCAAACTGTG

AGTCCCTGGAGAGACTAGATAGCTCCCTTCACAACCTGAA

[A/C]TCTACTACTTTCAGATTCATTAACTGCTTCAAACT

TAATCAAGAAGCCATACATCTCATCAGCCAGACTCCATGT

CGCCTAGTTGCAGTTTTACCCGGTGGAGAAGTGCCTGCCT

GCTTTACTTACCGAGCTTTTGGGAATTTCGTAACAGTAGA

GTTGGATGGGAGGTCTCTTCCTAGATCGAAAAAATTTAGG

GCTTG

70
N05490-1-Q1

CTTGANCCGCCAATAGATGAGTCTCCTGAACCGTTTCCTT

CATATTTCCACAGGCCAGTATCTTGGGATGCNAACTTCTT

GACTGCAGAAACATAATCTGGCTTGAAGAAAAACATATTA

TCCTTTCCAGCAGATTGGTAATGGTTCCCTGTTTGTTGCA

TATCATC[C/G]CCACCAAACTGTGCAGGGCTTAGGGCAG

GAAAATCCATGGGNGTNAGATTAGGAGCCGGAGCAGGAGC

AGGAATCTTAGGCCTCATGTTCTGATTCANNCCATTATCC

ACTTGTAGCTNNAGTACAAACATAACCAAATCACTCGAAT

GAACAAAACATTCATAAAGTAGCAAAACAACATGATTGGA

GCAAAAAAAAAGGACATCAACACATTCAATAACTTAAAAA

TTACAATTAGCATCAACATTGAACCATATGTTCACAAGGA

ATAATGAGTTTCATAGGAAGAGAAGCAGAAAGCTAGGGAG

AATANCTCAAGCTGAGTAAGCATCTCAACNGTCAACTGCA

AATCACACCCATTAGCAAAATAAACNTCNGCAAGACTCTC

AGCAGCAAANCCAGGGAACTGAGNAGCNAGAAACTCCACA

GGGTTCACCTCCATTTCCCCTATNCTTGGATTCTNGGAAA

CCATGAATCGACCAAAAGGGTTTCTTCTCTCC

71
N18849-001-Q001

CTTCATAAATAATTGACACAATATATTTGAATATATGATA

TATTTAATAAAAACCTCAATACATAAAAATAATTAATAGT

ATTAATTTTAATATATATATTTATATTCACTCTATTCATT

ACTATTAAAATTTGGATATTATATAAATTAAAAACTATGA

TTATATTTTTATTGATATATGATATTGTATTTTTTTTTAA

AGAAGGNAAGCGTGATTCCAAAACAGAATCATAAGCTTCC

AACATGTTTTTAAAGAGAATATTTTGNAAGCGTTTTGGAA

TCGAGATTCCGTAAGCTTCCACAAGGTTCCGATTCCNGTT

CCAAAGCGGGAAGCAGATGTCCGGATGAAGNTTCCATGCA

ACGTAGGAAACAAGTATCAAAAGGATAAGAGAAAGGAAAA

[G/T]ATAGAGAAGTTCACACATACCAAAACAATTCGACC

TCGAGGTTTAGACCTAATCTTCGGCCAGGTTCCTTCTATC

ATCCTTTGTTACTTTCCGTTAGATTGTAACCCATATTCTC

TGTAACCTTTCAGTTACTAATATATANACATTTCTTTTCG

TCAATCTTGATATGTATCTTGCCTAGCCAACAACTGGATC

GTGAGTGTTAAACGTCTTCCGATCATATTTATTCAATTTC

GCATCACAATTCGCTAGGCCCAATCCAAACGACNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNTACGTAGCCGCTTCCGTTTCCATGTAA

AGTAG

72
N08200-1-Q1

GCCGCCGAGCGAAGTTCCCTTGCCGGCGTTCTTCGCGAAG

AGAAGCGTCTCCTCGTTCGAACCAGCCGACGCAAC[C/G]

ACCGATCTGATCAGAATCTTGGGGATAGACGTCGATCGAT

CTCAGATCTCCGATTAATAAGAAAAAGNTATCNCT

73
N19827-001-Q001

ATAAAAGTTTGGATACTTATATAATAAAGTAATTATATAC

GAATAAAAGTGAAATATGATAAAAATCATGATGAATAGTT

GGAAACAAACATGACCTTGTCTCTTTTTTCCTGAACAACA

CATGACCTTGTCCTAAGAAATGAAAAGATAATTAAATAAA

ACGAACCAACAAACAACATAAAAAACTAAGAAATAGCCAC

[A/G]AATGAAAATATAGAGGGAGGAAAATCCTAGTAATA

AATGTTTGGATACTTAAATAAAGTGAAATGTGATATATTC

ATGATGAATGGTTGGAAACAAACATGACATTGTCCTAAGA

AACCAAAAGATAATTAACTAAAATGAAATTAAAAAAACTA

AGAAAAAGACCACAAATGGAAAAATATAGGGAGAGAAACT

CTAGT

74
N001R9W-001-Q001*

GGGTGACCCGGGTTCGATCCCCGGCAACGGCGAATCTTTT

TNTTACATTTTAAGAAATTGAAATGTTTTCATGAAAATGA

ACAAAAGATTATAATGGCTTCGCCCGGGTTCGAACCGGAG

ACCTTCAGTGTGTTAGACTGACGTGATAAC[A/C]AACTA

CACCACGGAACCTTTGTGCTTACATTGGGAACAAAGAGCT

TTGATAATTTTGATGTCTAAGAAATCTTTAGATGTTTGGT

CCGTAGAGTCATGTAAGCTGCTCTGTCTATTAAGCCATCT

CGACTGCTCTGATCTTCTGTAAAAA

75
N08264-1-Q1

CCTCATGCATNGTTCAAGNGGAAGATCAGAGACTTCAACA

ACTGGTTCATCTGGAATAGCATCTACCACAGACTT[A/G]

TCTGTTTCTTTGCATCCATCTTCACTGGCGTTTACACCAC

CATTCTCATTATTCTGAGTGGTCTGAGTTGAGTCC

76
N23132-001-Q001

GCTCAAATTGCTACAACGATTGAATAGAATTTCCCGTACA

NAACACTGTAGTTGGCCAGATACGTTGNAGGTTTAAGAAG

TGTAATGTATCTTGTATTCCTTATTTATTTTTCCTTTTTT

TTGCTAAAAATATGCTTTATTTTTTACAATTAATTTCTCT

TATTTTTTACAATTAATTTCTCTTATTTCCTTTTACAATT

AATTTCTCTTATTTCCCGTACATAATTATTTGTTTTATTT

TAAATTTATTATGTTTGTATTAATTTATTTCAGTAGTCAG

ATTGAAATAACAGTTAAAATGAATAAATTTCATTACTTCC

GATCATGTATTGTTANGTTTAGAACTACAAGCTTACTGAT

CATGTTAGTTGGCAGTTTTTTGTATCTTTTGAGTATGCAG

[A/G]TATGTCAATATTTTCAAATNATATATTTTTCTAAA

TATCATATTCAATTTCTCTTACAAACAATGGCTCTCATTA

ATCTACATTAATTTATTCTAAAATAATAATAACCCTTTGT

AAATATCAATCCTAACATTTAGGGAGGTGTATTCAATTGG

GAGTTTGAAGTGATTTTTATTAAAATGATAAATCTACTGT

TCTTAAAACATGATTTTTTATTAAAAAAACTACTTTGAAA

TCTAGTAGTATTGAACTTGTTATTTCATAAAACACTCTTA

AATGCACTATTATTGAATAAATTTAAGTTAGAAATTTTAG

AGTGATTTATTTCTATAGTGTTTGAGGANNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNN

77
N03615-1-Q1

TCTTTGACTCCTGTTTGACATCTCCTANGTGCATTTTGTA

NTGAAAGAGCTGGTNAGAAAGTCNATTTCAGAAACAGAGG

NGAGAAGACGCCTTCTGTTGTGACAATTCCTCTTTAACTC

TTTACGTTCTGTTTCAACCCTAAAATGCCATTTTTTTATN

AGGAGCTGAAGCGATTCCCAACTCTAGCNAGCGATATAGC

AGCNGCTGCAAATGAAGCTCTTGAAAGATTCAGAGACGAA

AGCAGGAAAACNGTTCTGCGTCTGGTGGACATGGAATCCA

GCTACCTCACTGTTGAGTTCTTCAGNAAACTTCACATGGA

ACCNGAGAAAGAGAAACCAAACCCGAGGAATGCCCCACAG

CCAAACGCAGACATATACTCCGACAGTCACTTCAGAAAGA

TCGGNNCGTCTCTGCTTACCTCACTATGAATGAATGAGAC

ATATCAAATNTGTGTTGACTTTTGAATATAACTCNGGATC

CAACGTGAGTGCATACATAA[A/T]CATGGTCTGCGACAC

ATTGAGAATCTCTCTTCCAAAAGCTGTTGTCTACTGCCAA

GTNAGAGAAGCTAAGAGATCGCTCCNTAACTTCTTCTACG

CTCAAGTNGGCAGGAAAGAGNNAAGTAATTTTCTAAACTA

GAGAATATCTGAATCATTTTAAAGAGTGAAGAACACTTTC

TAATGATCATTAAAAAAAATGGGTNNAGAAGGAGAAGCTG

GGGGCGATGTTGGACGAAGACCCACAGCTGATGGAANGAA

GAGGAACATTAGCCAAACGGCTCGAGCTTTACAAACAAGC

TAGAGACGACATCGATGCTGTGGCTTGGAAGTAAGGTGTG

ATCAAAAAGGGTTTCCTAAGAAAATATTCTTTATATCTTT

TAATTGCTTTGCTCGTGTGGGCACTTATGTTGGAAGTTCT

AACCTCCNATCCATNGCTGCACACACATACAGACGATAAC

TCGTATTTTNTTTNGCCGCTAATATTTGTTTCCCACTTTT

TTGGT

78
N001RWT-001-Q001*

TGATTTGCCTAGACCAATTTTTAGAACACTGGTAATAAGN

GACACTGTTTGTCTTTGGTT[A/G]TAGTTGATACTTCAG

CTTAACGGTTCATGTTTTAACCATTTCCTAACTATTATTG

ATTCT

79
N08465-1-Q1

AGTTTCCTTCTCCTCCGAGAAAGTTAGCTCNTTTCTCTTG

TTCTCTNTCNAAAANATCTCTCCTTTCAACGTTAA[A/G]

TCGTTTAGTTGTTTGAGTGATGTCTACGGATACGGAGGCG

AATTTACTTGCGTTAGCGGCTGTGTTCCGCAGGAG

80
N10774-001-Q001

TAATTAACGTGATATATTATTTCCATAATTATGCACAAAT

TATTTGTTAACATAANNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGAAAA

AAGGTGTATTGCACAATTATGTTAACAAATAATTTGTGCA

TAATTATGGAAATATTACATCACGTTAAGCCTTTTAGTCT

TATGTTTAGATCAATGTTATAAAAATTGGTAGACGGTGGT

TAAGCGTCTTGTAAAAGATTATTGTTTAGTTGGATATCTA

GGCGCCGCTTAAACAGATTTTTATAACACTAATTTATATA

GAAAATATGTAAAACTTTTTCCATCATCCATTTCTAACAT

[A/C]GTGGAAATAAATAAACAACTTAGACAATATAAAAT

TACAAATAGATTATTAGTTTTTACAAAAAAAGTATAAATA

GATTATTATGGCACCCATCATATATTGCATAGGCTATATT

CTTCTCGCCTTAAGCGTAAAAAAGAGACAAAATATTCTTT

AGTGAAGATGGGAACAGAGACGTCGACGATGACGGCGAAG

TTAGCATTCTTCCCACCAAATCCGCCGACGTACACGGTGG

TGACGGATGAATCGACGGGGAAGATCGATGAGGATATCGG

CGGACATGATTCGTCGTCATAGACGAGGAAATTGAAGTGG

TGAAGATAATGACTAGAAGAGGGAATGAGATCGTGGGGTT

GTATGTAAAGAATCCAACGGCTAAACTCACCGGTGTGTAT

TCTCA

81
N17035-001-Q001

NNNNNNNNNNNNNNACAACAAGCTATCGTTTTTATATAAT

AAGTCTTTGCATATTCATTTTAAATTCTGAACCGTTTGTG

TTTGATCTAGGTTGATACTTGGTACTGCCCGAGTGATATG

GACACTCCACACTGAAAGAATTGTGAAAAAAAATTAGATA

ATAAATTATAGTTTTTTTTTGTAACTTAGTAAATTATAAT

GTTTAGAAGCTAAAAACATAACACGGAGTTATGGACAGTC

GATACTGACTGAATCACACTTCAGACATATCCAAATATAN

TTTGACTTGCATTCCTATTTTTAAAAAATTAACGTTTTGA

TGTGAAAATATAAGTTTCACATCGAGATCGAANATTCANT

TGANACATGAATAATATATAAAAGATTTGGATCAATCCAC

[A/G]TATTACCAATTTATTTTAATTTGAAAACTCATGAT

AAATCCAAACTTAACATGATATTATAGTTCGAACACATGC

TAGCTGCCCAACCGACCGAAATTGATTAGGCTCATCGAAA

GAGGTTCAATTGATCCTAAACCTCATGGCCTGAATGGTCT

GGCGAACTGACCGAACCAAATCCTAAATTGAGATATTGTC

GAGGTTAATGGTTATAAGAATCACTATNTTGAAGAGGCGT

GTGANAATATAAACTCACATATTGGAAGTTTAAGTGGGAC

ATGAATAATATATAAATAGTTAAAGCTAATCCACTTATCA

TCAATTGGTTTTAAGTTAAAAGCCTATGATAAATCCAAAT

TTAACATATTTGCTTATTAATCAGTGTTTTAGATAAAAGT

GGATA

82
N20834-001-Q001

CTAGCTTCCTCACCATGAAATCAATACGGTCCAATCTCGA

AAGGATGGGTTCTTCCACTATAGCCATAATGTTGATAACA

CACCGTTGTTCTGTTGCACACTTCACAGCTTTGGTTGTTG

CCGTGGAGTAGTTAAAGCCTTAAAGACAAGACACTATATG

AAACGCCAAAACTGGTCATCTTGGAGATTGAAGACATGAA

[C/T]TACGACACGTTTAATATTACAGAGAGAGCTGGTTA

CGGTCTTACGCGTGTCGTTGATCACACGTACTGGGTTTAG

TTTGTGGACACCTCTTCTGTCTCACACGTCCAACATTATT

CCATCCTTCCTTATCTTAATCGCTGACGCCTCTCCGAGAT

TATATCAGACGCAAGATAAAATTTCTAGTGTTTATTGCAA

GTGTT

83
N22903-001-Q001

GTTAAAACTCGTGAGTGGTAATCTTGTAAATTGATGAAGA

CTTGGGTTCTAAGTTGGCTGAAACCAAATGGGGTTTCTGC

GCGCTTCGCCGTCGACCGATGACACAGGATGTGCATCGAT

CGATTATTATCTCTGAATGTCGACCGATGGTCTTGCTCGA

TGGTCAGCTCGGATGCTTTCTCCAAATATTTCCAAAATGC

TCCAAAATCATCACTTTCTTTCAAATCACTCATGATCGTA

TAAATATACTAAATAGATTTTATAATATAATAATTAGTTA

TTAAAACATCTATAAACCGTGGGTAAAAGTGGGTAAAATC

CATGGCATTCCAATACCTTCCGCCAAGTTTTAAACAAAAT

CAATAAATCATATTTTCTATAATAGAAGACTCGAATGGTG

[C/G]TTGAAACTTCAAACTAAACCACAAAGCTTNACGTT

TTTATATCGAAATTCAAAGATACACTATACAACTATTAAC

TGCACANCTAATCTCTAAGTTACTATAAATCCAGCAAAAC

GAAGTTGTTGACATGTTCGCGTCAGAGTGTGAGAGGTACA

ATTTTTAAGAATGATNCGAAACTAAGAAGATATGAGTAAC

GNAAGGTATTAAAAACATATATGAGGTTGNAGTGAAGATT

CAGTTTTGGTGAATAAAATGACTGATCAGAATATTTTGAA

CATGTACATATATAAGTGTGTATCAGATTCAGATTGTAGA

TTATAGATTTCTTTAATTGTGTGAGGATATTTATGAAGAT

CGCATTACTCTGGTTCGTATGTCAAAAACATAGTTGGTAG

CTTCA

84
N09920-001-Q001

TAAGAAAAGAAACTGAGATTAATCGCGGTCTAAGTGCTAG

TTTGAGGTGACTTTTATGAAAGTATACCATTACAGTAAAA

CTGACTCTTAGTCATAGCCTCATAGGTTTATCATTCAACC

CATAAACTAGAATAATATTACTCAGCCAATTTTAAGTAAA

TCATTGAATATTGTTAGTGTGAGATTACCCCTCTCTGAGT

[A/T]AATTGATTCTATAAACATAAATACCCATATCCCCA

TGCAATGATACAACACATACTATTTCGAAATCCCTCCGTG

ATTGAAAATATGCATTGTTCACGCCTCTTCTCTATTTTAT

CTCTAATTGTAACCATCGCCGGAGCTTTCGCCGCCGCTCT

AGAGTGTTCATGTAATGATCTGGTCTGTTTATTTAATATA

AATCA

85
N22822-001-Q001

AGTATAAGGAAGATGTTTGAAAACATGGAAACTCACATAT

TATTTAGAAGNCTGCCTTACCATTGCCAATTGTTAGCCAC

AACAAAAAATACCAATCTAACAAGGTACTTTCATGACTCG

ATCTCAACTTAGCAACCTAACCATTTTCAGCCCACAGCTC

ATAGGAGTTATCCAAATTCACTATCACTGACCTCATTTAA

GACTAAAAGTGCAGTCTCATCGAGGGAGTATCGATCATAA

GACACATGGACTCTTACCTATTCAAGTATCTGATCACACA

TATAATTTTACTCTGNTTTCTTTCCTCTCTCTTTCTCATT

TCCTCATTNGATTTCTCTATACTCTGTAGGGTATCATTTT

ATTTTTCTATCGACTGAGTTTATTAGACAAGATTCCATGA

[C/G]ACACAAGCACTGATGGTGGTAACCACTAAGCACGC

GATGCACTTCTTGTTGATTTTTATCTTTTTTCTTTCATTT

TTTTTGAAGAGAAGAGAGAGTAAAACCATATTCACACAGA

CTAGTCTTTTTAGACGTGAGCAAGAAGTCCAACCCAATGT

ATACCAAGATTTAAGACAAGAGAATCAAATAGATTAGGTG

AGAGGTCAGCTTTGGATCCTTCGAANGCCCAACAAACATG

GATGGCAGATCAATCCACTTTGGCAACAATGTCTTTTGTT

CAGTATTGCTACTTGGAAAGCAAAACACAAAAGCAGCTTA

TTTGTTTTATTTTCTTCTCATACTTGTGTGGTTCAACTGG

AAGTAGCTCAGTTTGGAAGGGTTCCATCATAGTGTTCTCT

GTAGG

86
N22688-001-Q001

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNCCTTTATTACATGCCAGTTCATCGTCATTCGTCAC

TGTATTGACACGTGTCGTAAGACCATTATATACTTTCTGT

AATCCCATGAAACAGTAGTTAATTACACTTTTAACTTCTG

CACGTAAGACTAAAGATGAAGGGAAGATGAATGACATCTT

ATGCATGTGCACTGCACGCGGCCGGCGACTCCCGCCGGTT

TCGGGTTTAAGTGGGTCTCTCTTCTGCATGAATAGTAGTA

GCTTTCTTTTTTTAAATGTGTATATAGTAGCTATATAGTT

TATTTATGNTTCCTAGTAATATATAATGTGTTTTATTTTC

[C/G]AGACTTCTCAATTACTCAATTCTTTATAATAATTA

GGTAACACTGTTGTTAATTTTTTATTTTCTTTCTTAAAAT

GGTTTTAGCAAAATTAAATAAGCTAGATAGATATAGCTAA

TATACATCTTTCATTTTAAGTGTTTGTGTGTATTTTTGTA

AATTTGAAGTTCAAAAACGAGTATTTAAATGATTGGATCC

GCTTGCAAGTGGTATNGACAATTAGCACAAAATTCCACTC

TAACTCTCAGTATCGTTTACAAAGAAAAGCGTATGGAAAT

AAATNAACCTTTACGTATTAGATTTTANGAACGAAAACTC

AAATCCTAATATGATAATTGTCTTTAATTATAAAAAGTTA

TAAATAATTACATATTACTAGCAACCTCCAGTGACTCTTA

ATTAG

87
N10074-001-Q001

ACCAGAACTAGATATTGATCCTTATCCCTGAATAATCTGG

TATATTCGGAAGGCTCGAAATGAAAAAACTCTTTAAGGGA

TAGACATGGATCCTTTGGAGCTAGTTCGCTATGCAGAGAG

TGAGTGTCAAGTCTGATTCAATGCAAACAAGATGGTAACG

CCAACTACACAAGAGCATAATTTTGAAGACCCTCAAGTCT

[G/T]AAACTTGGGTAATATTTGCATTGTAACTTCCACAG

CTTCGTTCAGCGGGTGTGGTTGGGTTTGGAAGGATATATA

GCTCGGGGAAGGTTCAACTTATGGGGACACGAAATTATAG

ACGGTGAGAGATTGCTTTGCTTTCAGAAGTGGAGGTCTTA

CAATGGGCGATGGAGAGCATGCTGCAGAATTCGACATGCC

AGAGT

88
N10057-001-Q001

AATATATCTTTTTTTGAAAAATCGTGTATCTTTAAAACAG

AAGCGCACGTAATCCACTTTTCTCTCAGTTTCGAGTCTCT

CTTTTCCTCGGAGCTTGTCACCAAGAGAGAGAGAGAGACA

TGGAAAGACTTCTGCAACCACCGTCTTCTTCCACAATCTC

TCCTTCCAAATTCACCTCGAGGAATCCCCCTCTCCTTCCT

[C/T]GTCTCCGGTTCGTCTCAACGTACAGACCCGAGTCA

CGCCGAGTGAGCTCCATTTCCTGCAGCAATCTCCAGAGCC

CATTTGTGGGATCTAATCAGACCAACATTTCCTTGAATGG

ATCTCCTTCCTCATCTCCTGTAGCAGGAGAATCGAACCCT

AATCATGGGTTTTTCCAACGGATCGTCACCACGGCTGATG

AGCAG

89
N10086-001-Q001

TCACAAGGGCTTTAAAGTAAACTCCCATCAGACCCATGAC

CATATCTACTAACTTCAAATCAGACCCTGAAGTGGTGTGC

ACTGTTTGGTCAACTGTCTTCATGAATCATGATGGCCTCT

CCCCTGGTGATTCGTTCTTGGTATTTCACCTCTCATGTTG

CTATAAGGTTATCTATCTTCAGTCAGATCTCATGGCAATG

[C/T]GAGAAGTTGTTAAGTTTCCTTTAGTTCAATTTGTG

ATTGCTTCAGAATATTGACTCTTTGGCTATGACTTTAATG

TGTGATTGATTCTGCGGATTTGAAGGGCCAGTCCTATTAC

TGGAACTATCAGAAGCTTCATCAGTTACTTCAGTTTCTGC

TTGTATTCTCTGTGTGTTAGTGGTTTTGTCTAGCTCCTTG

CTAAT

90
N11084-001-Q001

ATTTATTGAATTCTTAACAAGCGTGAACATTTTAGAAAAT

TTAACTTTTGAGAATAGAGGGAGTAGTACTGATATTTTTA

ACCAGTATCTACTATCTACATTGGTTTTAGTAATGTGTTA

TTCATGGCCGTGGTTAAGTTAATCTGGTTAATTAAAAATA

AGAAACAAATTACCGTGGTGTCTGATAGACACGGGCTTGA

GCGAATGAGTAAAATAAGAAAGCGTGGGGAGTGGAAACTC

GAGCCTCACAGAATCAATCACCTAGACTAAATATTCTTTG

AACAATGACAGTCACATCCTCTTATTATAGTGTATTTATA

ATTTACTAGATTAATTTATAGTTCTTTTTCTCACAAAGAT

CATGTACTCATTACTTCTTTTCCATGATATGGACAATCTT

[A/G]TGTTGCGGTTGGCCATCTCTTTTGGCTTGCAAGCT

TTTTGACTGAAAAAGTTAGATCCTCTTTCTAGGTGGTGAC

TTTTGTTGCAAGTGATCTGGATTATGGGTTTTCATCCTGT

ATCTGTAGTTTATAAATATATCTGTGAGGAAAAAAGAAGA

AGATCATGTACTCGTAATTCAGTATTNTTCTGCAGCACAA

TTCTGAATTTGGAAAGTTTAAAATAGACTTCTTAATTCAN

ATAAGTCAGCAAGGTAAGTTACATGATTACATATCTACAA

TTATGGAAAATCAACANATTTCAATTAATTGTTTGTTNCT

TAAACTCAATAATTTTTATAATAAAAACAAATATTTAAAA

ATAAAATAACGTGTTTACTTTTTATTATATATATGATTAT

AGTTT

91
N22814-001-Q001

AAGAGTATCCAAAAAGAAAGAGAGAAGAATGGATGACGCG

CGTCTCTGTTTTTAGTAGCAGAGANGAGAGAGAAATGAAG

AACAATGGTCGCTANTTTTTTGACGTCAGNGGCAACACCG

GAAATCTTAGCCTTTTTTGACGCTGCGTTGCTTGTTAATG

GCCGCTGCGGTCATCGGTGTTGTGTGTTGCCATGACTCCC

GTTTAAATTTTGGCCGCTGCCGCTGCGTCCTGCAGCTAAG

AAACGAACACGGCTATTGTTATTTTCGTTGTTGACATGGT

CAATGTAAGCTGCATGTTTCTCTTCTGTATTTCCATACAT

CTTTCTCGATCAAATGTTTGCAGCCATGGGTAAGAATAAT

CTCCCCTCCTTCTAGAGTCTGGGAATCGAAATTTGCGTCA

[A/T]TAAAATTTGTAACANAGAAAATAAAGCTTTTAATG

GGGGTCCAAATTTTCTGCAGAGCCAACGANAAGTAATCAA

ACACCATATATATCTAAACTCCAAATATCAATCATGATCT

AAGTGTTAAACAGCTCAAAATTTTGCTATAATTTTAGACA

TTAATATATATATATAAACCCAAAAAAAACTAACAATTTA

AAATATAATTTTATATTAATTATAATAATATGATTTATAA

TATTTACATAATATAAAGTGTTAATATTGTNCATTTATTT

TAAACTGATGTCAACCGGTTATAATTATCCCACAAACATA

CCAATTTCTAATTGATTGTACCAGTCGTACTAATCGGTTA

ATAACNTTTGAAACNGCATCCGCAAACTCGCATTTACACC

AGTTA

92
N01564-2-Q1

GGAATTTCTGGGTCGACGATTCCGTCCCAACGTCACCACT

TCCCTCCCAGATCTACTATCACCCTGCAAGTTCATCTACA

CTTAATCCGACACAGCGCCCTCGCCTATCAAAGCA[A/C]

GTCTCAGATGGTCAGATCTGTGGAATGAACTCACTTAGCA

GAAGCTCGATAACTGAAGAGAGGCAGGGAACTCCTTTAAG

ATGTGATTCTTCTGAGAGTGGACCATCTGAAGGTTGGTCA

CTGCAGGCCTTTTCTGAAATGATGTCATCTTCTCGCAGCA

CCGAGCCTTTGTCTTATGATAACGACCACTTTGGGCTTGA

ACGGGACATGATAGGCCATCACAGCAACCGAATGTCCAAT

CATCAGCAGCAAAGCTGTGGTGCGTGCTCTAGACCCTTGT

CAGAGAAATCCTTGTGGAGCAGCCAAAAGATGTTTATGAC

CAACGAGCTCTCTGTGTCTGCAATTC

93
N12902-001-Q001

ATAGATAGTTCATAATCAAGAGATTATAATTTGTAATATT

TCCATTTATTTATGACGGTGTAATCTTTTATATAAAGAAC

TTCTATGCTTTGAATAAAGATAAATTTTCTTATTTTTATA

AGAAAAATATAATTTTGTTAAAAACCGACATACAATGAGA

CTTGTGTCGCCGTCTCATCGTGTTTTTCTTTTCCCTTCAN

GACTATTTTATAAATCTTGCGTTAGACGTTAACGCTCCAA

TTGATTTGTGCGAGAAAATTTTACATAAACCCTAGAAAAC

TCTCTTATTGTTCGCGTTTAATTCTTCAGGTACGATTTGC

CATCCTCTCTCTCTCTCTCTTTTCAAAAGGTATCTGCTTT

TCATCGTCTACGATCGAGAGAAACTTCGAGACTTTGCCTC

[C/T]TTTGTTGGGATTGAAATTGGTTAAAGGTTTAATTG

TTTTTGGTGTTGATTGTTTTATCGGCGCGCAGATATGCCG

GTGATGAATCCGTCGTCGTTGTGTATTGGTGCACAACCAT

TGGTCTTCCTCCCTCCTCGCTTTAATCATCGACCAGCTAA

TGGTATCTCTCTACTTTTGGGATATACACTTCTTCTTGAT

TTGCTTCTTGCCACTAATTTATGATTGCTCATGAAAAAAC

TGATGTTCTTGTAGCATTAGATCCTTGCTTAATCAGATTT

CTCAAGCTTTTGATTTTCGTATTCTATTGAATGTTTCAAG

TGTTAATAAAAGCTCTTCTTCTTTTTGTTCTGTGGTAGGA

CAATTTCGTGGGCGTTACTACCCTACAAGAGTTTCTATGC

AATTC

94
N21144-001-Q001

ATGTAAAAGAACGTAAACAGATTAATAGTATATAAAGTAA

TTTGTATATAGAATATTTATTCCACTCAAGGCGCGGTTAA

GTAGTTATTATTCAGCATGTATATATTTGTTAACTATTAT

TAAAATGCAAAAAATATGTATAGTAGAATACTTAATGTTT

ATAATCACGAGATATAATTGTTTTCATAAATTCATCCCCA

[A/C]ATGATGCGGGTTATCACCTAGTGTGATATTATACA

TGGCCACCAGGTTGATCCGATCCAACAGAGTCCATCGGTC

CTCTTCAAAAAGAACAAAGCTTTATTGTACAACAGATAAA

ACGTAGCATATCGTGCTGCTTAGACTTATCTTCTCCATAT

GGTTGCAGAGCTCTCGGATACAGTCGTTGTTTTCTAGCAG

GCTGA

95
N07534-1-Q1

ACGACCGAGGAAAGCACCTGCGGAATAGAGACCAAGGCCC

AACAGCAGGAAGGAATCGATACTCTATAGATCCAC[A/C]

ACTAGCAACCTAGAATTTGAAGCAGAGATGGAGCGGAGAA

CCGCACCACACAAATAAGCCACCACCAAAATACCT

96
N22993-001-Q001

TACTAGCACTTGTTTGCAAGATCGGTGGACTCACACTTTG

ATGATGGAGTTTCTGTGTGTTGTATAGAGTCAATGACCTA

CATGAGTAACCCTGTTTGTAGTCCTTATTATCACGTAATG

GAAGGTTCCCCTTCTGGTGATCAAGGTCGATGTTATTGTG

CTAACAANGAAGGCGAGAATACTTGGATTTTAAAATAAAT

TTGAATAGTTAAACAATTTTGTATTTATCTTTGAAGTTTA

CATGTGTTTATAAAGAAATCTANGCCAACATAAGCCAAAG

CCCTCATCATATTTTCACTATGAAAGCAATACCCTTTTGT

TTAATCCTACTCTAACTTGTTTTACTACTTAAGGATGTAA

ACTGAGTAATTATAGTATTGTGCCAACCCTTTTGTTAGAC

[C/G]GACTTCTTTGTCCTTCTCCNCTTGCACAAGTCAAA

CAAAAGCCCCTAGGCCCATCCTCATGGCCTTAGCTCGTGA

TCTTTTTATCGGCCCANGTGTAAAAAGAAATAGATCTTTA

ACTTGGATGATAGACTCATTTTTGCCTGTGAAAAGCCCAT

TTNGACCATCTTTCTTTAAAGGTGGTTCTAGGTTCTCTTT

GACTTGTCCATGATGTTCATCGCTTCCAATTAGTCAAAAA

TGTCCTTACATAGTCATTTCCAGCTCCGAGTCATCTTTCT

CATTCTTATTCTCATTTCACGACCATAACGCATAGTCGAC

CTCATNTCAGGTTGATCGAAGATCGAATCGTCCATTNCCA

GCTAGTCCATGTTCANGTGTCGCATTTCCCGCCTACTCAT

GGTTG

97
N09963-001-Q001

TTGGTGATTGAATAATAAGAAAAGAACTTCTTATTGATAT

TGTGATTCTATAGATAACACTCCCATCATCCAGCAGGACG

CAACTCAGCAGTTCAATCCTGAAGGAACTCCCACACCACC

CCCAACTGGCAGTGTTACCAACGGCATCAACCATCAATCT

GAATGGTATGTTATCACAAATACGTGATAATTTGCAAAAA

[G/T]TTCTCTGTTTTGATTTAACATACAGGTCAGTAGGA

TGCCGTGGCACAGCAAGCTTTTTAACGCTTCAAGCAACTC

AATCAGGTCTAGACCAACCAGTAGGCGCAAATCAATTCCT

TAAGTGCATATCACCAAAGACTCCACGCCAGCCCTAATCA

CCAAAGACTCCACGCCAGCCCTAGGTGGACGTACAAACTC

AATAG

98
N11542-001-Q001

TGAGATTTGGGCCATGACTCGAAGATAAAGCTTTATGACG

ATCATCACACTACAAGAAGAGATCTGGAAACTTCTCCAAA

GGCCAGCATCGATCGACACCGCCAACCCAACATCGATCGA

CCCCGCCCACCTGATACCGATCGACATCCACCTAATGATA

TCGATCGACACCCATCGTTGGACGACCTGCCAAGGTCACA

GTTGGGCTGAAAGTAGTTGAGGAGAGAATGCACACGTCTA

CGACCTCACACCTTGCTGTCCCCGAACATCNGAGACCACC

TATATGCACAGAAGAAGCTGCTGGGTTTCACAAAAGAGTC

AAGAGGATACATGACCATGTGAAGTTTGTGGTCCCATGCA

TTGTATTTGAAGTTGAATCTCCTATTCCAACAAATAGAAG

[C/T]GTGCATCTAGGTTCTTACATTGGGAAATTTGATGA

TCATATGTATGCACTAGTTTTTGAGAGAGGGTTGAGACAT

ATAAGTGACGTCGACACAGCCCCAACAGAAACAACATCGA

TCGACACTACCACTTCATCGTCGATCGACATTGGACGTGT

ATCAGATCAGAAGGAGTTTGAAGTGTGTCGNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNCGGCGAAGAGTAGTAACTGTTAGCACCATC

AGAGATTTAAGATTTGTGCACCCGGGAAAAGCCATATTCA

ATTAGTCTTCTTTATCCGGAGATGCTAGGTCTGCTGGTAA

AAAAA

99
N14681-001-Q001

TTTAATACGAAAAAGAAAATTCAGTTTTTAACAAATAACG

ACCATAAAAATACTAAAATAAAAAACAAGAAACATTATTT

AACATTTCACTTTTTGTCTATACTTTATTAAATTTGNAAC

ATGTTCTCATACATAGAGGAAAAAACAAATTNCAAATACA

TAGTTAATTTTAACGTGCAAACATGTCATTATTTTAANTA

TTTCACTCTGCACAGGGCGCGGATTATTATTATGAATGAG

ATAAGTATGTTTGATACGATCATTCATATTTATGCAATTA

GTATATTTTTGGTATACGGTTTATATTATATGCATTCTTT

GTTTGGGATATGAATATTAAATAACTAGCCAGAAAACATT

ATCATAATGTCATGACTGCCTGGTTTTAATTAACATGATA

[A/C]ACATATGGGATGTGTAAGACCAATCNAATTAACGA

TGTGTGTAGTTGGTTTTTAAGANTGGTNTGGTCCAATGGT

TTTAATCTTTTATTGAATTAGAAATCTAATTTGATTGGAT

TTTAATAATAAAGTAAAGACTATCTGATTAGTAATTAAAA

ATTAATAATTTTAATACGATTAAAATTAGTTTAATTATTT

ATAATTCAGTTAAAATACATAAAAGATTTGTATTAGTTAT

ATTTATATTTTATATTATATATAAATTTTAAATGTAAAAT

TAAATTAGATTAAAATTGTTTCCTCAATTGATTTCAAGTA

TTTTTTATGTTTACACAGTCTTATCAACTATCAAAGCTCG

TTTCGTTGTAAGATGANTTTGGCAAAGNAAGGTGTCGTTA

GTTAC

100
N11636-001-Q001

AATTAATTTGTATAGTTACATTTTTTTAGTTGTATTACAA

ATCTCATAACGTAAAATCATTAATGAATCTTTAGTTAAAT

TATTAGACTAGACTCGCTAGATAGATTTCCACAAACCCTA

TTACATTTTCTTTATAATAACAGTGAAAATTACATGAAAA

TGTGAAAGGCTACTGCACATTTTCTTATGTGGTATAAAAA

TATTAAATTATAAATTTGGTATATATGCCGAAACTATTTA

TGTTGGTTCATATACGGTTACATATAAATACTTTTTATCG

GTATATTCCACTAAACACTAAAATATTGAAGATATTAAAT

ATTTAAGATATCATTGTCCGTTTAGAATTTCAAAGTTAAG

CGTGTTCGACCTGGAATATCGGAAGAATAGATGACTTATC

[A/G]GAAAGTGATTCGCGATATCGTGCAAGTGAATCNAA

AACACGGAGAAAAGTCACGTGGTNAACGGGTGGATAGTTT

GGTAGGCGGTCGGGCCGTTACATCTACCACGTCCTGTAAC

ACAGGTGCAGCCTCTGTGAANAAAAATGCTGGTTCCATAC

GGACAGGTGCAGCCAGTGGTATGGACGGGCAGGGCCGGGT

CTGAATAGAAGTNCATCGAGCATGTGTTTGGAGCCTGACG

AATATATAGATATTTTGGGGCCAATTATTTTTCATACAGA

ACATGCAGCTCTATTGGCTTGGGGTCCAACGAAAATATAG

ATGGACCTCTGTTCGCTTCTCCGCAATCGCATCTTATATT

ATTATCACTATTTTTTAGAAACAAGGGTCAAAAATATTTT

TTTAG

101
N13732-001-Q001

CTTGACTATTTTTATGTGAATTTAAGAAAAAAAATAAAAG

TAAAAAATTATTATTTTTTATTGTTTTCAGTTATTGTCTA

ATGAGTGATAACTCCTAACTTCTTAAGAAGTCTTAAATAA

GAATAATTATGAAAGCTAGTTATTTTTTTTGTCAACCGGA

TGTTTATTAAACAAGGTCTATAATATAAAACAGGTCCAAG

AAGATGGGCAGTAAAACTATTACAAAAANGTCCAACTGAA

AGGCAAATAAAACATAAATGAAAGGCCTANCATATAAAGC

CCAATATACAAAGCATCTTGAGGCCTTAAGCCCACGAGAG

AAAGATCTGTTGGGGAAGAGGGTCACACGACGCCATATAC

GATCACCCAACGATCAATGTACACGCGTCAAGACGCGGTT

[C/T]CAACATTCTTCCTCCGGAGCCAGCGAAGAGGCGTG

ATGGAACTCCGACGTCGACCAAGCCTATTAGATCGTTGAA

CGAAAACATCAATCGNCTGTAATCGATCAGAAATCCAATT

TTCTTGCATGCGCAAAACTCCATCTTTGATGATTTATATA

AGCTTGAAAATGGNGACAACCCTAACCGAGGGGAGGAAAG

AGACATTGAACCTTCAGATCAAGAGGTACGGTGCTATAGA

AGCCACCACTTCCCGTAAACCTAAAGCCGGCGAAGATGGT

GATAAGGGATCCACCGCTTCCAGAGGCAGAAACCCGACCA

TTGGGAGACTGAGTCCAAAGAGATCCGACAAACAAAACTC

GACGCTCTCTCNCTGAAAGATAGGANAGAGATAAGAGAGA

GCAGA

102
N11255-001-Q001

AGATCTCTTGTCCGAAGCAGAGCATTTTTTCGCTGAAATA

TGCTCTAAGAAGTTTTTTCGCCCTGATGTTCCAACGTATC

GAACGATGATGGATGCATATGTGAAGAAGGGTAGAGTCAG

CGATGCTGTCAAAACTGTGAACCAAACTTTGGATGCCTCT

CTAACCTATATTGCTAAGAAGGTCTTAGTAATGTAACTTC

CGTTTATGTGCATCACTATGCAATTCAGATTCTTAGTTGC

ATACCAACTGTTGTTTATCCACAAATCTGGTGGAACTTNA

TTAGTAGTGCTTAATCTTGATTGTTTATCTTTTAAGTCTA

GTTAACAAGATCGTTAATACTCCTTTCTTAAAGCTAGCAA

TAAAACAAAAACAATAATCTGACGCACATATTTGAACTTA

[C/T]CAAACTATGAAGCGGGCTTCCAGACTTTTAATTGG

GGCTATCAATCTGAGACTCCGTTTGGTCATAATCTGCCCA

CCATTAATTGGGCGTATAATGGCTCTTGAGGGAGAGTATA

GTTTATATTATNAGCAGTCACAATGAACCTTCTGCATATT

GAGTCAAGCTATAATGTGGTTTCATGTGTTCATAACGCAG

AAGGAGCTCGAAATGGGTCTATCCGAAAGCAACTGTTGTA

CCTAACTTCCATCTCCCAGTGCGCAGCTTTGGGNTTAATG

TAGAATATTTTGTTTTCAACGACGCGCGTGTTTGGTTCTA

CTATATGAAACGGCGCACATGTTTGATTATGTTGATAGAA

ACGGCATGGCTTTGCCGTCTTCGAACGGTCACGGCATGGC

CATGT

103
N15511-001-Q001

TCTCTGGTAAAATCACATATATACTATAAATAAATAGTAA

TCTCTCCGCTTCATAATATATGATGTGTTAGAAGANNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNATCTATTCTTCCCTCCTTAGTCATG

AAAATAAACTTTAGAAATGTTAGTTTATAGTATATAAATA

AACTTTTGTTATCGTATATTAAGATTGTTAGTGACTATAG

TAAATGTTATTCTGTAAAGATTTAGTGTTTATTAGAAGAA

ATGAGACCCACCTGTGTTTTTAGAGCCTGGTGCGTATGTA

TCATAAAATATTGTTGGCACGAAACAACTCTAAAACTGTG

[A/G]TGGTTTAAACTAATAAACTAGAGATGGTTATTAAT

CATTACTCTATGCATCTTTCGATGTATATTAAGATGGTTN

ANGTCCAAANAAAGAGGATTCTACGTAAAACGTTGAGTGT

TGCGTAAAGNATGTAACCTACAATACAGCATTAAAATATG

CATTCACTAGTGTTGGGGTGTAATAATATGATTAACACGT

ATTATAAGGAAAAAGAAATTAAAAATCTTGCCCCNAGATN

TAGGTTATTCGATAACAAAAAGAGTATGCGTTAACTCTGA

ACATTGGTGTATCAGGAAATCCTTAGACGAAATTGGTGTT

AGTTTAGGATTTTATGTAATTTTCTCAATGCTTATAAGGC

CTCTAAGAATGCGAGAAGGAGATAATATAAATATTACATT

CGTAT

104
N10536-001-Q001

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNCTTGTCATGTTATGATCAAGCAT

CTGGTCAAGAGAAGCGACCATATGAATGTACTTCTTTCTT

GGNAAACACTTTAAGAAACTTTTAACAAGCTTTGGCTCTT

CTATCATCTGTCCCAAAGAAGCCGATTTGGAGGAGATTTC

GGATAGCTTCCCGGAAAAGGTATCGATTGTATCTTCATAT

TTCATCTTGA[G/T]TCTATTGAATTCTGTCTTTAGGGTT

TGAAGCGTAGCCTATTTTACCCTCTCAGCTCCTACATGTC

TTGTNTTTATAGCATCCCATATCGCTTTTGTTGTGTCTAA

GTCGCCAACTTGCATAATCAAACCCTCCGGTATAGACTGG

AACAAAAGCACAGTGGCCATACTGTTTTTCTTCTCGTCTT

TGGTNCCAGGGTCTATTGCTTCCCAAACTTCGCTAACCTT

GAGTGCAATCTTCATTCTCA

105
N09862-001-Q001

CTTTAAGTAGTTGTACTATGACTTCTTAATAGTAGATAAA

AATAGGACCCTAAACTAATAGATTAGATATATTGTTATTT

ATAATAATGATAAAAAAGATAAAACATTATTCGAAATTTA

GAGATTTAAATGAATCATGGAAAATGCAAGACCTATTTGG

ACCAGGATCGATTTGAGATGAAAGCTAAGGATATAGCTAG

[A/G]CAATTCAATTGTTGAATGTTACCTTTTTTGTTCAA

AAGTTGACTGTTGAACAATCTATTGATTATTCAAGCCAAG

ATTAATACAAAACAAATCATCTTTGGCATCAACAGCTTCT

AGGATTTTAGATGCTTACATATTTTGTGCAAGGACCGCCA

GTGTTTTTGATAACAGTGTTGAAACACAAGTGGTCATCAA

AAACG

106
N23033-001-Q001

CTTTAAAGAATTCAATTTAAGGAATATCTGAAGAAGAAAA

TCAAAATGACTATGGATACTAGAAAGAATTAGAGAAGCAA

AGTGAGAAAATTCAAAATTGTAACGCTTTATGGTCTGAAA

ATATAAAATTTGACGCTGAAATGATCAAAGTTGACTCAAA

AGGTNNATAAAAGTCTTGAAAATTATGATAATAAATTTTG

AGAATAGTTATGATTGATGTGAAATAATGTCTACAAATAA

AAGAGAATATGATGTTTTCGGTGAATGAATGTCATATTTA

TATATTTTACCTTGATGAGGCTGAACTGAATTCGAGTCAT

GAAATTATTATTCTCATTGGATTTGGAGTTTCGGTAAACC

AGTACAAAATATCCAAATGTTTTTCATCTATCACTTGTGC

[A/T]AATCTCTTGACAAGCCATTATTTATCTTCAAACTA

TTTTAAATCCGTACAACTANCAAATTCGATGCTAGAAAGN

TTGGAAAAAACTCTCATACCTTATCTTTTGTTATATTTAA

TTAATCCAATGCTTTTATGTATCACATGCAGAAGATAANC

AATATCTGGTTTAATCAATNTCCCATGAGCAGAAGTTTTA

AAAAAGTAGTTTAGCAATCGCGGTGANGAGATATATAATT

ACAATTAACTTTTGTCTTTGACAAAAAAATACTACGAGTT

TCGTCCCAATTATTACAAAACAAAAGTTTTAAAAAGCATT

AAAATATATACATATTAAAAATCATTCTTCTGAAAATAAT

GCTTGCATCCAAAGTGAAAATGCGACACTTAATCTGAATT

TTTGA

107
N06039-1-Q1

GTCTTCAGGACTTCAAAATCACTCCCCAGTACCCAATGCT

CATCTCTTTTGCCAAGAAATGAAGAGAAACTGCCT[G/T]

TGTTTTGTTTGTCTTTTAAGATGATGACAGTGAGACTTGG

TGTGTATAACCGACGGTTTAATGTTTCGGTTCGAT

108
N10016-001-Q001

GAGGAATTATATACGCGAAAGCAATAAGCAACCAGACAGA

CACCTTACCACCACAGGGTGTATTTATTCATTTAATCTAT

TTTTTTAGTTAAACCTTGCAAGGTTTAACGAAACTTCTGG

TGTCTATCTGGCTTGACCGGCAGGGGGCCAAGCGGTTATT

TCCATGCTTTGCCTAAGAGATTGCTTCATGTGCAGCTAAT

[C/T]TTCGTTCAACCATAGTCAGATGACTACAAGCACAA

GCACAGTGCGGAAGAGTGTGACGCCGCTGTCCCTGCAAAA

GAATTGCAATCAAACTATAATTATGTTAAGATAGTATTGG

TTATAGAGGAAATTAAATAAAAAGTACCAAAAAGGGTATG

AGACAAGTCCTTGGAGATCGGTTTCAGCATGAAAAAACAT

GGGTA

109
N22743-001-Q001

TTCTTTCCCTCTAGTACTTGTCTCCTGAACCTAATCGTAT

GTATCTTTGACCAGGTGAAAGGTGTTAAGAGTGGTATGGT

TTAGTAAGGTCTACTAAACCAAACCGAGACTCGGGTTGTT

AAGGTTGTTGAGATAAGTACAATGTATTAGAAGATAAAGA

TGCACGGTAGATGTAGATAAAGTAGAATCTCCTTGTAAAG

ATACGAAGGTCGAGTATATGTATCCAACGTGATCAATGAG

ATCCACACACAGTTTACTAAATACAGTTTCTCTCTGAGTT

TACATGGTATCAAAGCGGGCCCAACATTTCCATATTCATC

TCCTCAGGAAACATCTTGATCATGATCAGAAGAAAAGCTT

CGGGGAGGATTTGTTATACCATTGCATCAAGCAATNAGCC

[A/T]TTGAGAAGTGTGCTACCGTTGTCCAACGACCCTCT

GTTTCCTCGGAGATCAGTAAAGGAAATGCTTTTGCAGTCT

TAAAATGTTAATGGCAATCAAAGTTGCCTCCTCATGAAAA

TATTATACGAGGCCTAAGTAGGTCCATATCTTTCAGTTAA

CAAGTTTGAAGTTCACCCTTTTGCATATGGAGTCTGAATG

ATCAACACCAAAATTTCATGTGGTTTCAGATTTATAGCGT

AATCCTTGATAAACAATATCAACAAGAATCATTTTTAAGC

AAGTTTTCAAAACTCTGCTTCCTGCTTTCTGATCTAATCA

GTGCTAAGATCATCACTTGTTAAGTAGTTCAACATCAAGC

TTTTCTGAAAATGAGAAACATCTCACATGTTCTGCCTGCT

TTCAG

110
N22953-001-Q001

TGTATCGATCGACGGCACTGGATGTGCATCGATCGATTGC

GTCTTCTTCGTATCGACCTCTAATGGTCAGCTCGGATGAA

ATCTATTTTAAGCTCCTAAATGCTCCATAGTCATCACTTT

ACTCCAAAATACTCCTGAACCTGAAAACATACCTAATATG

ATAGAATATATAATATATAGATAGTAAAACACTTATATAC

CATGGATGAAAATGGGTCAAATCCATGGTATATCAAGCAT

CCCAAATACTTCGTCAAATCCAACCTCANTGGTATTGGGG

AATACACTCAGTGCTGCTTTGTATGTCNTGTATTTCAATG

TAATACATCTAAGAATTTTCTTCACTAGTTTCTTTCCTTG

TACTTCCTTCCAGGAATAAGAGCCTATTCTGACAAGGAAC

[A/T]AAGTTTAGAGTGTGGGAACCGAAATNCGCACTGTC

AATTTCCGATAAAAATAGGAAAGCTAAGTAATCCTAACTT

TCCCAGAGGTCCCGGATATCTGCTAAACCACACGCCAAGC

GATCAAACAATGAGAACAAACNAAATAAAAAATAGTAGAA

AACGAAAAGAGAGCAGAGAAGATCTTATTCCGAATTGANT

GAACGAGCATTACAACAGATAAAGCCTCGGCGGTTAGAGA

TGTCGGNGAGTTCCTAGTTCTAACCTTGTGAGACTTGATT

AACCTAGTTGAGTCGCAGCTCGAAAACAGAAAACGGAAAT

ATGCCTAAGTTTCCCTAAGTGCTATGCTTTGTTCTNAATA

AAAAATGCNTCCCTTCAGCATCTGCAACCTCGACATCCTT

ATATA

111
N09987-001-Q001

TTTATTGATGAAATTCCCAAGAATCATTAAGCTTTTGTTA

ATGTACTTCCCTTCCGTCAAGCGTACTCCACCAGCACCAG

TTTTGGCAATTCTTTCAGATCCAGCCAAATCAACCAGATT

CTAGAATCCATTAAAAGAAGGCACATAAGAGAATAAACAG

CTTTCTACACCCGTAGAGAAAATGATCACTCACCAAGACT

[A/G]AGACACGGATAGGATCCATAGAATTGTTTCCTTTC

CCCCTGCTCTCAATCACCTTATCCCACCAGAGATATTGTA

TCAGAACAAATACTTCAAAAAAAAAAACAGTTGAAGGTTT

CATTCATAGGTTTCGAAAACCCCACCATTCTGAAGATGGT

GTGGGACGTGCTACTGTGAACGTTCATGTTTGTCTCACCA

AAGTG

112
N10092-001-Q001

GGCTCCCAACCACTCACCCGAGACCAGCAAACATCAATCA

AGAAAAATCTCTACCCAAATATCACGGTGGATTCTCTTAT

CGATTCAACATCCCACACGTGGAATTCCAAGGTTATTCGG

TCATTGGTGGAACCAGAGGACGCAAAGATCATAGAAAGCA

TACCTGGCATCGTCTGGTTGATCAAGATGCATGACATTTT

[A/G]CCATTAATGGAAAATATACGATAAAATCGGGTTAT

CAAGTGGAATGGGTATACCCAGATAGGGAGAAATCGTTGC

CGGTATTTGGACCTACAATAAACCTTTTAAAAGCATACTC

TTGGAAAATACGTTGTCCACCAAAAATAAAACATTTTTTA

TGGCAGTTAGTGTCGGGCTGTATATCAGTAAAGAAAAATT

TACGG

113
N10096-001-Q001

GACTATTTTAAGAGCTTCTAAAGGATGTCACATGGGCAGA

AATATTCTCACCAATCATTTTGCCATGTCGTTACGGGCTG

GGTTTCAAACTAATTTAAATAATTCCAGCCCAACCTTGCC

CCATTCCGATCCAACCTAGTTAATAAAGTTACATTTTATT

TCTTCATAGACCTCCACATCACCAAGAAAAAGGGAGACAC

[A/G]TGTCATTAAAAAATATTCACCAAAACTTTATTGTG

TTTGGGTTTTTGCTTTACACTTACGCATGGGCTTCAGACC

CATCGTAAAATTAAGTTTCCTTGCCTTCTCGCCTAAGGAC

AGGATTATCGGGGGTTTTAGTGAGGTTTTAGTGGGTTTTT

TAAGAGAGGAGGGACCTACAGGAAGGAAAAACCGGTGGCA

GAAAG

114
N22728-001-Q001

GTATGATCAAGGGTCGACTAAATAAAAATGTTGGCTTGAA

GAGCTTCAGCTGGGATCCGGTTAACGTTCGGAATGATCCT

ATAGGATCGGAGTTTGTTCTGTTTCCAATTGAGTTGTTGA

TTGTTTTACGAAGCTGACGAGCTCGAGTAAAACATGATGT

GTGCGTTATGACCTTCGAATCTTCGTCGCAATCAATTAAT

ATCAGTTTCTCTTGAGTTCCATAACTTCAACTTCAATGTT

TTTATTCTCTGCTTCTTATTCTTTCATTTAAATAATCGAG

ACACGTTACTTAACCAAAAGTATGGCTCGTTATACTGCAC

TACTGACAAATTGCTAAGAGTAAAGCACACAAAGGTTTCT

TAGGAAGATTATTATTAGACTCTCATGAGAATTAGGTCTG

[C/G]GCATTCGGGTTTCTGCCNAGTCCGGGTCTTTCGTG

TCCTAAACATTTGAACCTGACTAGGTATTTAAAAATTTTG

GTTCGGGTTCGGATCATTCTTGTGGGTCCGAATCGGTTCT

AATTCATATACCCGTAAAACCCTAATTTTCGGGTAATTTT

GAGTTCCGTTCGGTTTGGGTATTTAGGACCCGAAGTAAAG

TATCCGAACTGGATTTGAAAACCCGAAAATACCTTAAAAC

CAACAAAAAAAATCCGGAAAATACCCAATTTTTTTTACCA

TTAATCTAACACAAAGATCTAAAAATACCAAATTTTTTAT

TCAAATACACGAATTATATTTCTGAAAATTTTAAATTTTN

ACCTGAAACCTGAAACTATACACGAAAACCTGAACCCAAA

ACTNA

115
N22747-001-Q001

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCAG

AAAAAAAGAAACCGTTTCTAAATACACCTCTCTCTCTCTC

TCTCCGGATTTGCTTACTTGAAGTTTTTGAATCGCGTCTC

GTTGAAGTTATGGATGATTTTGCAGTCCCCCTTGAAAAGC

AATTTTGACCAAATATAGTATAATATATAATTCCTATTTT

ACAATATTCTAATATACACCGTTATTTTTTTTTCAGTAAA

TTGATGAAGAAGGATTATCCATATTGCATTTCAAGATTTT

TAGATATCCTTAAGACTCATAAAAATCTAACATAAATTAC

ATCTATTCCCAGAGAATACGTTTTTTCGAAATCAATTCCT

[C/G]GTCTCTGAACTAATCGGGTTTTATATNGTGTTACC

TCATTCTTTTCACTAGGGGCATTGCTGGACAGAGTTCTTG

TTTCATCTTAATATTTGCTAANGTTATTGTAGTTGTGAAT

TTTGCGTTTTGAGTTGTTTTTCAAGTTTTTTTTATTGTTA

TGGGATTAGAGTTGTGATGATGATCTGTGTATGCTTTGTT

CTCCACTTATGAAGGACTAAACTGTGTTAGNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNAAAAGTTTCTTCATTTTTTCTTTTCTTGAT

GATTTCTGGGAAGAGGAAGAGGTAGTTGAACTTGTTGGGC

TTGAT

116
N22840-001-Q001

TGNATATGTTGATGTGGTCGTGTTCAAGTTTCGAAGTTTC

GAACTCATTATCTAGATTGTTGTTTCTGGACATATTTTTT

TTTGTAACCGATTGNTGTGTTGATAATGAGTAATCAATCT

ACCTACATTACATTTTNAAAGGATGATAACATGATCATTG

ATCACTAACATACGTGTGTAGTTTGATGATTGGTTATGTC

ATGTTACATTGAGTNTTTACTTGTTACAAATGTTCTCGAA

TGGTGTACAAAAACGAGCTTCGACTCATCAATATGAAAAC

TNTATCCAAGGATTATGTTCATGAAATAACAGATCCGAAC

TAACTCTAATCGTGATCAATGTATTAAAATCGGATAATGC

TTACCCACCTCCATTCATCATTCCCAATCCCAACACCAAG

[A/T]TCTTACTTTCAGTGTGATTCTTAGCCGAGCAGGCT

CTCGGCTAATAAAATGCACAAATCACTAATAAAATCTGTA

AACTAATAAAATTCCAAAATTGAAAAAAAATTTAACCTAT

CCAAAATTGCATTATTTATAATACAACACTAAATTTAAAA

GTTATTAATTGTATTGAAGATTTATCATTTTAATCAAAGT

TATTAATCTACAACACTCAAATATTTTTTTAAATACACAT

AAACGTAAAATTCAATTAAAAGTATTTAATGTTTTAGAAA

CTTTTCTTGTAAACAACACATTTTGTTTTCTATTGTGATC

GCAATACTAAGTGAAAATTGTAGGTGCCACATTTTAAAAA

GAAACCTTGTTTTTGGCCGAGGAATAAACATCATAAGTAT

TAAAA

117
N23027-001-Q001

TTCCATGTTTGGCTCATAAGCCACCTATGTGTGTTTAGTG

GTTTTCCATTTTGTATTTTCAACAGGTAGAACAAACTTAA

CCAAGTATAATCTATTCTACTTTAGTTTTACTTTTACAAC

AAGTCCATTTACTCATTCAGTCAGTGACGGTCCTAAAAAA

ATTTGGGCTGGACGCAAATTATAAATTGTGTGACCTATAA

ATTTATAATAAAACAAAAATATGCTAATTATATCCCACAA

AAGGTTCGAACCTCTCCATACTTTTTNAAAAAATATCACG

ATTAACCAACAACGCTACTAAAGGTTTGGTGCAAAACACG

GCCAAAATATTACATACTGTAAACCGGGCNGGAAGCACAT

GNTTCTGCCGCTTTTGCCCATGGCCGATATTGCATTCAGT

[A/T]GTTGAATAATAAATTTAAAATCCAAGTGTATTCTT

AACGTTGGTAGTATAATGCTTTATCTTTTCTCTCTTTAAA

TNTTATTGAAAATGAGTTATTTTAAAAGTACTTCTAAGTG

TCTTACAAAATTGTTTAGGTTTTTCTGGAGCAAGGTGACG

AGTATGGTGGCTTCCATATGTGAACACAATATTCTTTTGN

ATTTTGTTCTCTTTTTGATGTTATCACTTCTTGGTAATAG

ATTAACCGGCTTCATCTCNTTTCTGTTTTGCTATGCCATC

ATTTCTTTTCTGGACTGTTTGTATATAGTCCCAACCGATC

CCTGCACAACGAANATCATGAATTTTCTTTTCTGAAAATC

AACTTGTTGGATCTGTAAAATGATGATAAGAATAGACAAA

GGAAA

118
N22777-001-Q001

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNCAAGTTTCTAGTTTCTT

GTTTAATTCTGAAAAGGGATAAAGATTTCTTGTCGACTGA

GAAAGAAGAGTCGAAGACAACGNTAGAGTTCTGTTTCTTT

TTTTTTCTTAATGTTTTTACTTTGTATTTCATTATAGTTT

AAAGTAAAATGTTAATTTTATTAACTGTATTTTAAAGAAA

ATGACAAAACTGGATTATTATTATTATTTATACTAAAGGA

ACAATGATTTTGGTTTTAGTTCTAAGATGATAAGAGTNTA

GTTGTTTTGTTACTTTTTACCAAATTTCCTTTTATAATAC

ATTGAACAACAGTTTGCCATTTCCTTACTATTTTACTTTC

[C/G]CTTTTACGGAAAGGTCGNGTCAACATAAACATCCA

AGAAATTGATAGGTAATGGATGCCTTTTNTAATGAACTGG

ACCNCTCCTTGAGGCATTTGTCTACTTTTGACAAAATATT

CACATGGTTTTGTTTATGGTTTTAAGAGCATGATTAACCC

TAGAATTCCATTAGAGTCTCTTAATGATTTTTTAAGTATT

AAATGTTAGTTAAGAACCTTAGTTAAGAGACATCTAGTTT

TTGTTGCTCCAATGCTATTCTTTTTAATTAAGGGTTCTTA

AAACACAACTAGATTTTNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAG

TGGGG

119
N09636-001-Q001

ATTTGACACTCTAACTAAAACAACTAAGTTTTTTTGTCAT

TTGTTGGATTAATAATCAAAATAGAAATGAGAGTTACACC

AAGCCGGCAAAGGTACACAACATTTACAGAGCCATAAGCT

AACATTTATATATGATTTAAAAGCGAAAAGATTACACTTT

GTTTCTTTGAAATATTTCAACGATCTTGTTTTGGTTTTTC

[G/T]GGTTTATGATTTGGCAATGAAACTAGAGTATACTA

AGGCTGAAAAGATTGTGTATGATACTTTAGCTTTTAATAT

TTCAAGATCTTGTTTTCGTTTTTCGAGTTTATGAATTTCA

TCCCAATTAAATTTTGGATTTTTGGGTTTTTTGAATTTCG

GCCTCATGAAAATACTATAAAATTTTTATTCAAATTTAGT

TCGGG

120
N09879-001-Q001

TCCATGGTATATTTTGATTTAGGATTGATTTATCTTAGAA

TATAGAATTTGGTATGGTCGATTTTTGCAGATATAAGATA

TTTATAGTAAAGATCAGTCGCAAACCGGATATGTTTTTAT

TATTGGTGGAACCACAATTAATAGAGTTCCCAAAAATAAA

CTCCAGTTGCAACTTCTTCAAATCATACGGAAACAATAGC

[A/G]TTTCATTAAACATGTATAGAATGTATATGGCTTTG

GTCTATGGGTCATCACATATAAGAAGCAAGCAAAATAGTT

ACAAAAAAGAGCCAACAAAATTGTTTGAAGATAATTGGCT

TGTGACGTTCAACTCAAAGAAAGTTATGTTAAGAATGATA

GAACAAAGCATATTCCTAAATATGAGATATCAAGAAAAAT

AAGAA

121
N10123-001-Q001

TCTTGAGAAGTGCGTGTATGCGCTTGGTATGCTAAACCCT

CCACAGATTCAGGAACACCTAACATTTCTTGCTTCGAATT

CAAATCGCACCCTGTAACAGAATCCTAGTGGAGATACAAG

ACATCAGTCATATACGTTACTAATCTACCAATGGAAGGAT

TCAGTCAAATGACTAACCTGCGCAAGTGGCGGCCCGGTTG

[A/G]TTCGACACAGATATCTACTTTCCCACTCCCTTGAC

TAGTAACAGAGAACACAGTAGTCGAACACGAACCATTATC

AACAAGAAGAAGTGAATAATCTTCTTCCGCATTGTTGCTT

TCACGAAAATCCTGCTCTTTCTCATCGAGATACTGCAATG

TAGGAGAGGCAACAAGCGGAGTTTCCAATATCGAATCGCT

ATCTA

122
N10316-001-Q001

TTTCGGAAGACATCTCTTCGTAAAGGTGAATTCAAAGCTA

TGATTAGTCTCAGTGGTCAACAATCAAAGTTTTATACAAG

TAAACATTTGTTTCAATATACCCAACCTTTACACCATTTT

CTTTGTACCAAAACTATCAATAATGATCGGGAAAAATACA

CTTACCAGGAAGGAGGTCAAGATCTTAAATTATTTGTTAA

[C/T]GGAGCATTAGTATAAATGATAAATATAAAAAGAGA

ACATAATGTAGAAAGTCGATGCTAGAGCATGATTATCGGT

TCAGGTGGGTTTTTAGTAGTAATTAGAAATTAAAAAAAAA

AAAAACGGGAAAATAACTTAAGCGACGTATTCTAATTAAG

GCACAAGAACCCTCTCTTGTAAGACACGCGTCACGTGGGA

GGAGA

123
N10507-001-Q001

ATGATCGACCGCTTATTTTGTGCATAACTTAGAGAGAGTT

TTTTTAATGAAATTATTTGATGATATTTCNCAATGGGGGT

ACACACATATTTATAAGCAAGGATAAGGCGCTGACATAAG

CGCTTACCTCAGCGATTACATCATCGCTTACATCACGCTT

ACATAAGCTTATAGCGATTACATCATCGCTTACATCATTA

TTCATTTTTTAGACACACTTATTTTATATGATATTTTACA

TAATTAAACA[C/T]CGATAATGGTGTGCTTAATGATCCA

TCTCGAANTCGANGATGTGCTTGTCGCAACTACCGTAGTG

ATCTTCTGGACATATGTAGTCTTCCTCTGGATATTCATTT

TGGGTNTTTTTTTTTGACTTCAATGTTGAACATTTTTCTG

ATTTCATCAATATTCATTGAGTCACTGGGAGCTGCGAGTC

ATGGAAGTATTCTGGTGGGCATTCTTGGGACAGAGATTCG

TGCNTTACTATCTTCTGANA

124
N09834-001-Q001

TGTCCGTGTAATAAAAAACGGGTTTTAAGAACAATAAACG

GCCAAGCCCATTTAAGACCTCTTTTTTTGAAAATACAAGA

ATTATTAGTTTGATGTTCTAGGTTAAACAAATATAATCAT

CTCAAATCGTCAGCCCTAGAAACTGACAAGACCCTTTTGC

TCCACTGTCTCTTCAGAAACGGAGAGACGGAGGTGGAATC

[C/T]AGCGAATCCGAGTCCAGGTTTCAGTTAAGGATTTG

AGATTAGGTTATGATTACTGCACCAAGCTTATTCTGTTAA

AATTTTAAGCTTTGTGGATTTGAGCTTATTTCTCTCTCTA

GAAATAGGTGATTACTGATCTGGCAGAGGTGTCTTCCAAA

ACTAGGGCAGACAACGAGGTATCATCAGAACCACTGTCTC

CGATT

125
N22934-001-Q001

GGAAAAAGCCTCTGTAACTCCTCGAGAACATCGCCTGAGA

GCGTTTTACATTTTTTTCCTTTTGGTATTTTAGTCAAAAG

AAAATTAACCAAATCTTTTAAATATTTTTTAAAATCCTNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNTATTGAAAACGTGTTTTAACAG

ATATGGCAATGTTTCTCATTTTAATTTCCAGAGTTTCTTG

CTCCTCGTTCTCTCTCTTATTTCTCTCTGTATCGAGCTTT

GCTTCAGGTTTGGGTGAAGGAGAAGGTTGTAGAGAGNGAT

GACCAGAGTTGATCCAGGAATGTAAGAGACACTGAGCTCA

[A/T]AGGCCAGAAACGGAGTCTTCTGATGTGTCCAAGAT

CACCGTGCCTGTTGAACCTCCTAAAGCTATCTGCAAGACA

CAACAGAGAAAGAGTTGGAGTTTAGACGGTCTAGAATGGT

TGGAAGTAGTTCAACANAAGCCTGCTAATGATTCTATTAC

CTTGCGTGGCATCATTCCTAGAAGCTCCATCATCAAAGGC

AAGTGCTCCTGTTAGACGCAACAAGTGAAGTGAATTAGCT

CACAAGCAACAGCCTATAGCTACAATAATGTTTCATGACA

GAATCAAGAAACCAAACCACAGTTACCTCATCTCTATCAT

AGTTGTCTCCNCTATGGGGATCGAACAGTACATCTCCAGT

GGCAAGTTCAANACATATACACGTAAACGACCAGAATCAG

CCGAT

126
N22700-001-Q001

TCAAAATTAATATGACCTAGTTGTGAATGTTATCTTCTTG

ACTCATTCTCAGTTGATGCTTGGAGACATGTGGTTGGCTT

TATAACCATATGAACTTTATACAATCTGTTTCGTGATCTT

CNAGCTTTGACGAGCAATTTTCTGTACTGATCTTGCATCA

TTAATTCTTCCCCTCTCATCCTGATGTGACACCCTGCTTT

TGTAGCTTACCCCAAGCTTATAATATTACTCTTTAGGTTA

GNAATGAAATACACGTCAGTCATCTTTCTCGAGTCNCTGT

TCATGTCGGTGAAGTATATTGTGCCTTTTCTTTTAATGTC

TCTANGAGAANCGTCACCAAACCGTACTTTCCCTATGATG

GTACTATCAATCNGTGAGAAGTACCTTCGATCTCCTGTCA

[A/T]ATGATTACTAGCTCCATTGTCGAGATACCATATGT

TCTCCCCTCTAGTATTTGTCTCATATTTCTCAAGGAGAAC

ATTCTTTTCGTTCAAAATACTTCTTCATACATCATAAGTT

CATCAGCTTCTTGCATCTCAGTGTTTCGGTCTCTTGAGCC

TCCTGTAACTTAAGCTTGANCTCCGGACATTGAGCCACGA

AGTGTCCAATTTTATCACACTGATAGCAAGTTATTCTCAT

TGCNTTTCGTCCANCATTAAAACGTCTTNGACCTCTTCCT

CTGTAATAGTTTGATCGACCGCCTCTGCCTCGACCTCTGT

ATGTATCTCCATTGTAATCTAGGTTTGCTTGTTCTTGATA

AGATCAGTTTGGTTGATCTTGAGAAGAACGATTTTGGTTG

ACTTG

127
N22725-001-Q001

ATCGTTATTTCCTTTTTTTCATTATGACGTATTATNATAT

CCTTTTCCTTATTNGGTTTGTCCAATCGAAGGGATATATA

AACAGAGCTTTGTTTCTTGTTTGAAGATACGTTTGATTTA

ATAGAAAGAGCTTTGCTTTATACCTTGTTTAGATTNGATT

AAATTCATCAAAACAGAAGTTGGTCTCAAGAAGCTATCGA

AAGAAANTTTTGATCGATCCAAGAACACGTCTCGAAGAAC

CCTAAATTCTTATATCGAGCGTTCACCCATTCGTACGCTG

CGCCATTATGAGATGCTTACTCTTATCCATGCCGGTCAGT

CTAGTACGTCTCGTTTTCATCAATTTCTCTGTTCCAGAGT

ACCTGCATTGGCAGGTTTTAAATCTCAGATTTGTGCATAA

[A/T]GACACCCNAGTAAGAAACACAAAGATTGTTTGTGC

GTTATGTTNATCTACAGTAACACTTCCATTGGTTTTGAGC

AAGGCCAAGTNATACTTATCAGTTNTCTCTTCAGAACTGG

AAAAATCTTTGTCGCTACTATTAGACACCAGAATGGGACC

GATACGTAAGTTAGATGACTCAGAGAAAACCACTTAGACG

ACAAGTTGCATATAAGAAAGCCGAATAATAAGAATGCATT

TTCAAGGGATGTACTAAAAATGAATGGAGGTCAATTTTGG

GAGTTGAATGTCTTTCTAAAGCAAAACAAGACTTATAATA

TTCTACAGCTTTGCATACCATTTCAGATATAGAACAGCCA

AATTCAGTCAGGTCGTACAAAGCGATCAAACCAATAGAAT

TCAAA

128
N22881-001-Q001

ATTCGTCCAAGACTTCAGGCAAGATATTTAAATTAGTCTC

ACACCTTTAGTTGCTTCAGTTTTCCTGTCCATGATTTCTT

GTCCATGATTTTCTGTCCATGATCAATCAGGTTCTTCATG

TTTTTAGTCTTGCTCTTTATTCTCTTCACGTCAACAGAAA

CCAGCTACTTAGGCAGTTAATCAATGTATTGAACATGNAG

AGATCATTTCGAATCCNTACAGCTTCCATCTTTTTCCACA

GAGAGATGACGACNTCGTACTTCTTCAGTTTGANAATGGC

GTTCAACAGTTTATTGAGATCGATGATTGAAGGGAAGGGG

CGAGATTTGGCCATGTCGTTGAACAAATCGATAGCGTCAT

CTAGTTTGATATCACGACGACGGTTTCTGCTCAGTCTCTC

[A/T]CGGAGATCAATCACGCTGGAGAAAGCTCGTACCCA

GCAACCNAGAGAAGGGAGAGCGCTTTTGCGTTTACTATTT

TCGAAAAGATTCCGATGAAGCAATGTCTTCGCCGTCATCG

CAATNGATCTCTGCATCGTTTTGCGATNGTCTCTCTGATT

CCGAATCCTAAAACGAGACATGTTTATGGAGAGAGAGAGA

GAGAGANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNATTCTCAGGGGTAA

ACTCTTTGCCGCTTGGAATAATTAAACTGTTCCTTGCGTT

GCAAGTAATCATGTTATGAAAGTTAATTCACGCAAATCCA

AAGTC

129
N23032-001-Q001

TGTGCAGGATAGAACGTTATATGGGGAGTTGGAATACTTT

CATTTGTTTGAAGCTGCCAATGAAGGAGGATGGTTGGGAC

ACTACTGTTTATAGATGNTCTGCTTTCTTTCTACAACGGA

ATACCCCTGCGTTACTGCCTTGACAATGAGTCTGACCTCA

AGACCTATCCCGACATTAGCCAAGCTGCGTCTGATACCAG

CAGACGTATCTTTGTTTCGGTAAAGGGATGTCTATCGTTA

ATTATTATCTTATATATAATACTATTGGTAATTCTATTTG

GCTATGAATGTTTTCGTTGCTTTATCAACCACAAAACGAA

CCTGAATTCTGCAAATGAATAAATATCTTATATAAGTATT

TGAAATCCTACACGATTTTAAGCATGTCAATTTTTAAGCC

[A/T]GGGGTGTATCGCGAGACGGAATGAGGGTGATGCGC

CTGTTCTCAAACGAGAAGGAGTATGTGTTAGCCAAGAAGA

AGGTGACACGCATCCATTGGGACAACGTCGCAACAGATCA

CATCACTGATAGTTCTGTCCAACAGAGAATGGGACTTTGC

AACGACGTGATGTACGAATATCTGTCTGTTTATTCAGNCA

CACCAGTTTGTAAGGTGTTGGGTGTGGTTCGGCAAATAAG

GCAAGCTTGGCCACAGCTTCTTCNGAGATAACATTGGTGC

AGCTACCGAAATCGATCACGAAACGACAAACCTTTCCCCG

GATGGTGCAAGAGGAGCGCGATTCGTCAATCACCTGAGGA

GTCAGCATATTTCGACGAATAACCAATAGTTGTCCAATAT

CTCCA

130
N22786-001-Q001

ATCGAACCATCGACGAAACCGAGCTTCTTCCTTGCTTTAA

GCGCCATACGCAAGTTAGTAGCCCATTCATTGTAGTTAGG

TCCTTTGAGNAATNGCTGGGAAATCACCGAGCCTGGATTG

TCANTAGAAGATAAGTCATACGGAGATATCCTCCTTCGTT

GGACTTCAACACGAGATTGTGAAGAAGCAGATTTCGTAGC

CGAAGCGTTAGTCAAATCGTCACCATCATTACTACCCATA

TTGGCAAACTTAGAGAAGAACAAAAGAGGATGAAAAACCC

CAAGAACAAAGAAATTTTTTTGTGTTTAATGCTCTGATAC

CATGTCAAGAAACCAGAGAAAGCATAGAAGAGTTTCTTGT

ATTCATCTAGGTCAACGACCATAAGTATATATACATGCTA

[C/G]GTTACCTAATACCGTAAGATATGTACATCGAGATA

AAAGGAATATTAACATAATAGATTACANCCAAATATATGG

CAAGATATGCATGTATATCCTCAATATTGCCGCTCTTCCA

GCTGAAGTATAGCTGCATTGACCTCATCAATAGTCGTGTA

ACGGGTCATATTGGTCTCAAATTGGCAAATAGATCCTGCG

TTGAGACAGAAGTTGACACATTAATTTGTTACAGATATGC

GGAACCNAAGTGAAAACTGAATAAACAAATGTTTTAAGAA

CTAACCTGCAAGTCAAATTCAATATCCAGAGGAAGATGAC

CCTTGCTCAGTATGTATCTCTGGAAATGTATCAAAAAATT

GATCAATCCTTTTCTTTGAAGAACCTTATATGATGTTGAA

GTTAA

131
N23014-001-Q001

ACTCAAGAAGTATCTTCTCAGCGCAGGTTTTCATANCTCT

CTTGCTGATACGTCTCTATTCATTCTCCGCCATGAAGGAC

AGTATGTCTACTTNCTGGTTTATGTGGACGATATTCTCGT

TACTGGTACTGATAGCACTCTGGTTCAACGAGGNATCNAA

CGTCTGGCTGCAAAGATCTCTATCAAGGATATGGGTCATC

TCAGTTATTTTCTCGGAATCGAGGTGATACGAACGAAACA

AGGACTCCATCTAATGCAGCGGANATATGTTACAGACTTN

CTGCAGAAGACAAACATGCTTCATGCAAAACCGGTTGCTA

CGCCTCTCCCTTCCTCACCAAAGCTAACTCTGCACTCTGG

TCCTCTNTTGTATGATCCTTNTGACTATCGACGTGTAGTA

[C/G]GCAGTCTACAATACCTTGCCTTAACTCGTCCTGAT

GTTTCATATGATGTTAACCGACTCTCGCAGTTTATGCACA

AGCCATCGGTGGACCATTGGAATGCAGTCAAGNGTATGCT

ATGCTACCTTGCCGGAACTCTAAGCCATGGGATCTTCCTT

CGCAAACAATCATCTCCTCAGCTCCATGCATTATCTGACG

CCGACTTGGCCGGNGACACAGATGATTATGGGGGTGATTG

GTTGGGCTGTAACTGTAGTAAATTTACTTTAGAATTTAGT

CTGTAGGATTTTTGATTTACCTTTAANGGATGTAGCTTTA

AAATTTCCTACACCTAAAAAGATGGGGCTTTAGAAAATAA

GATATTTACAACCATTTTTTGTTTGTTTTTGTTGNNNNNN

NNNNN

132
N10471-001-Q001

CAAGTTGATATGGATCTTGTTATTTCATCCATAAAGGGTC

AATTGATAACTATTATTGGACTACCAAAGCAAGTGTATCC

TTCTCATGCCTCGCTAACAAACTACTAAAGCTCAAGGACA

TAGTCTTTCCTCTCATTAAGCAAAGGCTGGAAAATGGCCT

CTCAGCTAGGTTCTGGTTCAATAATTGGACATCTTTTGGG

ACCTTAGCATCTTNCCTTGACTCCTCTACTACTAGGCTAG

GGATTCTTCT[C/T]ATTGAGTTGCTTCTATTTGTAGGAA

TGGAACTTGGCTAATCCCACCTTCAAGAACAGACAACTAG

CTTCAAATTCAGGCTTTTTGACCACTATCAACTTTTTTTN

ACCACTATCAACTTCTTGCAAAACTAGGGGTGGGCGTTCG

GTTCTTCGGTTCAGTTCGGGTCGGTTCTTTCGGTTCTCGG

TTCTCGGTTCTTTCGGTTCCTGNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNN

133
N11419-001-Q001

ACTTCCGTGTGGCAATAAGCGCTCGGAAAAAGTTTGCTTT

TANCAACGGATCTATACCAAAACCTGCACGACTCACCCGA

CCTTGAAGATTGGATAGCTAACAATCACCTACTGGTGAAT

TGGATCAAACTAACAATCGAACCAAAACTTCGATCGAATA

TCTCTCACAAAGAAATCACTCGAGACCTCTGGGACCACAT

CAAAAAGCGATTTNCTCTTAAAAGTGGAGCTCGTTACCAA

CAACTACGAGCTTCCCTGGCAACTTGTCGACATGTGGGAT

CTACGGTCGAAGACTACTTTGGACGCTTGACAAGAATCTG

GGATTCTATGGCTAAATGTATGTCAACCAAGACATGTGAC

TGCGGAAAGTGTGAATGCAACTTGGTCAGCACTCATGAAA

[C/T]AGAGCGCGAGATCATTCGTGCTCATGATTTCCTAT

ATGGTGTTCAATTTCAATTAATTTAGAGGACACTTCCAAA

ATTCTGAGCTTCCCCGGAGTTTGGCTGTGATCCCTACATC

TGGAAACCAAAGCATACTTAGTCTCAACAATCTAACACAN

AATGAAAAATGTATTAAAGTAAATGCACTTTGTAAGAAAC

AGGGGACTGTTATATATTTAAGTGAATGGGTGCAATATTA

TATATGAACCATTGCAATTGTTTATATGAACAATTGCAAC

TATATAATGAAACCTTGCAACCATTAATGATTGCAACATT

TGNTAATTAACCATATTGATAATTGCAACCCTTGGTGATT

AACCATTGCAACTTTTGGTTTAACCATTGCAACTATTGGT

TGCAA

134
N22724-001-Q001

TGAATTTGTTCACCAGAAATATATTAAACNAGATTACTGA

ACCAGGTTTAACCAGGTCAAACCATATTGAACCGTGACCC

AAAAATTATCCGGTTCAGCTNCCGGTCCGGTTTTAAAAAC

ACTGTCCAAAACTGATTAATAACGAGTTTCAGATTGTTAT

TAATAACGATATCTAATGTTTGCCAGCAGAGGACTTCTGT

ATATCACGGTGATCTTTATTTATTTTTAAGAATATTTTCA

TGCGACTGCTTACTTAGTTATATAAAATATCGAAGTCGAA

GACCATATAAGATTTTTTTTTTGGCCAACAATAACTAATC

TGCTACGAAATACTCACCGATTCGGAATGATCATATAAGA

TCAAACTTCAAAGTATGGTGATATGTTAATGTCTGCACTA

[A/T]ATCTATCCGCTTATTTTATTAACGNTTTAACCTAA

AATATTTAAAAACTATAAAGTCTTGCATCCGTAATTACCC

AGCTAAATTCATGAAATTGAATTTGATTAAAGTCTTTAAT

TATTTGAATAGTCTTAAAAGATGGTACATAGCTGATGTAA

AAAAGCGCGTTCTTGAAGAGAACAGGAAGTCGTACAAGCT

TTTAGTCAAAAAAAAAAAGTCGTACAAGCTTAATACTTCA

ATGTTTTTTTATTCNCGAGACGGTTGATTATGTCTGCTTA

AAACTATATATATACTCACTGCTGGTCAAGACAAGAACAA

CAACGAAACAATGACAAAGATTCTTCTAAGCTTGCTCCAT

ATACTTTTATGTGTGTCTTTACATGGTGTGGCAGAGGCTA

GTTTC

135
N12785-001-Q001

GACTNGTAAAAGCCTGCCCGGAGAATGTTTTTGACATTGA

AGACATGGGCAATGGTAAAAGTAAAATCCTTCCTTTAAAC

AATAGGAGAGGATGCNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGAATG

TCTGTAACAAGCTTGGGTTTATTGATGGAACGGTGATTCA

ACCGGCGTCTACTTACCGTGANTATGGTGCTTGGTCTCGT

TGCAATGATATGGTNGCAATATGGCTGATGAACTCTGTAT

CCAAGAANATTGGTCAGAGCTTGTTATTCATCAATACCGC

TGAAGGTATATCGAAGAATCTCTTGGCTCGTTTCAAACCG

[A/G]ATGATGCACCGANGGNTTTTGATATTAAACAAAAG

CTGAGTAAGACTGAACAAGGTTCAATGGATGTATCAACAT

ATTATACTGAACTTGTGACTTTGTGGGAAGAGCACCGACC

CGCGAACGAGTCGGTCGCTACCTAGCGACCGACCAAACCT

TTCGTTCGGTCGATACGTAGCGACCGATCCAGCGCGGACC

AGGTCGCTACGTAGCGACCGAACTATCTCGGACATCGATC

AACGGGTACGACCCAAATCTGTGCATTCTCGTTTATTCAT

CAATGCTATCTCCCATGTACCGCATCCATATCATTTCTCA

GATCATTCCGATCAAAGTTACCGTTGAAACTTTACGATAA

AAACCGCGAGAACTTGTTTTTGTCGAAAAGAAAATCGTAA

CAAAC

136
N09910-001-Q001

TTACTATTTTGGTTTATTGATACTTTGATTCAAAATTTTA

GTAGTTCGGTTTAATTTTTTTTTAATTAGAATTGAAATAA

TTAGGGTTTTGTGCCTTTAGTTAACAATCTATCGACAGCC

CCTCTGGAAGGAAAAAAGCAACCAAAGAAGTGTTTGATAT

AACTTATTTTTATTCGTACTTTTTGTTTTTTAACTTGAAC

[A/T]ATATGGACGATATGATTTTAAGTCTTCCCATGGAT

TGATGGAGGAGATTCTCTCAAGGGTTCCGGTGAAATCTAT

TGGAGCAGTACGATCAACTTGTAGAAACTGGAACGCTTTA

TCTAAAGATCAAAGTTTTGTCAATAAGCATATTGACAAAG

CAGCAGCATCATCAAGAGAAACGGAGGTTCATGTGATCAC

GGTGA

137
N21146-001-Q001

TTTGGTGGTTGTTTGGAATTGAATCGTGTGTAATAAATGG

TTAGGTTAGTGTTAGATTATGGTTGGTGTTAGTAATAAAT

AGAAGTGATGAATGAGTTAGATAAAGGTCTAGTATATTTG

TTGGGTTATGAATTCACTCGATGGTTGTTAGATATATGTC

TAGTCCTAAGTATATTTATTATCATATATTCAACGTTCAC

[A/C]ATCTCTACTCGGTTAAACACAAAAGTCTTCCTCTT

GTTCTTCTCTTCCATCAAAACACCTTCGTTCATCTTCTTC

TTCTCTTTTTCTCATATTTCACACACTAGAATATGGATTA

TAATCCATAAACGAGTGGGGAAAATTTTGTTGCTCTTTTT

CAAAGTCAACAACAAAGTGTCTTCGGTTCATCACAAGTTC

CTTTT

138
N17618-001-Q001

AAAGTTTCACCGCCANACGAATGAGTTTGAATGGCCAGGA

GCCCCGATACGGGATGGCCCCTAGTACCAGCCAGATTCGA

CCAATCACGAGCCAGAGGTGGTGCCGGTTAAGCGGGGACA

GGGTCGTCCCAGGAAGGATAAGCCGGCGCCCGGCATAGAG

TGGGTCCCTGAGCCACCTGTATAGGAGCCACCCAAGAAGA

AAAGGGGGCGTCCGCGCAAGGCCGCTGCCCCCAAGGAAGA

CGCTTCAAAAAAGAGTTTGGAATCGTGTGGGAGTGATGTC

ACGGTCCCAAAGTCTTGCTCAGTGGGTGAATACTTGGAGG

GGTTCCTGAATACGACTAAGGGGTGCGCACCCAAGCTCGT

ATAAGTGTGGTGCCAATTGTTTAAGGCAGGGATCCGCAAG

[A/G]ACATTCGGGGGGAGCCGGAGAGCGCCAAGGATCCC

CTGGAGTTCTTGCTGATGAAAAGGATAGTCGGGCAAACAA

TAACCGCAGGGGAGTGGATCGTGGGGTTGGGCGAAGAGGA

TCGAAGATNCGCACTTCGTCAGACAGAGGAGAACCCAGAG

CCGAGANATGATGACCTAGGACCTACACCTCGCGATGCNG

GGATGGTTGGTTTAGGACCGGAGGCCGACCGTGTATTTCC

ACATCCTTGTGGCTAGTTGAGGCTTTATGATCACTTCGTA

GGAGTAGGACGTGAGCTTTTCTATTGGATTTATTCTAACT

ATTGAGTGGTTGCTTTGGTGTTGTTTCTCCATTTCTAAAG

GCATTTTGTGTNTGTACATATGCCTTGANCTTTTCAAATT

ATTGT

139
N09776-001-Q001

AAACCGGAGTTCCAAACATAGACAACAGAAGCTCGGCTCC

AGCGTAAAATCCAAACTACAGAGGCCATGGGAGATCTTCT

TACAGAGGCAGAGGAGGCTACACCTCCAGAGGAAGAGGCT

TTATTCAACATCAGTCACAACCAACATCCTCTGGAGAGCG

TCTTGTTTGCCAAATCTGTGGTCGCACTGGTTATACAGCT

[A/C]TGAAGTGCTACAACAGATTCAACAACAACTACCAA

AGTAATGAAGCTTATACGGTTGTTCGTGTTGCTGATGAAC

ACGGAAGAGAATGGTACCCTGATTCTGGTTCCTCAGTTCA

TGTCACCTCGTCGACTCAGAATCTACAAACCTCCCACCCC

TACGAAGCTCATGATGCTGTCATGGTGGGAGATGGAGCTT

TCCTT

140
N19296-001-Q001

ACGAACTTATCATACAAGATGATTCGGAAATAGTATCACA

CTCACGAGGGTCATGGAAATCAAAAATATAATGGTCGAGA

TCCGAGAACTACATATAATTAACGTGGATGAGTTGCTCAA

AAGCATATGTACAGCTTGCAACGAACAAAATAAAAGATGG

AGATGTAACATTTTATAGAGATAAAATAAAAGGCTGAATC

[A/C]GTTTTTCAGAAATTGGAAGGGCTTAAAAGAAAGTA

CAGACAGATCGAGACTGGTTTCTTTATGTGCGGCAGATCC

AAACACTGTGGTCGCCATTAAACAATCTTTGTAATAATCG

CAAGTTCACAACATTTACACATAAATTTCCACATTATCAA

AGAATTAATTAGATGTTGAGAAAAGAAAATCAAAGTACTA

ATTAA

141
N05205-1-Q1

TACCTCAAACTTGAAGAATCCATCAAGAACATCAAGAAGG

AGTGGTTTGCTACCTCCGTTTCTGTTGAGCTCATA[G/T]

CCACTGTTGCTACTAAAGTCGNGTAACTCAGACTTCCATG

AATCTTTTGCCTTTTGCATAGGTTTCCAGAATGTT

142
N10406-001-Q001

CACATTAATGGAGTCATGCCGGAAGCCTTGATTTCACTCC

ACAGCCAAATACTTCCTCTTCTCCAACATGAAATCATGTT

TCTTGATGAACTAGAATGCAATATCGACATCTACTGGAGG

GTGTGTTTGCATATGTTGATCCTGTTCGTACAGTGTTCAN

TGGCAGAACAACAATTTCTTTGTGGCGATGTTGTTGGACG

GCTCATGTTGGATCGTGGGGTTTTGATAGCTTTTCTTTCN

ACGAAAAAGG[C/G]AAAACAAAATATTTGTGGCGACCAT

GGGTTTATGAATTCTTCATGGTTCTTCTTCAAACATTCTC

AGTTGAGTGGCAGTTCATACTTTTCTATTGGAGAGTTGGA

GACGTTGCTCAAGAGTTGGATTGTTATAGTAATGGTGCTT

GCGAATCTAACAACGTGTTGTGCATTCTGGTTTCGAATAT

TGTACCAAAACCTGTTTGGAAGTGCATGTCTTTGAGTACA

GCTGGCAAGAAGGTCGTGAT

143
N22941-001-Q001

TCTGTGAGCTCTTCATTGTCAACNACCCAGACATAGGTGT

CCTCAATTGTTGATAGAGAGGGTAGTTGCAGATAAGTCAA

ATATATAAGGAGTTCCTCAGCCGCCTCTGATCGAGCACCA

GGGAATATCCAACCAGCTCTGTTGCAAGCGTCAGAGACTG

TTGCTGTGTTTGGGATTTGAAGTTCCCTTGGGGCCAAGAT

CCCCAAACCTGTGAAAGAGCGGTCCTAGCGGTGTCCAACA

ATCATGCCAGAAGCTTATGACNCTGCCATTTCCCAGATTT

CCATGTTGAGGAGGAAGCGTTGTTTTCATCAACAGCCCAT

AGATTTTCACCCTTCAGTCTGTATTCNCGAGTCCANTCTG

CCCAAAGTGAATCAGTATTCAAAAATAGTTTCCATAAAAG

[C/G]TTCAGGCAGAGAGTTCTGTGACATATCCTCTTGCA

CAAATTTAGGCCCTGTGCAAGCTAATTCCTTACGTTTGGA

AGAAGCAAGAAAGTGGTTCAGCCCAGGATTGAAATGCCTA

ANTAATAAAAATTTGAGAAGAGAGACTTCACTATGACATA

TCCTCTTGTGTAAACAGGGGGGATTGCAAGAGCAGATTTG

AAGCGAGAAAGATTTGTTGCTAGGAATAGCCAAGAATCTG

CTTCAGAAAACAATAGATTGAACAAAATCATTTTGAATCT

GAGATCAAATGAAATTTAAAGAAAAAACATAAGTCACATT

AGACTCAACTGAAGCTCTCTAGGCAGGTATATATAGTCAT

GAAATATATTACTTTTTGTCTNTTAATATATAGNCTTCGA

AAGTG

144
N22875-001-Q001

TAAACACAACAAAGATCACAAAAATATGCTGAAAAACAGA

ATATAATTAAATCACATCGTAAACTGGTAGCAAATATAAA

TATGCAATTTCAAAACCATTTGCTATAAATTAGAAATGTA

AATAAAGCTTAAATAAACATAACAATATCAGATAAAGCAT

TTTCTAATTAGAGTACTAAAATCCATTTAATCAAATTACT

TTCACAGGGAGATAATTAGATAAGAAGATAACAAACTTGT

GACCAAAACTAGACGCGAGACAAGATTTCTTAGAATCTAC

TGTCAATACCATTCAATAGCGATCCTACAGCTGCAAATAC

AAATCGTCTAATTAACTAGAACAAAACAAAAGAAGGTTTG

AATCGAGCATTAATGACTTACCATCCTTCAATCAAACCAG

[C/G]GTTACTACTCTGCTTCTGATGTTCCTGAGGCTGTG

GGTATTGCGGTGCATACGGAGGTGGATAGCCTTGCTGAGG

ATATCCCTGCGGTGGTGGATAACATTGTTGAGGATAGGGT

TGTTGTTGTGCCGGAGGATTTTCTGGAGGCGTNGGAGAAG

CGTCCTTAGGGTTAGGGTGTCCATCTGACGGATTACCTGT

TCAACCATAATATGAAATAAACTAATCAGAAGAAAAAACG

ATTAGAAGTTAGGATGGAACATTTAAATTGACCCCCTCCC

CCCCCCCCCCCAAAAGAGTTATTATATGATATATAGATGT

CATGACACCTAATTATCAAAAAAATTAATTTATATAAGCC

TAAAAATGTTTATGATCTATGACAACAATACTTATATCTG

TCTTG

145
N13286-001-Q001

TTAAATTAANAATTAACCTTTCTTTTATGGAAAAACTGTC

CTGGTCGAACTTGGGAGACTTTTTGAGTTCGCCTAGAATG

GGTGAGAGGTAAAGAACATGATTGGTTTGTAACGAATGAA

AATAATTGATTTNGAAAGAAAAAAACATAGTTATATATGA

TCTTTGGGAATTTTAAATGTTAGACTGTGGCCTACCAATA

TGTAGTATTAAAGTTTAATTGGTTAAAGATGCTTTAGGTA

CATTATGCATCCTCTGTATAAAATGTTTATCAGTTGCACC

TAAGCCGGACGTATGTGGATTTTCGTTCACATTAACAAGT

AAANTGAATAAGCCATTACTTGTATCGACCGTGTTAAGCT

GTAAATCGATAACAAACTAAAACGTTTTTTTTATTATGAA

[A/G]GTCATGCAAGTGAGATACATTTCTTCACTACTAGT

TACTTATTTTAAGAGACCAGTTTCAAACATTCCACCAAGC

TTTTCCATTAGTNATATATATTGTCCAAAAACACTAACAA

TCACCCACCAAAATAATTTTATATTTCCATCTAAATCTAC

CTTTACTATATACATTCATGGACGAATTAGTTTGGAGTTA

AAATCCTAATCGTACCACCACATTTTCCCAAGCATAAATA

AACAACGAACGAAGTTGATGCTACTTTGTGGTAAACCAGT

GGTAAGAGTTGATTTGAATTAAAGCGAAAATCCATCGATT

CTGTATTNAAGTTTTGGTAAAACGAAATTACTGTCAATCA

GACGAAGATGGGATTATACTTTAGACCTCGTTAAAAATCT

GTACC

146
N04503-1-Q1

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAAAATTAATT

ATTCGTGACCATTTTATTTTGAAACAAAAGAACGACAGAG

ACATAACGAGATTACATTTATTACAAGCGAAAATACTACT

AGTCTACTACTACAAAACATCTATAACAACAATATAACAT

GGGAATAACAAAATGGTAGTAAAAAGAATAATAAGCCGAG

CCACCAAGTAAACCAAGCTTCT[C/G]TCGCTTACACAAG

AATCTCAACACGCCATCTGAACATTTCCTCCCTCGTCACA

GGTAGGTTCAACGTCACAAGCCCAACCTCCGNGTCGTAGT

TGAACTCAGTCTCGGTNCNATCAACAGCGCATCTNAGNGG

ACGCTGAGAAGAGTAAGCCCCAAAACGACCACAACCTCTA

ACACCTAGAGATATCAGAGCTGTTGGAGAACGGNTTTCGC

TGACCACTGAAGAAGAAGAGAGCTCAGGTTTCTCGTCTGT

CACGGTATTGATCTCCATGGACTGGATAGCTCCACTTGAG

TTGAACATGTCCAGGAGTCCAATAGGTGCGAATGAGATGC

TTGCAGTGATTTCCTTTAGAGGAGAGATGTGGAAGAGTTC

ATATTCAAGAACCTTGAGAGTGAGTGGGATTGATGCACCC

TTTGGTAGTCTAACCAGCTCCCCTGATTTGTAAGCGTAGA

CTATTGAATCTCCACTCCAGTCTTCACCAGCCACTTCAGA

GATGAGA

147
N22925-001-Q001

GCTTTTTGTGAAATGAATGGTTGGCTGGTTTTTGCAAGTC

ATGCTTCTGAAGATAACCTTGCTTTCATTAGGTAGCCGCG

AATCACCCCTCTTGGACATTGTGGCATATGCAGGCTATGC

TTTCACTGGTCTCTTCTTGGCAAGATCATTTGGGGATATT

CTTACTATGTTTTGATTCCGTGGACTTGCTTATGCACCGG

AGTTCTCTTGGTGAAGACAATGAAGCGAGTTCTCTTTGCA

GAAGCTAGGAGTTATGACTCAAGCAGAAAATCATTACCTC

TTGATTTTTTAGCATTAGCACAGTTTCCTCTTTTGATCTG

GCTTGGTAACATTAGTGTCGATTGGCTCTTTTGAGATTCA

TTAGTTGTGAATNAAAAGAACACTTATGATGTTATGAGAT

[C/G]TACGATACTCCTGATGAAGTAGTACACCTCTCTCT

TTTCATAACTTCTTTTTAATGTCAATTTTTTTTGCATAGA

CTACATTTCCAACATGATTTNAAACCAAACAAGGACATGA

ACTTTGGTCATATAGTATTATTATCTATACTCCAAGCCCT

CCTNTTGCATTGCATTGGTGCATTGTGATACTCCTTGGCA

GATGGTTCNTTTTAAACCTGAATGAAAGACATGGAACCCT

TTCNTTTAAAGATAGTATTCTTATACAAAGAAAAAGAAGA

AGNAGGTCGTCGTGTTGAACCTAATTAGGATCTAATATGC

TTCCCATCTTTGTAGCGTTGTTGATAAGTTACATCAAANC

AAATATTGACCGACCTCAAAAACTAGTTTTAAATCATTTT

CACTT

148
N05656-1-Q1

CGCTGTAACTTCTCAAGAGTTTCTCGCTAACATCACCGTT

GCTTTCTCCTTTAGCCACTCTCCTAAGCTTCAAGAGAGAC

TCNGCNAGAGAACCCTCCANTGCATTTCCTCCAAGTTTCA

AACTTTGCTCGGTTTTAACCTCGTTGTTCTTCGCT[G/T]

TATCACCTTTCCTCTGAATCAACCCCCAAAGANTCCATCC

TTTCCCCCATTTCTTTCCANNCTTCTTCAAACCAAAACCA

TCCTGCTTCACCTCNCCACNACCAGCAACACAAACTACGC

CTTTGGAAGCTAACTCTAAGCTCTCCGGTTTGTAGTTTTT

GATGGAATACCAATTCGAATCNCTCAGTTCNCTCTCNGTA

ACAAGCAACTTAGCTCCATGAAACAAACCAACACCTTCAG

GTGACAACTTNGNTTTNACATCCACCAATCCATGCNTNGA

TGACCGATCAAAGCTTCTTCTCCGCCGTGANTCCAAGTAN

TAATCCCTCGTCTGAGCTGTCCCNCCTGGCTTCTTCTCCG

GCGAGGCTTTANCATCTTCGGTCACGGATGANAAAGGTAN

CAGCTTTGGATACGTCTTCCCGATCAAGCATCCGTCCCAT

GACGCTCTANGCTCCTCGAAAGAGACTCGNCACAACCTNG

GATCNACATCACATGANCGA

149
N17581-001-Q001

ATATTTAATTATTATATATATGGAGTAAAAGTATAAGAGT

CTTTTTCCTCTTAATGAAGTAGATATTTTTGAAAATATTT

ATTTAGTGATGATAAACATGAATAATGATACGAGCAAAGT

GTTAAACATGAAAATTCCCCTTAAATATTCTCTTTGTTTT

ACAAAGTATTATTATTATTTTGACATATTTTTTTGTTACA

CAAAGAATATCATTTTAGAATTTAAGTGTGATTTATATTT

ATTTTAAACTTAATCTTTATTTCTAAATGCATTGATTTTA

TAAANTATTTTACTTATCTCAAATATGATTTGTTAGATAA

ATATGATTAATAAAAATATAATTTTTTTGTTTGAATAACC

TGAAGGTTTCCTCGTGGAATGACTCCGATTAATCCCTAAG

[A/C]AGAGAAGTAACCCAAAAATAAACTATTNCTTCGTG

TATTTAAATAGACCGCAAGGACCCATATCTATATAGGTGT

CTAGGATAATGTAACTTAATTTCACACATAAGATATATCG

AATTTGAAATGTGTTGGCATTCTAATTCATTTNTCCTCGT

CACTCGACCACACAAAAACATAAATATTCAAATCATATNT

TTAACCGGTGTGAGAATTAAAATTGAGAAATTGCCACAAA

TACCACATTCATAGTACCACTTTTNATGTNTACACTAATC

ACTTTTATCCTCAATTTTAATAAAGGGTAAAAGACATTTA

TACCNCTATGGTTAACTAATCTAAACTTAGGGTTTAGAGT

TGAGAGANGGTAGGTTTTTTGGCATCTGAAATTTAGGATT

CTAAT

150
N001NVH-001-Q001*

AATACGTCTACAATTTCATTAGTCTCAAGAAAAACAATAT

AAAAACAAAATAAATAGCCA[A/G]ATTACATCCCAAATT

CATCAAGTAGNCTTGAGTGGNGCCCCAATCCAATTATCCA

GAAGC

151
N22928-001-Q001

TGTGTTTTGCTTGTGCAAATGTGCANCTTTGAGTCTGTGT

GATGGACCTTTAGGGTTTGATAANGGGAAGGGAAAGATGG

TAGATGCAGGGGTGTTGCAGGACAGCCAAGGAGCAAGGGC

TTATGAAGGTTCTTCCCAACTTCATGGCAGGTTTGCGCAT

GGGGGAAAACTGACCATCGCAGAATGTACGGCCATGNATA

ATTTGTGAAATTTCATGCATCTTCAGTCAATAAATTCCCG

TAACTGTCATTACAACTTACTGTACTGGGACATCAGTTGG

CCNTCTTCTACTCGGCTTCATGAGTATAAGTATGAGTTTG

TTGAGATCTCGTCAGAATACGCTGAAGAGTTGCCAATAAC

GATTGCGTTTTTGCGTGAAGTCAAAGGCTTTGCAAGTGTC

[A/T]TTCATGACTGGTGGTGGTGGTAGATCAAACACGTT

TCAGTATGAGTTGCTTAGATTCTCTCNCAGCATCCCTTCA

ACTAAATTGANAGGGAAAGAAAGGAACTGTGTTCCTGTTG

GTTCTTATGAGCTTGATACAGCTGCGTTACCACAAATGAT

AGAAGATGGTGAAGAGGAAGACTGGTGATATTTGGGCAAT

GTACANNAACTGGAGGAATGAAATCAGGGTAGGGANCTTG

AAGAAGTGTGCTTACGAGGTTGTTGATGTTGGAGTGGGTG

GACGGGTTTGTATATTGTGCAAAGTCTCTAGCTTTTAATG

TTTTAGATTCAGACATTAACTTGCATCGGATCTGTCTTTT

GACTCTAGTTTTAGTCAATCTGGTGAAATGTTCTTTTACC

TCTTC

152
N08219-1-Q001

GACCGACGGCGTTCTTCAAGAGCTTAGGNGGACAGGTGGA

CATCGTCAAAGACGGGAAGCCTTACGTGATGTTCGGAGAC

GGGAAGCTNTGCGCTTGCAAGCCCATCAGCGAGGAGGATT

TAGCTTCGTTCATAGCGGACTGTGTCTTGGAAGAGGATAA

GATCAATAAGGTTTTGCCTATCGGTGGACCGGGGAAGGCC

TTGACGCCNTTGGAGCAAGGNGAGATTCTGTTTAGGATAC

TTGGGAGAGAGCCTAAGTTTCTGANAGTNCCTATTGAGAT

TATGGACTTTGTGATTGGGGT[G/T]CTTGATGGTGTGGC

GAAGGTGTTTCCTAGTGTTGCGGAGGCTGCTGAGTTTGGG

AAGATTGGGAGGTATTATGCTGCGGAGAGTATGTTGATTC

TTGATCCNGAGACTGGGGAGTATAGTGAGGAGAAGACTCC

GAGCTATGGGAAGGATACTCTTGAGGACTTCTTTGAGAAA

GTGGTTAGAGAAGGGATGGCTGGTCAAGAGCTTGGTGAAC

AGTTCTTCTAGTGGGGAGAAGTTTTTATGCTAATGAGTTT

GAGCTGTGTTGAGTGTTGTTAGCTGTTGAGATTATAAAAA

CTGTGAATTTGAGAGATTTGTTGATCCAAAAAAAACAGTT

ATAAAACACATATTTCACANGTNCAAAAAAAAAAAAAAAA

AAAAAAAAAAAA

153
N05710-1-Q1

GAAACAATATGTTCATCTACATTTACACTTCCATTTGTTT

TGAGATAGTAATCTCTTCAAAACTGGAAAAGCATTGTCGC

TACTGTTAGACACCAGAATGGGACCGATATATAAGTTAGA

TGACTCAAAGAAACAACACTTAGACGACAAGTTGCACAAC

AAAGACCTATAATAAGAATGCATTTTCGAGGGGTGTTCTA

AAAATGTGAACCAAGAAGCGTGATCTGATATAAGTAGACA

TAAATTNNACCTCAAGCGTCGACCGAGATGAGGAGGAGGT

TGATGAAGAAAGCTTATCGGCACCAGATATAGCGCTGATT

ATGATGATGCTGANGGCAAAGCGTAAAACNCTTNTGGAGG

ATCCCTTTGATANGCAAAGTCCATATACAGAATCTCTAAC

TTGGAGTGAAGCGATTGAGCATCTCTCAACTCNANATCTC

GAATTAGAGACATANCCAAGTGCCAGTGATTCCATCACCA

CTGAAACAGATGTGTTCCCA[A/G]GACAAAACCCAAGCA

TAACATCATAGTGGAAATGCAAAACCCACAGTAATAGTTA

CAAGAGAGAAACAGCNTTTTTGCANCCCCCGGATGGTGCA

TAGCCACGGCTCAACATTTCTTTGATAAGCTCGGCCGATA

AGNTTATATCACCATCTCTAAGACATAGCGTGCCATCATT

CAGCATAAGCCCATCTCCTTTCATCTTTGT

154
N15338-001-Q001

TAGACGTAATAAATGTAGATCTGTTGAGGTTCTTTTTATA

TATATTTTCCGTATTTGGTCGGTCTTTGAGATGAGTTTGT

TTAAGAAAAACGTAAAATGAAGTTGCTTGGGGTCTANGTG

ATTNCCTGATTGGCACGAAGAGCGACGCATCCCCGAAGCG

CGGCACTCTCCACGTGCTCTTGTGATCCACTAAACAAGCC

CATACTAATATAAAGTCATCTTAAAACCGACGCTGCATAA

TCTTTAGGTTCATGCACATTCTTGAATAGATTTCGAGGAA

CATACTGTAGTTAATATTTNAGCCTGGACCAGAAATAATA

TACTCGGATTGCATCTCAATAAAGAGTATTAAAAAACAAA

AACAAAAACAAAATCATTCTGAGACTTTGAAACGAAAAAG

[C/T]AATTAGTTTATTGAGCGCGGGGAAGAGTATATACA

TTATTAAACATACATCTCTCACTTNCTTTTCTATACGGCG

AGTTCATCTTCAAGATAACTGTATTCAAACGTGAACTCGT

TTTTGATCCTTTGAACCCTACACAGAGTTTTCATAAAGAA

AAAAGATTAGGATCATCACCTCGAACGACAAAGAGAACAA

GNAGACGAGAAATCGATAGATCATACCATCCTCCTTCTTG

GCTCTCATCTTTGGGGGTTTGGAAATCAAAGTAAACATAG

GCTCCTTGTTCATACTCATCAGTTGCAATCTTTGGCTCCT

TGTGTCTCTGAAACCATGTGTTGAACTTTCTGTGGTATCT

CCACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNN

155
N10424-001-Q001

CTTGTTCTCGCTGAGACTGTCTTTCTTTTCTAGGAAGAAG

ACGCTAATACCGCGGAGCCGCCACCTCCTCTTCCTTGCCG

ATACAGGTCTCTGCGGAGCCTGTTTAAAACTTGTCCTCTA

CTCGGCTGTTTCCTGAAAGTTGAGCAAAATCGCAGCCACT

GCTGCTACGGCCATTTTCAGTTGGCACGAGAATTTGCCCA

ACGAGCTGTGGCTTTTCATTACTTCGACGGATTCCTTGAT

GGAGAACATC[A/G]AAAGAGGGTTTGTATCTTGGCTCTG

CTGAACAGTGTCCGAGGCTTACAAACAGGTGTTCCAAATC

TGGATCACGTTGCTGGATTATTCACCACACTTCCTTGGCA

GCTAAAGATCATACCAGAAAATGCAGNAAATTGGCTATTT

TTCAAGACACCCCCACTACGCTACACATCGATGAGCTACA

CANCGACGAACTGTGGCANTAAGCCGNCGNCCATCTGGTG

GAAAAAAAATCCATTCCTAA

156
N16006-001-Q001

TTCTNTGCAATATGGGAAGTTGTATCTCTCTCTCGCTATC

ATGTGATCAGTGTACGAATCAAGTCTTTCAGTGGTTATGC

ATCAGAAGGGGTTATATTCACAATCTCGAGGAGAATCTCA

AGGCTTTGGAGACAACCATGGAGGAGCTTCAGGCAAATCG

GGATGATTTGTCAAAAAGGGNGGAGAGAGAGGAGGGTAAA

GGACTAAAAAGGCTNTCCCAAATCCAGGTATGGCTTACGA

GAGTCGACACAATCAAAACACANGTGAATGCTATATTTNG

TGCAATACCTGTTGGAAGTCAAAGGTTGTCTCTCTGTGGG

TTTTGCTCTANGAATTTAAAATCTAGATATCGTTACGGGA

AAAGGGTTTTTCTGATGTTGAAGGAGGTTGAGAATCTAAA

[C/T]TCTGGTGGAGACTTTGAAGTCNTTGCCGAGCAAGC

TCAAGCATCNGAGGTAGAGGAGCGGNCTATCCAACCGGGA

ATTGTTGGTCGGGACACGATGCTCAAAAAGGCATGGGTGT

AGAAGAATTTAGCTTAGGTTACTTAGGTTTCAAGTTAGCC

TTAGAACAGTAAATACATAATCAATTTAACGAGTTCCCGG

CCCTCGGCACGGTACGTCTCGTGGGAGAACTTCTGCTCCC

AAACTTGACTAGATCAAAAGAGTCACCAGCCACACCAAAT

AAGTGTGCTAGTTAGTTTACAATGAACCAAATACTCCAAG

CTAGAGAATACAACAAGCCCTTAGATAATAGACTTAAGCC

TAAGCTAGTTATCTTGTATGTTGTCTTCTTTCTCTTGCTA

ATCTC

761
N07278-1-Q1

AGTGTCAATATCAAAYGAGYCCTGATTCAAGAAGTCGCAG

AGTTTCTGTGAATAYCCAACSGAGTTCTTAGTTTC[A/G]

TCTGTATTCTTCCCATTTGCATATCCCTTGTCTAGCTTGA

GCTTGGGAATGTAGGTGGTGGATCTTATCGGAATC

762
N16343-001-Q001

CTAAGCATGATACAAGAGTCTGCCTTTCGGTCCTTTTCTT

TCAAGTGGGCCACTTTTTTTTCCCGAACGCAGTCCGAATA

TGAAGCGCATGGATACAAGTTATGACTTGGAATGAAAGAC

AATTCCTTTAGCTTTGATTTGGTTTGTTTCTTACGAATAT

TTTATATTATTATTTGATTGTTTTTTACAATTACGAATAT

CCATATTTTTTTATTCGAATCGAAATGGATAATGAATCAA

ATCAAAAWWTTTGAATATTCCGCCTAGTCCTAGTTTGGAG

CCTAGTGAAAATTAAAATAGCTTGACATTAGTTCTCAACA

TTTACGGTAATGATTATTAAGGACAATCGAACTTTTCAAT

ATAGACGATTACCAATTTACCATATAGGGATATATAGAGC

[A/C]GAAGACTCAAAACCTTTAGTTGGACAAATGGTTAA

GTCACGAGTGCAATAACATATTTTATATACAAATGTCAAC

AATTCTGTTAGTAGTCGTTCGTGCCACATAATTATATATC

TTTGTGATTTTCTGTTTCGATTAATCTGTCTCATAGTTGC

AGTCGTTAGCTAGTATTAAGATCAACAAATTTTCGGGTGC

ATATTTTTTTAAGTTTCTTAATTAAGGATATAAAGTACGC

GCGCTCAACTATTTGTAAATTAAAACATCACTGCAAGAGT

TCAGTCAAACAGGGGCAGACCCCCACTATGGTCCAAGGGA

GTCACTTGACACCCCTAAAATAGTTAAATAAATGAATTGC

ATAGAATATCCAATGGATTTTTGTAGCAGAAATAGTTACT

TTGWA

763
N23417-001-Q001

AAACTATGGTTCTGTTACACTTGTGTGTTCTTTAACTTGA

AGCATACGAGTCTCCAACGAGTTAGGAGCAGGAAATCCAN

AAGTGGCAAAACTGGCTGTATATGTCATTGTAGGCATAGC

AGTTGCCCAAGGTATCGTGGTGGTAACGGTTTTGTTGTCG

GTTCGAAAGGTCCTAGGCCGAGCTTTTAGCAGTGACCCGA

AAATCATCTCTTATGCTGCATCGATGATACCTATTGTCGC

ATGTGGAAACTTCCTTGATGGTCTCCAATGCGTTCTCTCA

GGTTCTTATTGCTTGCA[C/G]ACTCGACCATATGTTTCT

GGAACAAGAAAATATTGGTTTAGGCTCCTCCTAGCTTATA

TATATATATGTCCGGTTAATCCTGGTTTGGTATGGTTTAG

GGGTTGCTAGAGGATGTGGATGGCAGAAAATTGGAGCGTG

TGTAAATCTTGGTTCATATTATCTCGTTGGAGTTCCGTTA

GGGCTATTACTTGGTTTCCATTTCCACATCGGTGGTCGGG

TAATAATCTTT

764
N22902-001-Q001

AAATTCATATTATTTACTTTGTTATTTATTTTTATTTTTC

TACAATTTTTATGTATTNTGGAACAAGTATAATTTTAATA

AAATATGACGATATAAGTTTGATAATACGANAAGTTTGTC

AACCATAATAAAATTATTGTTATTCAGATACTCCGGTAAT

TTTATTTTTTGACTGAAAATTCCAGTAAGGTTCCAATCCC

AGATTAGATAATTTTATTTGCATTAATCTGTATATCAAAC

ATTACTTTTGCTAATCTATACCTTGTATAATATGTCANAA

TTGCTTTTATCTTTTCAATATCTATAGATTAAAATTTATA

ATCTTTTATAGTCTATTCAACTTGCGCAGCATCATCTCGT

AGGTGTTAAACCCTCTGGGTTTTCTGAACTTTGTTAAACC

[C/G]TCTTTATACGATAGACTAGTAGTGCGCACCAAAAT

CTTGAACATTATTTTATAAAATCAAAAACAAAATAACCAC

CACCTTATCTCGGTCTTGTATCTGCAGATGACCCGGTTTA

AGTGCAGAGGATTAGCTTTCGGCCTGAGCTGGGCCAACAT

CATGGGAGATTCNTTCTCTTTATTCTATGCCTTCAACTTA

TGGGTTAAGGTCCTTTCAGGAGAAAAAATCTATGCCCCGG

AAACTTCTATNATAGACAGAAGGTTTCAGAATCCAAATCC

AACGGTGAAAGACCCGGAATCAATAAAACAGGTTGACCCG

GTTGGAGATCTATGGATCACTCCAAGCAACAAGAAAATGG

TGAGTTACTGTTTCAACCTCACCGTCNCCGACCAGATGTC

ACCGC

765
N23063-001-Q001

GGGTAAGCTCAAGCAACTAGAGACTGAGGCAAAAGCTAAA

AAGGAGAGGGTCAAAGAGCTCGAGGATTCTAAATATGGTA

TCCGAGCTTGCATCAATGTCGTAAGGAGGGCTCTATCAGA

CCAAGGTTGCAGCTGAGAATTTTGACACCGATGTCATAAA

GGATCTAGCAGACACTCAGAGGGAAAGTAAGAAGCTCGTT

AACCGGCTTAAACTGGCGCAAAGAGCTCATAAGGAGGAGT

TCCAGAGGCTGAATGAATCAAGATCCAGAAGGTTTAGAAA

AANGGCTTGTCAAGCTGCAGNGGTGAAATACCAGTCGCAT

TTTGATCAGATATGTGAGCTTTGGCCGATTCTAAGATCGT

AAAGGAGAATGCCTTGTTGATGTCTCAGGCGGCTAGTCAG

[A/T]CAGGTTTGATCGATAAGATGACACAAAGGGGACTC

GTGGGTCATGTGGCAGATCAGGAGATATGGGTGCAGTCTC

TCAAGAATTTCTAAGTGAAGATCGACGAGATAGACATCGT

CCAACTAGATCCATAAAAGGATCTAAGNGTCTCACCCGTT

TCTGATGTCTCCAGCTGAAATAGCCTCCCTTTGGGGCCAA

TTCCTGAGCTAAATCCTATCGATGATTAAGAGGTCAGGAA

TTAGACNGTCATGTCAGAAACTGTGACCATTCCAGGATGG

TCGAGTTGAGGATCCTCCTGCTGACCAGAAGAAGACCGAT

GCAACAACNGCTGAGGAGCCTCTGGATATAGATCAGTTTG

ACTCGAAACTCGTCTGTAACTCCGTTGTGGATGTTTCGGG

CGCTG

766
N22723-001-Q001

CACTTATGCAGTTTGCGATCTTTGTGGAGACAGGGCAGTC

TCTAGACTCAGACNACACTTTGCCCACAAGAGCTCTTACC

TCTTCATTGTGGCGTAATCTCCTATCGTAGCAGAAACTAT

ACGGTCGTTTCTTCCCTGTAGCAGTGAAGGACGAGATAAG

AGGCCTGTGATCAGAGCCTTCAAAAGGGAGATAGACACNG

TTTCCTTTCGGGAAACAGTCAGACCAGGAGATATTGGCTA

GAGCTTGATCCAGTCTGCAGTGCACTCGATGAGTATGTCT

CGTTCCTCTCCAGGAGAGAAAATTTCCACGGTGTCTCAGA

TCAAAGAGATCATTCGTTGCGAGGAAAGTCCTAAAGTTAC

TGAAGGAGCTTTCCGGTCTATCTCTGCCTCCAGTTTTCTC

[C/G]GAGTTGTCAGTGATTTCGTTGAAATCTCCAGTCAT

TAACCAAGGATCGCTTCTCGAACCACCTAGCTGTGTAAGT

TGGTCCCATACTTCTTGTCTATGGGATACTTCAAGAGCTC

CATAAACGAAGGAACTGAAGAAGTTTGAATTTTTGTAGGA

GATATGGGTATNTATGTAGTTAGGAGTCGCCGTCAGCACC

TGGACCTTTACGTCAGACTTCCAGAGCAGGCAGAGCCCCC

CTGAGCTTGGACTGTTCGGTGAAACAAGGAAGTGAAACTC

AAGAAGTAAAGGTTCCAGGTCTTTAAGGACAAAGGCATCT

GGATTCTTTGTTTCCTGGATAAAGATGATATCTGGAGAGA

ATTTTTTGTTGAGAGCAATGAGCCTTTGGACTGTTCTGGG

ATTCN

767
N23049-001-Q001

AAAAAAATGGAGAATAACGACTAAAATGTCCGTGAGTAAA

CATCAAATCAACATACTATTTAGATGTTTTAACGTGCGGT

TGGATTTGGCCTTACCTCCGCCCAGATCCCCCCTTTTTGT

AACTGTGATTGCCCATCAACACCTTTCTGACCTGACCCGA

CATATTAACATAACTTT[A/T]ATTTGGGGGTAAACTAAA

ATTGGGTTTTGATCCGTTTATTTCTCAGTTACAGACATGA

CGAAGAACATAAGAAGAGAGAGAGAGAGAGGACGTACAAC

AGAGAAGACGATGACGAAGAGTCGAAGACCAGTAACAGAG

AGAGAGAGACGATGACGACGACCAGTAACAGAGAGAGACG

ACGTAGNAGAGAGAGAGAAAGACGACTTAGAACAGACAGA

GAGAGACGACAACCAAGAACAAAGAGAGAGATGACAATGA

ACGACGAAACGTGGAGAGCGAGACGAAGCCAGAGAGAG

768
N10321-001-Q001

TGGAAAATTCTATAGGTTTTATTGTAATCTTTTGTTTGAA

AATCATTTGTGTGTCCATAATTGCTCTTTGAGAGAATTGT

TTTCTTGAATGCTTTGATGGAACACTTTGCCGCATATGAA

AAGGGATGTAGAGAGGATATGTGGCCGGAGTACCGTTTGC

AAAACAACCATGGCAAGGCACAAACACCATGTAAGTACAC

[A/T]TCTTTGTCAATACCTAACGAGCCTTCAAACTGATA

TCTCATTGAATTTCGTATTAGGCTTGCCTAGAATCAGGGA

AGGGAAGGACTCAATCTATGTAGTCGTACATAAAATGACA

CATTTTATTCCTTGTTAAAACGCCAACGAATAATTGAATC

GCACGCGAGAACACTTTCTTTAAAAAGTATTTTGACTCTA

TTCCA

769
N15374-001-Q001

CCTAATTTCCCTAATTTTTACTATGTAATACACCAAACTA

TAAAAAGTTTTATCAAACGGACAATGTTGAGATTTATTAT

TCTAAATAGATGTTTGTCTCCCTACATTTTCATATTTTGT

ACCGAAACATTTATTTAAAAGATATGCAAAGTAGTGTGTA

GCCAAATGCTATCATGTTTAAATTTGCCAAGAGCATATCC

ACCTATACAGTGCAGATAATTGTTTACTTTTGTTAAGCTA

CAAAAAGATGTTAAGCTGTAAGAGTGATTCGCATGAAAAA

TCTATTACAATAAAAGAGAGTTACCTCTCTCTAYATGYCA

CCACGTCATCAGGAAATATAAGACGTTTTTGGACACTTGG

CACTTCACCAAACCAGCCCGCGTCTGATTTGATTTTGTAT

[A/C]ATCCGGTTTWTCTCATGCCTCTATCCTGGACTGGG

CCATTATTACAACTTTTCCTTGGGCCTTTRAAAACACTAT

GACAAGCTCACTAGAATTTTATGTTTCTTCTTCTTCTTTG

CGGTTGCGACAGTGGACAATTTTTTTTTCAGAACTAAAGG

GACGTTCCAATCTCTCCCGTAACGTATCAATACTTCATTA

ATCTGTTCTTAACTCTGAGGTTTTGCGTGATTAGCCMACT

CCTCCCACTGGAAAGCATTCAATCGACATCTTTAATCTAA

CTTTAAGATGATATYGTTTATTTTTCTTCKTGACCTTTGT

GATTTCTCCATCTTCTTCCACTCACTTCATCTGTTATTGA

CATCATAATTCCTCTTCAACCCYCATTCTCTCTATAGAAA

GCCAG

770
N22802-001-Q001

GCATCTTGGCATTACTAGTTTGTATTTTTATTTTGCAAAG

AGAACCAAANCTTAGTTATTACAATATGATTATCTTGCAA

CAGTATTTCCAACAGCGTTATTTTAGAGTACTAATACTCA

TGGCTGCTTTTTGGAGTGTCTCTCTGCAAAGTTATTTAGC

TGGAAAATCAGTTTATGATGAGGTCAGTATCAACCGCAAA

GGTGATACATAACCAGTTCTTAAGCAACAGCTCGACCTGC

CAAACCACCATCTGTTATTTTAAATGTCAAACAAAGCTCG

TTAGCAAGACACAAATATANTTACCGGGTAGAATGATTAC

GGCAACTNTAAGCCTGCAGCAAACCAACACGAAGTCTCAT

TGTTCACATTAATGAAGAACACGCCGCCTTTTCTGCTGAA

[C/G]TTGTACTTCCATTTGTCTTCAAAGAAATTGGTATG

AAGACAATAAGCAAAGATGTGATGGAGTCGTGCTTTAGAA

ACAATTGTCAAAGCCCAAAAACTAAGAACAACAAATGCCA

AAATTGTGAAAGAACAACGAACTGGTTTTGCNAGCCAGGA

AATCTGATCATGACAGTTGTCCAACACAGCTGACGCCAAC

CGCTTGCATTGTGTGTTTTGAGAAACAGCCGCNTCGTCAA

ACATTGTCCATGAGAAGAGACATCAATGTGTATGAATGTG

GTGGAGCAATGAAATAATTACCAAGTGATAAACTCCCAGT

GGATATGCTGATAATTATTATGAAANTCAAATTGAGAACC

AGCTTTCTCAAACCCATGCTCTCAGTTTCTTGCCACCAAA

CATTA

771
N22803-001-Q001

TAGATTGTTATGTTTTGATATCATCACAACCAAATAACAT

GCCATGTATTTCCATCATCATTATCGTTATCATTATTTTG

CCAACATTATTATCAATTTATCACTGTTTTGTTAAATAAC

GTATTTCCATTATACGGTTCCTTTGTTGTTGATATTATAT

AATATAAAAGGTCACATGTATTACTATTATCGAACATGAC

ATAATATAAGGATTATTAGAGCGGAAGCTAAATAAAATTT

NGCTNGATATTGTAATGGTGCTGCATCATGTTTTCTTTTT

TGGTAAAATTTTAATTGATTACACCAAAATGTTCGTATGT

TACAACTTACAAGGTTGGGACTGAAATGGCTCGTTGCTTC

TCTTTCCAAAGAGGCATCTTAGCTCTCCTAAAATCGCTAT

[A/T]CTTGGGCAGATGATCATCATGAGGATGGTTTCACT

TTTGATTTCCTCCTGCTCTGTTTTATGCTTTTTGCCCACT

TGTTTTGGGTTATTATTGNCTATTGGCATTTGTTTTTGCT

TGAACTACCTCTAGTCGTTTTGACTAGAATACTTTATGAT

TTTTAATCAATGANAAAAAAAACAGTTACAAAAGGATATT

AATAGCAGAAAACTGGATGAAACTAAACAACAGAGCTACT

CAACCACATCTTAACTTTGAACAAAGACTAGAAGGTAGAC

AGTAGAGAGGGAAACCAACGAAACAAGCTTAGACCAAGGA

ATTGGTAAGGACCGCAAGAACAAATGGGTCGCAGCGAGAA

AGAGAGAGNTGGATGCCCTCAAACCATGTTGCAACACCTT

CCTAC

772
N18929-001-Q001

TAAGACATTGTTTTATAATTTATTCAAACAATTATTTCCA

TTTTAACCAAAGTTGAGCAATTATTGTGGAAGTTTGAAAA

ATTGGTAGATTTGTCAGTAATGTACTATCTAAACCCATCA

ATAGAGTGATGGTTGAAGAGAGTATTGGTTTCCACCCGCA

ATGCAAGGAAATCAACTTATCTCATTTGAGTTTTGCCGAT

GATATTGTGGTTTTTAC[A/G]GATGGTTCTCCAATGTCG

CTTCAGGGTACTCTAAAGGTCTTTGAAGACTTCACTGCTA

TGTCTGGTTTGCAGATAAACATAGCAAAGTCCACGGTCTT

AACTGCTGGTAGAGGAAAGCATGTACTAGAGGATGCAGCA

GCTGATGCGGGTCTCTCCGTTTCTGCCCTGCCTATTAGGT

ATCTTGGACTACCGCTTACCACCAAGATAATGTGCAGGGA

TGATTATGAGCCGCTTATTACTAAGATCAGGAACYGGTTC

CTCTCTTGGACGAGCAAAGCTCTCTCATATGCAGGTCGAT

TACAACTTATAAAATCAGTTATTGCTAGTATCACAAACTT

TTGGTGTGCTGCGTTCTGTCTTCCGCAAAACTGTATAGCG

GAAATCGAGAGTATGTGCTCTG

773
N16041-001-Q001

CCATTAACATTAGGACATTACATCTTTTCTTTTTTGTTGC

TTTTTGTCCGTACGTTATCGTCGTCCGTTTGAAACTTTTT

AATCTTATCCCTAATTTTTTATCATCGTAAGCAAATCTAG

TTTTATGTTCGAGATATGATGGTTATTGAAATATATATGG

GTTGTGCTTTCAATAATCTTGTCCATAGTTTTTTTGTCAA

AAAAACCCATCCATAATCACCAGCTACCGCCACAAAATCT

TTATACGTTAGCTTGTTGCAGTGAACTTTAAAAAAAATAT

TGTTGTTATTTCAATAAACACGGACATGGGCCATTATGCT

TAATAGACTTTAGTCTACAGCTTTATCTCTCAAAACCCAT

AGTAAAACACAAGTCTAATGATACAAGCT[C/T]AGAGCC

TAAGCATTTACAAATAGAAATTTAAGGTTAACTTTTATAA

TTGTTATCAAACATTTCATAGGCAAAAGATAGAAAATGGA

CAAATTAACAGGTAGTATACGATTTCTACATTTTAGCAGT

ATTTTATATTAATCATGCTATTAATTTAGCTGATTAAATC

ATTTTATGTAGTTTATCTTCTTTTTATCAACTTATAGTTT

ATCTTTGTTGGCAAATAATTTGTTTTCTTTTTAAATCAAA

ATCGTTGATTTTATCCATGGTAAACTTTGAGTATCTAACG

CATTATAATTTTTTAAGGTCTGGGAAAATAAATCGAATCC

AAAAATCCAAGCCGAACCCGATCCAATAAAAATGAATATC

AAATGGATCTTATTTTATGATATTTTGGATTATG

774
N18401-001-Q001

CTTGTCTTTCTTTTTTGATAGAGAGAATATTGCTCTGCTT

TCTKATTAGAGGCTTTGATWACATACTTTAGGTGGTAAAG

GGTCTGATCCAGATTTTCTGAAGAGGTAACTTGTATTGAT

TACATACTTTAGATCCAGGGTGCGTTTATAACTGATACCT

TTGCTTGTGGTGGATTTTCTTTTGCCAGCGAAGAGCTGTA

AGTTTTGTACTTTAGTCAATTTTGCAGCTCTACAGAACCT

TCTAAGAAGCTACCTCACTAGACAGGTGATTYTAAAGATT

CTCTTGYGTCTGCTACTGTTCACTCTTAGATCTCAGTCTT

TCCTGTTTCTGAAGTTACCTTTGCCAACTCTACTTATTCT

CGTCTTTCTCGCTTTCTCACTTTTTTTGTTCTTAGCTTTG

[C/T]AGACGGGTTAGGTTAGTAAAAGCCAGTGGTAAAAA

ATATGACTCTGTAGAAAGGAGTATTATATCAGTAACAAGG

AGATGCCTTTTCATTCTGAGGACGACAARAGTGAAGACTA

CCTCTTCAAGATTCTGCAGCTCGGAAATCAAATTTTGCTC

GCAAGATTTGCTAGGGATTAGTTATACCCCAGTTAAAAGT

CGACGACTAGAGTGGAGTTTCAGACGGAGATCAAATAACA

GATATGGGACACAGAGGTGCTGTTGGAGCTCTTCTGGTTC

ACAACATCAGCAGACAGAAAACTTTTCAGAGCATTGGTAG

ATGGCTTTAACTAGCTGCATAGTAAGAAAACTACTCGTGG

GTAACAAGTCGGATCTAAAGTACATAAGCACTAAACATCG

GAAGG

Table 8 below sets forth exemplary sets of forward and reverse primer sequences for each polymorphic region in the above-listed markers. For genotyping carried out via the KASP genotyping assay (LGC Genomics, Boston Mass.), two forward primers with alternative 3′ ends are shown. For genotyping carried out via the TaqMan SNP genotyping assay (Life Technologies, Inc., Grand Island N.Y.), additional differentially dye labeled probes (VIC and FAM) are shown as well as the forward and reverse primers.

TABLE 8

List of SNP markers and primer sequences

used for amplification of loci

associated with resistance to shatter

Forward

Reverse

Primer
Seq
Primer
Seq
FAM
Seq
VIC
Seq

Marker
SNP
Sequence
ID NO
Sequence
ID NO
Probe
ID NO
Probe
ID NO

N20003-
G/T
ACGTCGGTACTCTT
157
CCCTTACTCTAGG
158
AACTCAGGAATT
159
ACTCAGGAATGA
160

001-Q001

TTCTGTTGTC

ATGGGTGATACA

AATGAT

ATGAT

N03491-
C/G
TTCGGGGATTAGA
161
TCATCAGTACCGT
162
TCGATCTCTCAC
163
TCTCTCACTAGG
164

1-Q1

GCTTTCC

TTGATTTCG

TACGG

GACTAC

N0017NR-
A/G
TAGCTATTCATAAT
165
GCAGTATCATATG
166
ATTTTACACCTT
167
AATTTTACACCTT
168

001-Q001*

TAATCAAAAAGGTG

TTCCACTCTAGAG

AGTGCTGTGA

AGTGCTATGA

GTCC

ATG

N10336-
A/G
CCGCTTAGCTCTCT
169
AACAGCACATGAC
170
CTGAATGTGGTC
171
CTGAATGTGGTT
172

001-Q001

TCGGTTATTTT

GAGATGACATAT

TATCAC

TATCAC

N23133-
C/T
AACAAGCCCTCTC
173
ACCACCGCTATG
174
ACTAGTTGATTA
175
CTAGTTGATTGT
176

001-Q001

ATGTACAATGT

CATCAAATCT

TGAAGAAA

GAAGAAA

N16487-
G/T
CCGTACGATGAAT
177
GAAACGAATAAAT
178
AACAGAGAGATT
179
ACAGAGAGATGA
180

001-Q001

CAGACGAAAGTA

TATAGAACGAAGC

AATTGG

ATTGG

TACTAATGG

N23426-
A/G
TGATGCTTCCCTTC
181
ACATGTTACCAAT
182
TTTTTGTGCAAC
183
CTTGTTTTTGTA
184

001-Q001

AAAGAAAGACA

CAAAGCCTATATT

TTC

CAACTTC

ACATTTACA

N05671-
C/T
CCCATCAAATGAAA
185
CTATGGCGATGTT
186
ATGGTTCCATAA
187
CATGGTTCCGTA
188

1-Q1

AGGAGGA

GCTCAAA

CTC

ACT

N12643-
A/C
CCTCTTTGAGCTAA
189
ATCCAAGGGACA
190
ACCCATGGTGG
191
CAAACCCATGTT
192

001-Q001

CACTAGTCACA

AAATGCTACCAA

TTCT

GGTTCT

N05943-
A/C
TTGCAAAACTCCAG
193
AAAGCTTGTGTCG
194
ACGTACATAACA
195
AACGTACACAAC
196

1-Q1

GTCAGA

AAGCAAAT

CGCTT

ACG

N06007-
C/T
CACAATAAAACCAG
197
GCAACGAACCAA
198
CTTTTCCAAATG
199
CTTTTCCAGATG
200

1-Q1

AGCTTCCA

AAATCACA

ATTACAC

ATTAC

N10105-
A/T
CACCTGACCGAAA
201
TCTTTCTGTAAGA
202
AAGATAAAATTA
203
AAGATAAAATTA
204

001-Q001

GAAACACTAGTT

ATTATTCTTCATTT

CTGTTATTAGC

CTGATATTAGC

AGCTATGCT

N08181-
G/T
TCTTTTGTTGAATG
205
CGTCAAAAGAAAA
206
TCCAACTTCCAA
207
TCCAACTTCCAA
208

1-Q1

GGGATTTT

TAGAAAAAGACAG

ATTA

AGTAT

N06675-
C/T
AACGACATAGACG
209
TCTCCATCACTTC
210
CACAAGAATCCA
211
ACACAAGAGTCC
212

1-Q1

ATCGTTGG

GTTAGTATTCG

CAACT

ACAAC

NO01K1-12-
A/G
GAGAGAAAGAGTG
213
GCTCTCTGAAGAT
214
CTCCACGCCTTA
215
TCTCCACGCTTT
216

001-Q001*

GGAAAGAAAAGAG

GGGAAGAAATGA

GCT

AGCT

T

N29313-
G/T
ACAATCTGGCCAC
217
GTTATTCATAAAG
218
AGAGAAGAATCA
219
AGAAGAATCAAC
220

001-Q001

ACAGACG

CCAAGGTTTTCAC

ACTAGAAGTA

GAGAAGTA

TTCT

N88512-
A/C
GAAGGTGACCAAG
221
GGTGGAATAAAGT
223
0

0

001-K001

TTCATGCTCAACAA
and
GTTCTTTGACGAA

CTAGCAAATCAAAA
222
CTT

GTGACC

and

GAAGGTCGGAGTC

AACGGATTGATCAA

CAACTAGCAAATCA

AAAGTGACA

N88514-
C/G
GAAGGTGACCAAG
224
TCCCAACCAATTG
226
0

0

001-K001

TTCATGCTAATGTA
and
GTCGCCAGTAA

AAGGAGAAGAAGA
225

AGAGCAC

and

GAAGGTCGGAGTC

AACGGATTAATGTA

AAGGAGAAGAAGA

AGAGCAG

N88515-
A/G
GAAGGTGACCAAG
227
GAGGTAATCTACA
229
0

0

001-K001

TTCATGCTAACAGA
and
CCGCCCCTTATA

TCATTCTAACTCAT
228

TGCCG

and

GAAGGTCGGAGTC

AACGGATTGTAACA

GATCATTCTAACTC

ATTGCCA

N88516-
A/T
GAAGGTGACCAAG
230
GTACCATTTTATT
232
0

0

001-K001

TTCATGCTGTCGTG
and
GTAATGAACTATC

AAAAAATAATTTTC
231
TTTT

TATATTTCCAA

and

GAAGGTCGGAGTC

AACGGATTCTGTC

GTGAAAAAATAATT

TTCTATATTTCCAT

N88517-
A/G
GAAGGTGACCAAG
233
TCAGCTCCGGTG
235
0

0

001-K001

TTCATGCTGTTGAG
and
AAGAAAACAGAGA

CTGATCTTACAGGT
234

CCATTA

and

GAAGGTCGGAGTC

AACGGATTGAGCT

GATCTTACAGGTC

CATTG

N88518-
A/G
GAAGGTGACCAAG
236
CTCAACTAGTTTC
238
0

0

001-K001

TTCATGCTTTAAAG
and
TTTTAATTTCTGTT

TTTACTTTTATACAT
237
GAA

CACGAGATTAA

and

GAAGGTCGGAGTC

AACGGATTGAGCT

GATCTTACAGGTC

CATTG

N88519-
A/G
GAAGGTGACCAAG
239
CAGCCCATTTCCA
241
0

0

001-K001

TTCATGCTGAGATA
and
AAAGTTTTTGGGT

ATTCAAGGTGATTA
240
TTT

AGTGATATTG

and

GAAGGTCGGAGTC

AACGGATTATGAG

ATAATTCAAGGTGA

TTAAGTGATATTA

N88520-
G/T
GAAGGTGACCAAG
242
GAGATCTGATCAA
244
0

0

001-K001

TTCATGCTGAATCG
and
AATTGACTTTGCA

ATCAGAATCTAAAC
243
CTTA

GGTTATG

and

GAAGGTCGGAGTC

AACGGATTCGAAT

CGATCAGAATCTAA

ACGGTTATT

N88521-
A/G
GAAGGTGACCAAG
245
GTTGGAAGCTTTA
247
0

0

001-K001

TTCATGCTCCAATA
and
ACGGTTATGGAAT

TAGAAAAAAACAAA
246
GTA

ACACTCTTCG

and

GAAGGTCGGAGTC

AACGGATTAAACCA

ATATAGAAAAAAAC

AAAACACTCTTCA

N001KFE-
A/G
CAACGCGTTGCCC
248
TTGAAGTACTAAA
249
TTTAAAGAAGGA
250
TGATTTAAAGAA
251

001-Q001*

GAAAA

GTGGATAGCGGA

AAATTC

AGAAAATTC

AAA

N88522-
A/C
GAAGGTGACCAAG
252
CTCAGACTTCATT
254
0

0

001-K001

TTCATGCTTTTATT
and
GCAAAGCTGAATA

TTCTTCTGGATACA
253
GAA

GATAAGAATAA

and

GAAGGTCGGAGTC

AACGGATTTTATTT

TCTTCTGGATACAG

ATAAGAATAC

N88523-
A/G
GAAGGTGACCAAG
255
GGAGTCCGGACT
257
0

0

001-K001

TTCATGCTACAAGT
and
GAAATGCAGATTA

CATGTATTTGTAAC
256

GACTTGAAAA

and

GAAGGTCGGAGTC

AACGGATTCAAGT

CATGTATTTGTAAC

GACTTGAAAG

N88524-
G/T
GAAGGTGACCAAG
258
CAATGTTTCTGAA
260
0

0

001-K001

TTCATGCTCTGAAG
and
CAGAAACTTCTCA

AGCTGATGTCTTTT
259
GTTT

GGTG

and

GAAGGTCGGAGTC

AACGGATTCTGAA

GAGCTGATGTCTTT

TGGTT

N88525-
A/T
GAAGGTGACCAAG
261
CCTTTTGTGTTAT
263
0

0

001-K001

TTCATGCTCAATAG
and
TAAAAGCGGGAG

CCTTTTAAGAGTTT
262
TGTT

TCTAACCA

and

GAAGGTCGGAGTC

AACGGATTCAATAG

CCTTTTAAGAGTTT

TCTAACCT

N88529-
C/T
GAAGGTGACCAAG
264
GTCCCGGATATAT
266
0

0

001-K001

TTCATGCTCCTCAA
and
AACTGCTGTATAC

CAGCCTGAAAAATA
265
ATA

TAACATAAT

and

GAAGGTCGGAGTC

AACGGATTCCTCAA

CAGCCTGAAAAATA

TAACATAAC

N88530-
C/T
GAAGGTGACCAAG
267
GGTATCGTTCGTG
269
0

0

001-K001

TTCATGCTCATTGT
and
AGAGATTGGCTA

TGCGTATGACAAG
268

CTCGT

and

GAAGGTCGGAGTC

AACGGATTGTTGC

GTATGACAAGCTC

GC

N88531-
A/G
GAAGGTGACCAAG
270
GGCTTTGTAAATT
272
0

0

001-K001

TTCATGCTCAATAA
and
TCCGTTTTCAAAC

GACAAAAATTCAAA
271
GTTT

ACAAGAAAAAATG

and

GAAGGTCGGAGTC

AACGGATTCAATAA

GACAAAAATTCAAA

ACAAGAAAAAATA

N88533-
A/G
GAAGGTGACCAAG
273
ATCTCGCTGATCC
275
0

0

001-K001

TTCATGCTGATTAC
and
AGTTTGTTCTTGA

ACGCACAAATTCG
274
TTT

AGAAATG

and

GAAGGTCGGAGTC

AACGGATTACACG

CACAAATTCGAGAA

ATA

N88535-
A/C
GAAGGTGACCAAG
276
GAACCACTGTGTT
278
0

0

001-K001

TTCATGCTTGGTTC
and
AACAAAACAACAA

ATTTTATTTAATGG
277
CGTT

ACCTTTGC

and

GAAGGTCGGAGTC

AACGGATTCTTGGT

TCATTTTATTTAAT

GGACCTTTGA

N88536-
C/G
GAAGGTGACCAAG
279
CCTTTCTTGCTTC
281
0

0

001-K001

TTCATGCTCCCATT
and
TCCAGATACAATT

ATCATTGTGCAATT
280
TGTT

TCCGAG

and

GAAGGTCGGAGTC

AACGGATTCCCATT

ATCATTGTGCAATT

TCCGAC

N88537-
C/G
GAAGGTGACCAAG
282
TCCCCTTCCGATC
284
0

0

001-K001

TTCATGCTGTTTGA
and
CTCATCATCTTA

TCCTCAGTTCGCTC
283

GTC

and

GAAGGTCGGAGTC

AACGGATTGTTTGA

TCCTCAGTTCGCTC

GTG

N07541-
C/G
TTCTTCCATCGTCT
285
TGCAATTCAGTGT
286
CTCCCACCTATC
287
CTCCCAGCTATC
288

1-Q1

CTCCTGA

TTCGATTTT

AAA

AAA

N23413-
A/T
ACTTAAAGACCTCT
289
GCTGGTAATTGAA
290
CAAGCCAAAGT
291
CAAGCCAAAGAC
292

001-Q001

CCTTACTCTCCAA

AAGGATTGATCTT

CTTAAC

TTAAC

TGA

N08344-
A/C
CTCGTCTCCGTTG
293
GGCAACCCTTTCA
294
ACAGGTCTCCTC
295
ACAGGTCGCCTC
296

1-Q1

GTGGT

AAACAGA

CAC

C

N23533-
C/T
GGAAAGTTGAATTT
297
CATTCCTCAACAA
298
CTTGATCATCAC
299
TCACGAAACTCG
300

001-Q011

GATTCGCC

CAACCCCTAA

AAAAC

ACTGG

N14649-
A/C
TCCTGTACACACAA
301
GGGTCTTTGAAGT
302
CAAAGAGCAAC
303
CACAAAGAGAAA
304

001-Q001

ATTCAAGACATCA

TTAATACTTGTTTA

AACG

CAACG

GTTCTC

N23310-
A/G
TGCGCTTGTTTCTA
305
GGGTCTTCCTAG
306
CCTGAAACCGC
307
CCTGAAACCACA
308

001-Q001

AGACTCCAA

GAGTGCACTA

ATTGC

TTGC

N10526-
G/T
CAATCTCAAACTGC
309
TCCTGTCATGTGA
310
CCATCACGTTAC
311
CATCACGTGACT
312

001-Q001

CACTCTTGGTA

GCTTTGCA

TACCG

ACCG

N23373-
C/G
TTCTGGCCATTCAC
313
AATTAAAGCACCA
314
CCGAGTGA
315
CGGAGTGA
316

001-Q001

AGTTAATACGT

ACTATCTTACAAT

CAACGAATTGAA

CAACGAATTGAA

GTTGAAC

N23353-
C/G
CCTATCTGGAAGTT
317
CTCAACAAGATGT
318
AAGTTGAGCTAA
319
AAGTTGAGCTAA
320

001-Q001

TGAGCTTGCT

CACCGTCATAGA

GAATATA

CAATATA

N23206-
C/T
CAGTTTTTCAGTTT
321
GATAGTTCATGGT
322
CGAAAAGTAAAC
323
CGAAAAGTAAAC
324

001-Q001

AAGAAATATATAAA

AGACCTCAGCAT

TAAACCGA

TGAACCGA

CCGCTTAGAT

N11025-
A/G
CCACTGCACCCCG
325
GGGACCAGAGAC
326
AACATCTGCGTC
327
TTGAACATCTGC
328

001-Q001

TAATCT

GTCTGAG

GCTAG

ATCGCTAG

N09969-
C/T
CGGCTCCAAGTTG
329
GCTATACTTACGT
330
TTATCAAACAGA
331
ATCAAACGGAGT
332

001-Q001

CTTTTAGTTTG

AAAAAAAAGCCTT

GTAAATG

AAATG

GAGA

N09882-
A/C
AGATCTTGTTGACT
333
AGGGTCTTGGCA
334
TCGGTGACACTT
335
ATTTCGGTGAAA
336

001-Q001

TCTCGGTTTAACTC

TGTTCCTTTT

TTA

CTTTTA

N10389-
C/T
ACCTTTTTATATAG
337
GTTGACTCTGTGA
338
TTTGCTTATGTT
339
TTGCTTATGCTA
340

001-Q001

AGTAGTCGAGATG

AGTTAGATGGATC

ATTCTC

TTCTC

GTTTGA

TAAA

NO9940-
A/G
AAATGTTATATTTT
341
AAAACGTGGGCTT
342
ACTTGCGACGG
343
TGACTTGCGATG
344

001-Q001

CGTTTAATTGTCTG

TTTCACAGG

TCAA

GTCAA

CTGGTT

N23409-
C/G
AACCAACACAACTA
345
CCGCGTTTTAGAA
346
CAATGAGAGTCT
347
CAATGAGAGTGT
348

001-Q001

TTACCCAAACCT

CATGGAGTAGAA

CCACTTT

CCACTTT

N23119-
A/G
CAATGTTAAATTCT
349
GTAGCACTTGAG
350
ACTGCAGGTTCA
351
AACTGCAGATTC
352

001-Q001

GGTGGCCAACA

GAATAACCCTGAT

CCG

ACCG

N09861-
A/G
AACTGGATGATCG
353
TCGATTGTTCATA
354
CCTAATTTAGGA
355
CCCTAATTTAGG
356

001-Q001

TTTACCACTGAAA

GCTGCCTTTTGA

TATGTCCCAC

ATATATCCCAC

N04807-
A/G
TATCTTCACCGACG
357
GAAGTGCCGACT
358
CTCTTTTGTTTC
359
ACTCTTTCGTTT
360

1-Q1

GCTTTC

CACCAAGT

TCC

CTC

N06778-
C/G
CGATGCACCATCA
361
CCTCTAATTTCAC
362
CAGTTCTCCTTT
363
CAGTTCTCGTTT
364

1-Q1

TGTGAG

TGACACTCTTGA

CCTATT

CCT

N09897-
C/T
TGGTAGAGCTGAA
365
AGCCAACCGCTTA
366
TTCGGTATGACA
367
CTTCGGTATGAC
368

001-Q001

AGATGATGTTCTC

TTACCACTATG

AGATAA

GAGATAA

N10499-
A/C
TGGTCTCTGCATCT
369
CGATATACCAAGG
370
CCAGAAGCATTT
371
AACCAGAAGAAT
372

001-Q001

TCGAATCTG

TTGCTGATGCT

GC

TTGC

N23447-
A/G
GAACCTAAACCAAT
373
TCGAACTAAAACT
374
CTTAGGGTGTA
375
CCTTAGGGTGTA
376

001-Q001

GGATAGAAACTTG

AGATGATTATGAT

GGTTAAT

AGTTAAT

ACT

TAAAGCAGTAAA

N19834-
A/G
GTAGACTTTTCCAA
377
GGTGTTTATTGAA
378
CAAATACTTTCA
379
CAAATACTTTCA
380

001-Q001

GCTAATCTTCAGAC

GGCACTAGAAGA

GTATCCC

ATATCCC

AA

TCA

N23362-
A/G
GGCCTTGGGATTA
381
CGAGCCTGCCTG
382
CAGATCAACCTA
383
CCAGATCAACTT
384

001-Q001

AGAATCTTTCGA

AAAGAAAAGTA

AGGCA

AAGGCA

N23266-
C/G
GCAGCTCTTTGTTT
385
GTACCGATGAAAC
386
CACATCAATTCA
387
CACATCAATTGA
388

001-Q001

CAAACCCATTA

GCGAGAAG

GTTTTT

GTTTTT

N19862-
A/C
GTGGCACAAGATG
389
AAGTTTCATTAGT
390
AAGATCCGCAT
391
AAGAAGATCAGC
392

001-Q001

CGATGAG

TTTGATGGGTAGT

GGCT

ATGGCT

GCTA

N22187-
A/G
CATCTGGATCACC
393
CCGTCACTGTCTC
394
AACGCCCGCCA
395
CAACGCCCGCTA
396

001-Q001

GGTTGGT

AGGAGGAA

GAGA

GAGA

N08651-
A/T
GATAACTATATTCA
397
GGACCATCTGCG
398
ACATAAGTTACC
399
ACAAAAGTTACC
400

1-Q1

TCGACTCCCAAAC

TGGTAGA

ACCAGTT

ACCAGTT

N23296-
A/G
CTTATACACTTAAG
401
CCACAACAAACAG
402
CTTCGCATCCTA
403
CTTCGCATCTTA
404

001-Q001

TCTTTGAATTTCAA

CTCTACTTACAAT

ATCG

ATCG

ACTATGCA

AC

N17314-
G/T
CGCTTGAGAGCTT
405
GCCCATTTAAGCA
406
ACTGCATGCAAT
407
CTGCATGCAATG
408

001-Q001

TTAAAGAGAGATAG

CCATACCAATC

TATATAT

ATATAT

T

N20380-
A/C
AAACTGTGAGTCC
409
GTATGGCTTCTTG
410
CACAACCTGAAC
411
CACAACCTGAAA
412

001-Q001

CTGGAGAGA

ATTAAGTTTGAAG

TCTACT

TCTACT

CA

N05490-
C/G
CAGATTGGTAATG
413
CATGGATTTTCCT
414
TTGCATATCATC
415
TTGCATATCATC
416

1-Q1

GTTCCCTGT

GCCCTAA

CCCA

GCC

N18849-
G/T
CCATGCAACGTAG
417
CGAGGTCGAATT
418
AACTTCTCTATA
419
TGAACTTCTCTA
420

001-Q001

GAAACAAGTATC

GTTTTGGTATGTG

TTTTCC

TCTTTTCC

N08200-
C/G
AAGAGAAGCGTCT
421
TCGACGTCTATCC
422
ACGCAACCACC
423
ACGCAACGACC
424

1-Q1

CCTCGTTC

CCAAGAT

GAT

GAT

N19827-
A/G
GAAAAGATAATTAA
425
CCAAACATTTATT
426
AATAGCCACGAA
427
AGAAATAGCCAC
428

001-Q001

ATAAAACGAACCAA

ACTAGGATTTTCC

TGAA

AAATGAA

CAAACAACA

TCCCT

N001R9W-
A/C
CGGAGACCTTCAG
429
GTTCCCAATGTAA
430
ACGTGATAACCA
431
ACGTGATAACAA
432

001-Q001*

TGTGTTAGAC

GCACAAAGGTT

ACTAC

ACTAC

N08264-
C/T
TCAACAACTGGTTC
433
CCAGTGAAGATG
434
CTACCACAGACT
435
TCTACCACAGAC
436

1-Q1

ATCTGGAA

GATGCAAAG

TATC

TTGTC

N23132-
A/G
CTTACTGATCATGT
437
GAGCCATTGTTTG
438
TTGAGTATGCAG
439
TTGAGTATGCAG
440

001-Q001

TAGTTGGCAGTTTT

TAAGAGAAATTGA

GTATGTC

ATATGTC

ATATGA

N03615-
A/T
CGTCTCTGCTTACC
441
GCAGTAGACAAC
442
ACATAAACATGG
443
ACATAATCATGG
444

1-Q1

TCACTATGAA

AGCTTTTGGAA

TCTGC

TCTGC

N001RWT-
A/G
TGATTTGCCTAGAC
445
GGTTAAAACATGA
446
TTTGTCTTTGGT
447
TGTTTGTCTTTG
448

001-Q001*

CAATTTTTAGAACA

ACCGTTAAGCTGA

TGTAGTTG

GTTATAGTTG

C

A

N08465-
A/G
TCCTTCTCCTCCGA
449
CCTCCGTATCCGT
450
ACGTTAAATCGT
451
CAACGTTAAGTC
452

1-Q1

GAAAGTT

AGACATCA

TTAGTTG

GTTTAG

N1C774-
A/C
CACTAATTTATATA
453
GATGGGTGCCAT
454
TTTCTAACATCG
455
CCATTTCTAACA
456

001-Q001

GAAAATATGTAAAA

AATAATCTATTTAT

TGGAAAT

TAGTGGAAAT

CTTTTTCCATCA

ACTTTTTTTGT

N17035-
A/G
TGTGAAAATATAAG
457
TCATGTTAAGTTT
458
TCAATCCACGTA
459
ATCAATCCACAT
460

001-Q001

TTTCACATCGAGAT

GGATTTATCATGA

TTACC

ATTACC

CGA

GTTTTCAAATT

N20834-
C/T
AAACGCCAAAACT
461
ACCGTAACCAGCT
462
ACGTGTCGTAAT
463
ACGTGTCGTAGT
464

001-Q001

GGTCATCTTG

CTCTCTGTAATA

TCATGT

TCATGT

N22903-
C/G
CCGCCAAGTTTTAA
465
TGTGCAGTTAATA
466
TGAAGTTTCAAC
467
TGAAGTTTCAAG
468

001-Q001

ACAAAATCAATAAA

GTTGTATAGTGTA

CACCATT

CACCATT

TCATATT

TCTTTG

N09920-
A/T
AGCCAATTTTAAGT
469
CGGAGGGATTTC
470
CCTCTCTGAGTT
471
CCTCTCTGAGTA
472

001-Q001

AAATCATTGAATAT

GAAATAGTATGTG

AATTGA

AATTGA

TGTTAGTGT

T

N22822-
C/G
GTAGGGTATCATTT
473
CGCGTGCTTAGT
474
AGTGCTTGTGTC
475
CAGTGCTTGTGT
476

001-Q001

TATTTTTCTATCGA

GGTTACCA

TCATG

GTCATG

CTGAGT

N22688-
C/G
GCATGAATAGTAGT
477
AAATTAACAACAG
478
TTGAGAAGTCTC
479
TTGAGAAGTCTG
480

001-Q001

AGCTTTCTTTTTTT

TGTTACCTAATTA

GAAAAT

GAAAAT

AAATGTGTATA

TTATAAAGAATTG

A

N10074-
G/T
CGCCAACTACACA
481
GAAGCTGTGGAA
482
CCTCAAGTCTTA
483
CTCAAGTCTGAA
484

001-Q001

AGAGCATAATTT

GTTACAATGCAAA

AACTT

ACTT

N10057-
C/T
TCCACAATCTCTCC
485
ACTCGGGTCTGTA
486
CGGAGACAAGG
487
ACCGGAGACGA
488

001-Q001

TTCCAAATTCAC

CGTTGAGA

AAG

GGAAG

N10086-
C/T
TCACCTCTCATGTT
489
CTGAAGCAATCAC
490
AACTTCTCACAT
491
AACTTCTCGCAT
492

001-Q001

GCTATAAGGTTATC

AAATTGAACTAAA

TGCC

TGCC

T

GGA

N11084-
A/G
TTTTCTCACAAAGA
493
AAGCTTGCAAGC
494
CGCAACACAAG
495
ACCGCAACATAA
496

001-Q001

TCATGTACTCATTA

CAAAAGAGATG

ATT

GATT

CTTCTT

N22814-
A/T
CTCCTTCTAGAGTC
497
GCAGAAAATTTGG
498
TTTGCGTCATTA
499
TTTGCGTCAATA
500

001-Q001

TGGGAATCGAA

ACCCCCATTAAA

AAAT

AAAT

NO1564-
A/C
AAGTTCATCTACAC
501
GAGTTCATTCCAC
502
CCTATCAAAGCA
503
CTATCAAAGCAA
504

2-Q1

TTAATCCGACACA

AGATCTGACCAT

CGTCT

GTCT

N12902-
C/T
CGATCGAGAGAAA
505
CCGATAAAACAAT
506
CCCAACAAAAGA
507
CCCAACAAAGGA
508

001-Q001

CTTCGAGACTT

CAACACCAAAAAC

GGCA

GGCA

AATTAA

N21144-
A/C
AGTAGAATACTTAA
509
CCTGGTGGCCAT
510
ATCCCCACATGA
511
CATCCCCAAATG
512

001-Q001

TGTTTATAATCACG

GTATAATATCACA

TGC

ATGC

AGATATAATTGTTT

TCA

N07534-
G/T
TAGAGACCAAGGC
513
TTATTTGTGTGGT
514
TCCACAACTAGC
515
CCACCACTAGCA
516

1-Q1

CCAACAG

GCGGTTC

AACC

AC

N22993-
C/G
CTGAGTAATTATAG
517
GGGCTTTTGTTTG
518
CAAAGAAGTCC
519
CAAAGAAGTCGG
520

001-Q001

TATTGTGCCAACCC

ACTTGTGCAA

GTCTAAC

TCTAAC

T

N09963-
G/T
GCATCAACCATCAA
521
GGCATCCTACTGA
522
ATTTGCAAAAAT
523
AATTTGCAAAAA
524

001-Q001

TCTGAATGGTATG

CCTGTATGTTAA

TTCTCT

GTTCTCT

N115C2-
C/T
GTCCCATGCATTGT
525
TGCATACATATGA
526
CTAGATGCACAC
527
CTAGATGCACGC
528

001-Q001

ATTTGAAGTTGA

TCATCAAATTTCC

TTCTA

TTCTA

CAATG

N14681-
A/C
ATCATAATGTCATG
529
TCTTAAAAACCAA
530
TCCCATATGTGT
531
CATCCCATATGT
532

001-Q001

ACTGCCTGGTTT

CTACACACATCGT

ATCATG

TTATCATG

T

N11636-
A/G
GTTCGACCTGGAA
533
CACTTGCACGATA
534
TAGATGACTTAT
535
ATAGATGACTTA
536

001-Q001

TATCGGAAGA

TCGCGAATC

CGGAAAGT

TCAGAAAGT

N13732-
C/T
GATCAATGTACAC
537
GGAGTTCCATCAC
538
AAGAATGTTGAA
539
AAGAATGTTGGA
540

001-Q001

GCGTCAAGAC

GCCTCTTC

ACCGC

ACCGC

N11255-
C/T
CAATAAAACAAAAA
541
GAGTCTCAGATTG
542
CTTCATAGTTTG
543
TTCATAGTTTGG
544

001-Q001

CAATAATCTGACGC

ATAGCCCCAATT

ATAAGTTC

TAAGTTC

ACAT

N15511-
A/G
AGCCTGGTGCGTA
545
ATCGAAAGATGCA
546
CTAAAACTGTGG
547
CTCTAAAACTGT
548

001-Q001

TGTATCATAAAA

TAGAGTAATGATT

TGGTTTA

GATGGTTTA

AATAACCA

N10536-
G/T
CTTCCCGGAAAAG
549
ACGCTTCAAACCC
550
ATTTCATCTTGA
551
ATTTCATCTTGA
552

001-Q001

GTATCGATTGTA

TAAAGACAGAAT

TTCTATTG

GTCTATTG

N09862-
A/G
CCAGGATCGATTT
553
TCAACAGTCAACT
554
ATATAGCTAGGC
555
AGGATATAGCTA
556

001-Q001

GAGATGAAAGCT

TTTGAACAAAAAA

AATTCA

GACAATTCA

GGT

N23033-
A/T
CGGTAAACCAGTA
557
CGGATTTAAAATA
558
ACTTGTGCTAAT
559
CTTGTGCAAATC
560

001-Q001

CAAAATATCCAAAT

GTTTGAAGATAAA

CTCT

TCT

GTT

TAATGGCTTGT

N06039-
G/T
CCCAGTACCCAAT
561
CCGTCGGTTATAC
562
AACTGCCTTTGT
563
CTGCCTGTGTTT
564

1-Q1

GCTCATC

ACACCAAG

TTTGT

TGT

N10016-
C/T
GCTTTGCCTAAGA
565
TGCTTGTGCTTGT
566
TTGAACGAAAAT
567
TTGAACGAAGAT
568

001-Q001

GATTGCTTCATG

AGTCATCTGA

TAGC

TAGC

N22743-
A/T
GGATTTGTTATACC
569
ACAGAGGGTCGT
570
ACACTTCTCAAA
571
CACACTTCTCAA
572

001-Q001

ATTGCATCAAGCA

TGGACAAC

GGCT

TGGCT

N22953-
A/T
CTAGTTTCTTTCCT
573
GCTTTCCTATTTT
574
TGACAAGGAACT
575
TGACAAGGAACA
576

001-Q001

TGTACTTCCTTCCA

TATCGGAAATTGA

AAGTTTA

AAGTTTA

CAGT

N09987-
A/G
AGAATAAACAGCTT
577
TCTGGTGGGATAA
578
CACCAAGACTG
579
TCACCAAGACTA
580

001-Q001

TCTACACCCGTAG

GGTGATTGAGA

AGACAC

AGACAC

N10092-
A/G
GGCATCGTCTGGT
581
CCCATTCCACTTG
582
CATGACATTTTG
583
TGCATGACATTT
584

001-Q001

TGATCAAGA

ATAACCCGATTT

CCATTAA

TACCATTAA

N10096-
A/G
CATAGACCTCCAC
585
TGTAAAGCAAAAA
586
AGGGAGACACG
587
AAGGGAGACAC
588

001-Q001

ATCACCAAGAAA

CCCAAACACAATA

TGTCAT

ATGTCAT

AAGT

N22728-
C/G
AGCACACAAAGGT
589
GGTTCAAATGTTT
590
CCCGAATGCCC
591
CCGAATGCGCA
592

001-Q001

TTCTTAGGAAGATT

AGGACACGAAAG

AGACC

GACC

ATT

AC

N22747-
C/G
ATCTATTCCCAGAG
593
GCCCCTAGTGAA
594
TCAGAGACCAG
595
TCAGAGACGAG
596

001-Q001

AATACGTTTTTTCG

AAGAATGAGGTAA

GAATT

GAATT

A

N22840-
A/T
ACCTCCATTCATCA
597
GCTCGGCTAAGA
598
CAACACCAAGTT
599
CAACACCAAGAT
600

001-Q001

TTCCCAATCC

ATCACACTGAAA

CTTAC

CTTAC

N23027-
A/T
CCGCTTTTGCCCAT
601
ACTACCAACGTTA
602
ATTGCATTCAGT
603
ATTGCATTCAGT
604

001-Q001

GGC

AGAATACACTTGG

TGTTGAA

AGTTGAA

ATTT

N22777-
C/G
GAACAACAGTTTG
605
GGCATCCATTACC
606
CGTAAAAGCGA
607
CCGTAAAAGGGA
608

001-Q001

CCATTTCCTTACT

TATCAATTTCTTG

AAGTA

AAGTA

GA

N09636-
G/T
AGCGAAAAGATTA
609
GCCTTAGTATACT
610
TTTTGGTTTTTC
611
TTGGTTTTTCGG
612

001-Q001

CACTTTGTTTCTTT

CTAGTTTCATTGC

TGGTTTAT

GTTTAT

GAA

CAAA

N09879-
A/G
CTCCAGTTGCAACT
613
GATGACCCATAGA
614
TGTTTAATGAAA
615
CATGTTTAATGA
616

001-Q001

TCTTCAAATCAT

CCAAAGCCATAT

CGCTATTG

AATGCTATTG

N10123-
A/G
CAAATGACTAACCT
617
AGTCAAGGGAGT
618
CCGGTTGGTTC
619
CCCGGTTGATTC
620

001-Q001

GCGCAAGTG

GGGAAAGTAGAT

GACA

GACA

N10316-
C/T
ACCAGGAAGGAGG
621
AGCATCGACTTTC
622
AATGCTCCATTA
623
ATGCTCCGTTAA
624

001-Q001

TCAAGATCTTAA

TACATTATGTTCT

ACAAA

CAAA

CTTTT

N10507-
C/T
CGCTTACATCATTA
625
GGTAGTTGCGAC
626
CACCATTATCGA
627
ACCATTATCGGT
628

001-Q001

TTCATTTTTTAGAC

AAGCACATC

TGTTTAA

GTTTAA

ACACT

N09834-
C/T
TGCTCCACTGTCTC
629
AATCCTTAACTGA
630
ATTCGCTAGATT
631
ATTCGCTGGATT
632

001-Q001

TTCAGAAAC

AACCTGGACTCG

CCA

CCA

N22934-
A/T
GACCAGAGTTGAT
633
GCACGGTGATCTT
634
TTTCTGGCCTAT
635
TTTCTGGCCTTT
636

001-Q001

CCAGGAATGTAA

GGACACAT

GAGCTCA

GAGCTCA

N22700-
A/T
CAAACCGTACTTTC
637
TGGTATCTCGACA
638
ATCTCCTGTCAT
639
TCTCCTGTCAAA
640

001-Q001

CCTATGATGGT

ATGGAGCTAGT

ATGATT

TGATT

N22725-
A/T
GCATTGGCAGGTT
641
CTCAAAACCAATG
642
TTGTGCATAATG
643
TGTGCATAAAGA
644

001-Q001

TTAAATCTCAGA

GAAGTGTTACTGT

ACACCC

CACCC

N22881-
A/T
TGATATCACGACG
645
GAGCTTTCTCCAG
646
CTCAGTCTCTCT
647
CTCAGTCTCTCA
648

001-Q001

ACGGTTTCTG

CGTGATTG

CGGAGAT

CGGAGAT

N23032-
A/T
GAAATCCTACACG
649
GCGCATCACCCT
650
TACACCCCAGG
651
ACACCCCTGGCT
652

001-Q001

ATTTTAAGCATGTC

CATTCC

CTTAA

TAA

AA

N22786-
C/G
GAGTTTCTTGTATT
653
TGAGGATATACAT
654
CGGTATTAGGTA
655
CGGTATTAGGTA
656

001-Q001

CATCTAGGTCAAC

GCATATCTTGCCA

ACCTAGCAT

ACGTAGCAT

GA

TATATTTG

N23014-
C/G
CACCAAAGCTAACT
657
CAGGACGAGTTA
658
ATTGTAGACTGC
659
ATTGTAGACTGC
660

001-Q001

CTGCACTCT

AGGCAAGGT

CTACTAC

GTACTAC

N10471-
C/T
GACATCTTTTGGGA
661
GGATTAGCCAAGT
662
AAGCAACTCAAT
663
AAGCAACTCAAT
664

001-Q001

CCTTAGCATCT

TCCATTCCTACA

AAGAAGA

GAGAAGA

N11419-
C/T
TGCAACTTGGTCA
665
CACCATATAGGAA
666
TCGCGCTCTATT
667
TCGCGCTCTGTT
668

001-Q001

GCACTCA

ATCATGAGCACGA

TCA

TCA

A

N22724-
A/T
CGATTCGGAATGA
669
GGGTAATTACGG
670
ATGTCTGCACTA
671
TGTCTGCACTAA
672

001-Q001

TCATATAAGATCAA

ATGCAAGACTTTA

TATCTAT

ATCTAT

ACTTC

TAGTT

N12785-
A/G
CCGCTGAAGGTAT
673
CCTTGTTCAGTCT
674
CAAACCGGATG
675
TTCAAACCGAAT
676

001-Q001

ATCGAAGAATCT

TACTCAGCTTTTG

ATGC

GATGC

NO9910-
A/T
AAAGCAACCAAAG
677
CTCCATCAATCCA
678
TCGTCCATATAG
679
ATCGTCCATATT
680

001-Q001

AAGTGTTTGATATA

TGGGAAGACTTA

TTCAAG

GTTCAAG

ACTTATTTTTA

N21146-
A/C
CGATGGTTGTTAG
681
GGAAGAGAAGAA
682
AACGTTCACCAT
683
AACGTTCACAAT
684

001-Q001

ATATATGTCTAGTC

CAAGAGGAAGAC

CTCT

CTCT

CTAAGT

TTT

N17618-
A/G
CGTATAAGTGTGG
685
GGATCCTTGGCG
686
CGCAAGGACAT
687
CCGCAAGAACAT
688

001-Q001

TGCCAATTGTTT

CTCTCC

TC

TC

N09776-
A/C
GCGTCTTGTTTGC
689
CTTTGGTAGTTGT
690
CTGGTTATACAG
691
CTGGTTATACAG
692

001-Q001

CAAATCTGT

TGTTGAATCTGTT

CTCTGAAGT

CTATGAAGT

GT

N19296-
A/C
GCAACGAACAAAA
693
TCTGTACTTTCTT
694
AAAGGCTGAATC
695
AAGGCTGAATCA
696

001-Q001

TAAAAGATGGAGAT

TTAAGCCCTTCCA

CGTTTT

GTTTT

GTAA

A

N05205-
G/T
TGGTTTGCTACCTC
697
TGCAAAAGGCAAA
698
CTCATATCCACT
699
CTCATAGCCACT
700

1-Q1

CGTTTC

AGATTCA

GTTGC

GTTG

N10406-
C/G
ACGGCTCATGTTG
701
ACCCATGGTCGC
702
ATTTTGTTTTCC
703
TTTTGTTTTGCCT
704

001-Q001

GATCGT

CACAAAT

CTTTTTC

TTTTC

N22941-
C/G
TCTGCCCAAAGTG
705
TGCAAGAGGATAT
706
CTGCCTGAACCT
707
CTGCCTGAAGCT
708

001-Q001

AATCAGTATTCA

GTCACAGAACTC

TTTA

TTTA

N22875-
C/G
CGAGCATTAATGA
709
ACAGCCTCAGGA
710
CAGAGTAGTAAC
711
CAGAGTAGTAAC
712

001-Q001

CTTACCATCCTTCA

ACATCAGAAG

CCTGGTTT

GCTGGTTT

N11286-
A/G
GACCGTGTTAAGC
713
GGTCTCTTAAAAT
714
ACTTGCATGACC
715
CACTTGCATGAC
716

001-Q001

TGTAAATCGATAAC

AAGTAACTAGTAG

TTCATA

TTTCATA

TGAAGAAATGT

N04503-
C/G
TAATAAGCCGAGC
717
GACGAGGGAGGA
718
ACCAAGCTTCTC
719
CCAAGCTTCTGT
720

1-Q1

CACCAAG

AATGTTCA

TC

CGC

N22925-
C/G
TGTCGATTGGCTCT
721
AGAAGTTATGAAA
722
AGGAGTATCGTA
723
AGGAGTATCGTA
724

001-Q001

TTTGAGATTCA

AGAGAGAGGTGT

CATCTCA

GATCTCA

ACTACT

N05656-
G/T
TTGCTCGGTTTTAA
725
AAGAAATGGGGG
726
TCTTCGCTTTAT
727
CTTCGCTGTATC
728

1-Q1

CCTCGT

AAAGGATG

CACC

ACC

N17581-
A/C
GAAGGTTTCCTCG
729
GATATGGGTCCTT
730
TCCCTAAGCAGA
731
ATTAATCCCTAA
732

001-Q001

TGGAATGACT

GCGGTCTATTT

GAAG

GAAGAGAAG

N001NVH-
A/G
CGTCTACAATTTCA
733
GCTTCTGGATAAT
734
TGGGATGTAATC
735
TTTGGGATGTAA
736

001-Q001*

TTAGTCTCAAGAAA

TGGATTGGG

TGGCTAT

TTTGGCTAT

AACA

N22928-
A/T
GATTGCGTTTTTGC
737
GCAACTCATACTG
738
TTGCAAGTGTCT
739
TGCAAGTGTCAT
740

001-Q001

GTGAAGTC

AAACGTGTTTGA

TTCATG

TCATG

N08219-
G/T
TGGGAGAGAGCCT
741
CGCAACACTAGG
742
ACACCATCAAGA
743
ACACCATCAAGC
744

1-Q001

AAGTTTCTG

AAACACCTT

AC

AC

N05710-
A/G
TTCCATCACCACTG
745
CGTGGCTATGCA
746
TTCCCAAGACAA
747
CCCAGGACAAAA
748

1-Q1

AAACAGA

CCATCC

AAC

C

N15338-
C/T
AAACAAAAACAAAA
749
TGAAGATGAACTC
750
TCAATAAACTAA
751
CAATAAACTAAT
752

001-Q001

TCATTCTGAGACTT

GCCGTATAGAAAA

TTACTTTTTC

TGCTTTTTC

TGAAAC

G

N10424-
A/G
CTTCGACGGATTC
753
CTGTTCAGCAGA
754
CCCTCTTTCGAT
755
AACCCTCTTTTG
756

001-Q001

CTTGATGGA

GCCAAGATACA

GTTC

ATGTTC

N16006-
C/T
GTTTTTCTGATGTT
757
GTGTCCCGACCA
758
TCCACCAGAATT
759
CTCCACCAGAGT
760

001-Q001

GAAGGAGGTTGA

ACAATTCC

TAGA

TTAGA

N07278-
A/G
CCTGATTCAAGAA
775
TCCCAAGCTCAAG
776
CTTAGTTTCATC
777
CTTAGTTTCGTC
778

1-Q1

GTCGCAGA

CTAGACAA

TGTATTC

TGTATTC

N16343-
A/C
AGGACAATCGAAC
779
TCGTGACTTAACC
780
TAGAGCCGAAG
781
ATATATAGAGCA
782

001-Q001

TTTTCAATATAGAC

ATTTGTCCAACT

ACTC

GAAGACTC

GATT

N23417-
C/G
CCAATGCGTTCTCT
783
AGCTAGGAGGAG
784
ATGGTCGAGTCT
785
ATGGTCGAGTGT
786

001-Q001

CAGGTTCTTAT

CCTAAACCAATAT

GCAAG

GCAAG

N22902-
C/G
ACCCTCTGGGTTTT
787
AAGATTTTGGTGC
788
TCGTATAAAGAC
789
CTATCGTATAAA
790

001-Q001

CTGAACTTTG

GCACTACTAGT

GGTTTAA

GAGGGTTTAA

N23063-
A/T
GGAGAATGCCTTG
791
GTCCCCTTTGTGT
792
CTAGTCAGTCAG
793
CTAGTCAGACAG
794

001-Q001

TTGATGTCTCA

CATCTTATCGAT

GTTTG

GTTTG

N22723-
C/G
AAGGAGCTTTCCG
795
CGATCCTTGGTTA
796
CAGTTTTCTCGG
797
CCAGTTTTCTCC
798

001-Q001

GTCTATCTCT

ATGACTGGAGAT

AGTTGT

GAGTTGT

N23049-
A/T
TTGCCCATCAACAC
799
CTGAGAAATAAAC
800
CATATTAACATA
801
CATATTAACATA
802

001-Q001

CTTTCTGA

GGATCAAAACCCA

ACTTTTATTTGG

ACTTTAATTTGG

AT

N10321-
A/T
CCATGGCAAGGCA
803
CAGTTTGAAGGCT
804
ATGTAAGTACAC
805
ATGTAAGTACAC
806

001-Q001

CAAACAC

CGTTAGGTATTGA

TTCTTTG

ATCTTTG

N15374-
A/C
ACTTGGCACTTCAC
807
CAGTCCAGGATA
808
ATTTGATTTTGT
809
ATTTGATTTTGTA
810

001-Q001

CAAACCA

GAGGCATGAGA

ATCATCCG

TAATCCG

N22802-
C/G
GAAGAACACGCCG
811
TCACATCTTTGCT
812
ATGGAAGTACAA
813
ATGGAAGTACAA
814

001-Q001

CCTTTTC

TATTGTCTTCATA

CTTCAGC

GTTCAGC

CCA

N22803-
A/T
GCTCGTTGCTTCTC
815
CAGGAGGAAATC
816
CTAAAATCGCTA
817
CCTAAAATCGCT
818

001-Q001

TTTCCAAAG

AAAAGTGAAACCA

TTCTTGG

ATACTTGG

N18929-
A/G
ACTTATCTCATTTG
819
GTACCCTGAAGC
820
TGGTTTTTACGG
821
TGTGGTTTTTAC
822

001-Q001

AGTTTTGCCGATGA

GACATTGGA

ATGGTT

AGATGGTT

N16041-
C/T
TCTCAAAACCCATA
823
TGCCTATGAAATG
824
TTAGGCTCTAAG
825
CTTAGGCTCTGA
826

001-Q001

GTAAAACACAAGTC

TTTGATAACAATT

CTTG

GCTTG

T

ATAAAAGTTAACC

N18401-
C/T
TCGTCTTTCTCGCT
827
CAGAGTCATATTT
828
CCCGTCTACAAA
829
ACCCGTCTGCAA
830

001-Q001

TTCTCACTTTT

TTTACCACTGGCT

GC

AGC

TTT

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques, methods, compositions, apparatus and systems described above may be used in various combinations. All publications, patents, patent applications, or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted concurrently with the specification as a text file via EFS-Web, in compliance with the American Standard Code for Information Interchange (ASCII), with a file name of BB2360sequencelisting.txt, created on Jul. 10, 2015 and having a size of 307 kilobytes. The sequence listing filed via EFS-Web is part of the specification and is hereby incorporated in its entirety by reference herein.

Standard IUPAC notation is used in the text file submitted via EFS-Web. Standard IUPAC notation is shown in Table 9.

TABLE 9

Standard IUPAC notations.

IUPAC nucleotide

code
Base

A
Adenine

C
Cytosine

G
Guanine

T (or U)
Thymine (or Uracil)

R
A or G

Y
C or T

S
G or C

W
A or T

K
G or T

M
A or C

B
C or G or T

D
A or G or T

H
A or C or T

V
A or C or G

N
Any base

. or -
Gap

	Number	Date	Country
	62024686	Jul 2014	US
	62162301	May 2015	US

QTLs associated with and methods for identifying shatter resistance in canola

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

CPC

Field of Search

US

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

PCT Information

US Referenced Citations (1)

Foreign Referenced Citations (1)

Non-Patent Literature Citations (14)

Related Publications (1)

Provisional Applications (2)

Entry
Dong et al (2014 Nature Communications pp. 1-11, ncomms4352 (Year: 2014).
Brassica Information, Linkage Groups Assignments, 2016.
Jose R. Dinneny et al., A genetic framework for fruit patterning in Arabidopsis thaliana, Development, 2005, pp. 4687-4696, vol. 132.
S. Hossain et al., Breeding Brassica napus for Shatter Resistance, Jan. 2012, Plant Breeding, pp. 313-332.
Zhiyong Hu et al., Discovery of Pod Shatter-Resistant Associated SNPs by Deep Sequencing of a Representative Library Followed by Bulk Segregant Analysis in Rapseed, PLOSOne, Apr. 2012, pp. 1-7, vol. 7, Issue 4.
G. P. Kadkol et al., Breeding Brassica napus For Shatter Resistance, DSIR Plant Breeding Symposium, 1996, pp. 58-62, Paper 11, Special Publication No. 5.
Gururaj Kadkol, Brassica Shatter-resistance research update, 16th Australian Research Assembly on Brassicas, 2009, pp. 1-6.
Jia Liu et al., Multigenic Control of Pod Shattering Resistance in Chinese Rapeseed Germplasm Revealed by Genome-Wide Association and Linkage Analyses, Frontiers in Plant Science, Jul. 2016, pp. 1-14, vol. 7, Article 1058.
Mikihiro Ogawa et al., Arabidopsis Dehiscence Zone Polygalacturonase1 (ADPG1), ADPG2, and QUARTET2 Are Polygalacturonases Required for Cell Separation during Reproductive Development in Arabidopsis, The Plant Cell, Jan. 2009, pp. 216-233, vol. 21.
Shyam Prakash et al., Reconstruction of allopolyploid Brassicas through non-homologous recombination: introgression of resistance to pod shatter in Brassica napus, Genetical Research, 1990, pp. 1-2, vol. 56, Issue 01—Abstract Only.
Harsh Raman et al., Genome-Wide Delineation of Natural Variation for Pod Shatter Resistance in Brassica napus, PLOS One, Jul. 2014, pp. 1-13, vol. 9, Issue 7.
Y. C. Wen et al., Identification of QTLs involved in pod-shatter resistance in Brassica napus L., United States Department of Agriculture, National Agricultural Library, Crop & Pasture Science, 2012, pp. 1082-1089, vol. 63, No. 12—Abstract Only.
Y. Zhang et al., The basis of pod dehiscence: anatomical traits of the dehiscence zone and expression of eight pod shatter-related genes in four species of Brassicaceae, Biologia Plantarum, 2016, pp. 343-354, vol. 60 (2).
International Search Report and Written Opinion, PCT/US2015/040559, dated Jan. 5, 2016.