QTLs ASSOCIATED WITH AND METHODS FOR IDENTIFYING WHOLE PLANT FIELD RESISTANCE TO SCLEROTINIA

FIELD OF THE INVENTION

The present invention relates generally to plant molecular biology. More specifically, it relates to quantitative trait loci (QTLs) associated with whole plant field resistance to Sclerotinia in Brassica, and use of those QTLs to identify whole plant field resistance to Sclerotinia in Brassica and other plant species.

BACKGROUND OF THE INVENTION

Sclerotinia infects over 400 species of plants throughout Canada, including numerous economically important crops such as Brassica species, sunflowers, dry beans, field peas, lentils, and potatoes (Boland and Hall (1994) Can. J. Plant Pathol. 16:93-108). Sclerotinia sclerotiorum is responsible for over 99% of the disease, while Sclerotinia minor produces less than 1% of the disease. Sclerotinia produces sclerotia, which are irregularly shaped dark overwintering bodies that can endure in soil for four to five years. The sclerotia can germinate carpogenically or myceliogenically depending on the environmental conditions and crop canopies. The two types of germination cause two distinct types of diseases. Sclerotia that germinate carpogenically produce apothecia and ascospores that infect above-ground tissues, resulting in stem blight, stalk rot, head rot, pod rot, white mold and blossom blight of plants. Sclerotia that germinate myceliogenically produce mycelia that can infect root tissues, causing crown rot, root rot and basal stalk rot.

Sclerotinia causes Sclerotinia stem rot, also known as white mold, in Brassica, including canola. Canola is a type of Brassica having a low level of glucosinolates and erucic acid in the seed. The sclerotia germinate carpogenically in the summer, producing apothecia. The apothecia release wind-borne ascospores that travel up to one kilometer. The disease is favored by moist soil conditions (at least 10 days at or near field capacity) and temperatures of 15-25° C., prior to and during canola flowering. The spores cannot infect leaves and stems directly. They must first land on flowers, fallen petals, and pollen on the stems and leaves. Petal age affects the efficiency of infection, with older petals better able to effect infection (Heran et al. (1999) “The Effect of Petal Characteristics, Inoculum Density and Environmental Factors on Infection of Oilseed Rape by Sclerotinia sclerotiorum” The Regional Institute Ltd. http://www.regional.org.au/au/gcirc/3/428.htm). The fungal spores use the flower parts as a food source to germinate and infect the plant.

Brassica can also develop root rot under certain conditions. For example, winter and spring canola occasionally develop root rot during mild winters in Europe (winter canola) and in Georgia, US (spring canola).

The severity of Sclerotinia in Brassica is variable, and is dependent on the time of infection and climatic conditions (Heran et al., supra). The disease is favored by cool temperatures and prolonged periods of precipitation. Temperatures between 20 and 25° C. and relative humidities of greater than 80% are required for optimal plant infection (Heran et al., supra). Losses ranging from 5 to 100% have been reported for individual fields (Manitoba Agriculture, Food and Rural Initiatives, 2004). On average, yield losses equal 0.4 to 0.5 times the percentage infection. For example, if a field has 20% infection (20/100 infected plants), then the yield loss would be about 10%. Further, Sclerotinia can cause heavy losses in wet swaths.

The symptoms of Sclerotinia infection usually develop several weeks after flowering begins. The plants develop pale-grey to white lesions, at or above the soil line and on upper branches and pods. The infections often develop where the leaf and the stem join because the infected petals lodge there. Infected stems appear bleached and tend to shred. Hard black fungal sclerotia develop within the infected stems, branches, or pods. Plants infected at flowering produce little or no seed. Plants with girdled stems wilt and ripen prematurely. Severely infected crops frequently lodge, shatter at swathing, and make swathing more time consuming. Infections can occur in all above ground plant parts especially in dense or lodged stands. Once plants are infected, the mold continues to grow into the stem and invade healthy tissue. New sclerotia are formed to carry the disease over to the next season.

Some varieties of canola with certain morphological traits are better able to withstand Sclerotinia infection. For example, Polish varieties (Brassica rapa) have lighter canopies and seem to have much lower infection levels. In addition, petal-less varieties (apetalous varieties) do not provide the initial infection source (i.e., the flower petal) and avoid Sclerotinia infection to a greater extent (Okuyama et al. (1995) Bulletin of the Tohoku National Agricultural Experiment Station. National Agriculture Research Center, Tsukuba, Ibaraki 305, JAP 3-1-1an. 89: 11-20; Fu (1990) Acta Agriculture Shanghai. Economic Crop Research Institute, Jiangsu Province Academy of Agricultural Sciences, Nanjing 210024, China 6 (3): 76-77. Other examples of morphological traits that confer a degree of reduced susceptibility to Sclerotinia in Brassica include increased standability, lower petal retention, higher branching (both extent and position), flowering (early start and/or short duration) and early leaf abscission. Jurke and Fernando (“Plant Morphology of Canola and its Effects on Sclerotinia sclerotiorum infection in ICPP” 2003 8^thInternational Congress of Plant Pathology, New Zealand) screened eleven canola genotypes for Sclerotinia disease incidence. Significant variation in disease incidence was explained by plant morphology and the difference in petal retention was identified as the most important factor. However, these morphological traits alone do not confer resistance to Sclerotinia and most canola lines in Canada are considered susceptible to Sclerotinia.

The primary means of controlling Sclerotinia in infected canola crops is by spraying with fungicide. Typical fungicides used for controlling Sclerotinia on Brassica include Rovral™/Proline from Bayer and Ronilan™/Lance™ from BASF. If infection is already evident, there is no use in applying fungicide as it is too late to have an effect. Accordingly, growers must assess their fields for disease risk to decide whether to apply a fungicide. This can be done by using a government provided checklist or by using a petal testing kit. Either way, the method is cumbersome and prone to errors.

Numerous efforts have been made to develop Sclerotinia resistant spring Brassica plants. Built in resistance would be more convenient, economical, and environmentally friendly compared to controlling Sclerotinia by application of fungicides. Since the trait is polygenic it would be stable and not prone to changes in efficacy, as fungicides may be. Winter canola is also susceptible to Sclerotinia.

Spring canola (Brassica napus subsp. oleifera var. annua) differs from winter canola (Brassica napus subsp. oleifera var. biennis) primarily in the absence of an obligate vernalization requirement. Asiatic rapeseed and canola versions have a low to intermediate requirement for vernalization. While winter canola cannot finish its reproduction cycle when planted in the spring, Asiatic material cannot finish its reproduction cycle if planted in late spring, but early spring planting and exposure to cold enables Asiatic material to flower and set seed. In controlled conditions winter material requires 12-14 weeks of vernalization while Asiatic material requires 2-8 weeks. Table 1 summarizes the differences between winter, semi-winter (Asiatic) and spring canola varieties.

TABLE 1

Main determinations of growth habit in Brassica napus materials

Semi

Type
Spring*
Spring
Winter
Winter

Growing
Canada,
Australia
China, Japan
Europe

areas
Europe

Vernalization
None
None
2-8 weeks
12-14 weeks

Requirement

Intermediate
strong or

full

Time of
Spring
Fall
Fall
Fall

seeding
(Increasing
(Decreasing
(Decreasing
(Decreasing

Day Length)
Day Length)
Day Length)
Day Length)

Number of
30-90
90-150
120-180
150-270

days until

flowering

*Canadian, European and Australian spring materials can be planted and grown in any environment or seeding time for spring canola.

Some Chinese cultivars of rapeseed/canola are partially resistant to Sclerotinia. For example, ChunYun et al. ((2003) Acta Agronomica Sinica 29 (5): 715-718); HanZhong et al. ((2004) Scientia Agricultura Sinica 37 (1): 23-28); WeiXin et al. ((2002) Chinese Journal of Oil Crop Sciences 24 (3): 47-49); YongJu et al. ((2000) Chinese Journal of Oil Crop Sciences 22 (4): 1-5) describe partially resistant varieties of rapeseed. However, some of these varieties are not canola quality and all of them require vernalization. The partial field resistance in Chinese varieties originated from the rapeseed variety Zhong you 821. Despite improvements in partial resistance in Zhong you 821, its reaction to pathogens is less stable under environmental conditions favorable for development of Sclerotinia (Li et al. (1999) “Breeding, inheritance, and biochemical studies on Brassica napus cv. Zhongyou 821: Tolerance to Sclerotinia sclerotiorum (stem rot)”. Proceedings of the 10th International Rapeseed Congress, Canberra, Australia).

Some Japanese cultivars of rapeseed have partial stem resistance to Sclerotinia. Partial stem resistance was detected by indoor tests in comparison with winter canola (Brun et al. (1987) “A field study of rapeseed (Brassica napus) resistance to Sclerotinia sclerotiorum.” 7^thInternational Rapeseed Congress, Poznan, Poland). However, these varieties are not canola quality and are semi-winter types (see Table 1).

Breeding for Sclerotinia resistance in canola has been very difficult due to the quantitative nature of this trait. Further, the incorporation of physiological resistance with morphological traits that avoid or reduce infection multiplies the complexity of breeding for resistance. In addition, it has been very difficult to screen for resistance because of the direct environment by genetic (GXE) interaction (i.e., temperature and humidity requirements, as well as microenvironment requirements) with the plant. As stated above, there are few Canadian spring Brassica varieties with resistance to Sclerotinia, this despite many years of co-evolution and environmental pressure to select for this trait. A level of field resistance in rapeseed (and recently some canola materials) was attained via breeding efforts in China as described with Zhong you 821 (Li et al., supra). However, the levels of such partial resistance or tolerance are relatively low and fungicide applications are still recommended on all rapeseed and canola materials in China (verbal communication) (Hu et al. (1999) “Effect of cultural control on rapeseed stem rot (Sclerotinia sclerotiorum) in Brassica napus.” Proceedings of the 10th International Rapeseed Congress, Canberra, Australia). Other breeding efforts included quantitative trait loci analysis (Zhao and Meng (2003) Theoretical and Applied Genetics 106 (4): 759-764), mutagenesis breeding (Mullins et al. (1999) European Journal of Plant Pathology 105 (5): 465-475; Wu et al. (1996) Sichuan Daxue Xuebao (Ziran Kexueban) 33 (2): 201-205; LiangHong et al., 2003, extensive screening efforts (Sedun et al. (1989) Canadian Journal of Plant Science 69 (1): 229-232; Zhao et al. (2004) Plant Disease 88 (9): 1033-1039); and screening for expressed sequence tags (ESTs) (Li et al. (2004) Fungal Genetics and Biology 41 (8): 735-753) to name a few. Several spring canola varieties with moderate tolerance to Sclerotinia have been developed (Ahmadi et al. (2000) Seed and Plant 16 (1): Pe127-Pe129, en14; Ahmadi et al. (2000) Introduction of rapeseed (Brassica napus L.), cultivar Esteghlal. Seed and Plant 16 (1): Pe127-Pe126, en13; BaoMing et al. (1999) Chinese Journal of Oil Crop Sciences 4: 12-14; and Liu et al. (1991) Scientia Agricultura Sinica 24 (3): 43-49), however the level of tolerance is low and the lines cannot withstand high disease pressure. Recently, transgenic canola has been developed carrying an oxalic oxidase gene (U.S. Pat. No. 6,166,291 and divisional patents thereof) however there are regulatory and social problems associated with transgenic plants. Accordingly, significant technical human intervention is required to breed canola varieties that are resistant to Sclerotinia.

More recently, Brassica and canola varieties with high levels of resistance to Sclerotinia were developed after a long and intensive breeding program (See, for example, WO 2006/135717, the entire teachings of which are hereby incorporated by reference). This approach is very time and labor intensive, and requires a long time to determine whether the breeding program is successful. The difficulty in breeding for whole plant field resistance to Sclerotinia is due, at least in part, to the multigenic nature of this trait.

What is needed in the art and industry is a means to identify genes conferring whole plant field resistance to Sclerotinia, 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. The present invention provides this and other advantages.

SUMMARY OF THE INVENTION

The present invention provides methods and markers for identifying Quantitative Trait Loci (“QTLs”) associated with whole plant field resistance or improved whole plant field resistance to Sclerotinia in plants.

A first aspect of the invention features a method of identifying a Brassica plant or germplasm that exhibits whole plant field resistance or improved whole plant field resistance to Sclerotinia. 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 whole plant field resistance or improved whole plant field resistance to Sclerotinia, wherein the QTL is localized to a linkage group selected from N1, N3, N4, N7, N8, N9, N10, N11, N12, N13, N15, N18 or N19, wherein each linkage group comprises at least one marker that is associated with the whole plant field resistance or improved whole plant field resistance to Sclerotinia with a statistical significance of p≦0.01, thereby identifying the Brassica plant or germplasm that exhibits whole plant field resistance or improved whole plant field resistance to Sclerotinia.

In one embodiment, the QTL is localized to a chromosomal interval selected from: (a) an interval flanked by and including (i) markers CA0614 and PE0177 or (ii) markers AG0093 and AG0482 on linkage group N1; (b) an interval flanked by and including markers CA0410 and AG0023 on linkage group N3; (c) an interval flanked by and including markers BG1442 and BG0106 on linkage group N4; (d) an interval flanked by and including markers AG0510 and CA0105 on linkage group N7; (e) an interval flanked by and including markers CA0837 and BG1286 on linkage group N8; (f) an interval flanked by and including (i) markers CA1034 and AG0441 or (ii) markers AG0378 and KK66 on linkage group N9; (g) an interval flanked by and including markers BG0228 and PE0131 on linkage group N10; (h) an interval flanked by and including (i) markers CA0120 and CA0163 or (ii) markers CA0120 and CA1097 on linkage group N11; (i) an interval flanked by and including (i) markers BG1321 and CA0991 or (ii) markers CA0753 and PE0250 on linkage group N12; (j) an interval flanked by and including markers CA0603 and CA0736 on linkage group N13; (k) an interval flanked by and including markers PE0286 and AG0369 on linkage group N15; (1) an interval flanked by and including (i) markers BG0278 and CA0636 or (ii) markers UB0315 and CA0739 on linkage group N18; and (m) an interval flanked by and including (i) markers CA1107 and CA0221 or (ii) markers UB0307 and KK98G on linkage group N19.

In another embodiment, the QTL is localized to a chromosomal interval selected from: (a) one or more intervals on linkage group N1, flanked by and including markers (i) AG0093 and PE0203, or (ii) BG0111 and BG1392, or (iii) BG1090 and AG0482, or (iv) BG1090 and PE0203, or (v) CA0614 and BG1392, or (vi) BG0988 and AG0482; or (vii) AG0243 and AG0482; or (viii) AG0243 and BG1453; or BG0988; (b) one or more intervals on linkage group N3, flanked by and including markers (i) BG1197 and AG0023, or (ii) CA0410 and BG1368 or (iii) CA0410 and BG1197; (c) one or more intervals on linkage group N4, flanked by and including markers (i) BG1442 and BG0106, or (ii) UB0181 and BG0106; (d) one or more intervals on linkage group N8, flanked by and including markers (i) BG1449 and BG1062, or (ii) CA0837 and AG0328, or (iii) CA0837 and BG1062, or (iv) CA0837 and BG1101, or (v) CA0837 and BG1286, or (vi) CA0837 and BG1449 or (vii) PE0281 and BG0647; (e) one or more intervals on linkage group N9, flanked by and including markers (i) AG0323 and BG0295, or (ii) CA1034 and AG0378 or (iii) BG1123 and AG0441; (f) one or more intervals on linkage group N10, flanked by and including markers (i) BG0228 and AG0047, or BG0255 and PE0131; (g) one or more intervals on linkage group N11, flanked by and including markers (i) BG0031 and BG1149, or (ii) BG0031 and BG1230, or (iii) BG0031 and BG1513, or (iv) CA0120 and CA0328, or (v) PE0283 and CA0163, or (vi) PE0324 and PE0283 or (vii) CA0328 and PE0324, or (viii) CA0226 and BG0713, or (ix) CA0233 and CA1080, or (x) CA0233 and AG0370; (h) one or more intervals on linkage group N12, flanked by and including markers (i) BG1321 and CA0991, or (ii) BG1321 and CA1027, or (iii) BG1321 and PE0133, or (iv) PE0063 and CA0991, or (v) PE0133 and CA0991, or (vi) CA1027 and PE0063, or (vii) CA1027 and UB0331, or (viii) CA0423 and PE0250, or (ix) AG0359 and PE0250, or (x) AG0359 and CA0896; (i) one or more intervals on linkage group N13, flanked by and including markers (i) BG0516 and AG0148, or (ii) CA0488 and AG0148, or (iii) CA0488 and CA0736, or (iv) CA0603 and AG0504, or (v) BG1288 and AG0504; (j) one or more intervals on linkage group N15, flanked by and including markers (i) CA0719 and AG0369, or (ii) PE0091 and PE0187, or (iii) PE0286 and AG0369, or (iv) PE0286 and PE0187, or (v) PE0286 and CA0719; (k) one or more intervals on linkage group N18, flanked by and including markers (i) AG0285 and CA0636, or (ii) BG0278 and CA07739, or (iii) CA0739 and CA0636, or (iv) UB0315 and CA0636, or (v) UB0315 and CA0739; and (1) one or more intervals on linkage group N19, flanked by and including markers (i) CA0552 and CA0221, or (ii) CA1107 and CA0552, or (iii) CA1107 and CA0221, or (iv) CA0221 and KK98G, or (v) UB0307 and BG1241, or (vi) BG1241 and KK98G, or (vii) CA0221 and BG1241.

In a particular embodiment, the QTL is localized to a chromosomal interval on linkage group N1, N9, N11, N12, N18 or N19.

In other embodiments, 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 whole plant field resistance or improved whole plant field resistance to Sclerotinia, and the detecting comprises identifying the polymorphism. The polymorphism may be, for example, a single nucleotide polymorphism (SNP) or a simple sequence repeat (SSR). In another embodiment of the method of the invention, the detecting comprises detecting at least one marker comprising the polymorphism, selected from AG0023; AG0045; AG0047; AG0070; AG0086; AG0093; AG0125; AG0148; AG0171; AG0203; AG0239; AG0243; AG0272; AG0304; AG0323; AG0324; AG0328; AG0359; AG0369; AG0370; AG0378; AG0391; AG0410; AG0441; AG0477; AG0482; AG0504; AG0510; BG0031; BG0106; BG0111; BG0119; BG0181; BG0228; BG0255; BG0278; BG0295; BG0452; BG0516; BG0647; BG0651; BG0713; BG0864; BG0869; BG0988; BG1062; BG1090; BG1101; BG1123; BG1127; BG1149; BG1182; BG1197; BG1230; BG1241; BG1244; BG1286; BG1288; BG1321; BG1368; BG1392; BG1442; BG1449; BG1453; BG1513; CA0105; CA0120; CA0163; CA0221; CA0226; CA0233; CA0328; CA0410; CA0423; CA0456; CA0488; CA0546; CA0552; CA0603; CA0614; CA0636; CA0681; CA0719; CA0736; CA0739; CA0753; CA0834; CA0837; CA0896; CA0991; CA1027; CA1032; CA1034; CA1035; CA1066; CA1080; CA1090; CA1097; CA1107; PE0012; PE0017; PE0063; PE0091; PE0131; PE0133; PE0177; PE0187; PE0203; PE0250; PE0281; PE0283; PE0286; PE0324; PE0340; PE0355; UB0015; UB0126; UB0163; UB0181; UB0196; UB0307; UB0315; UB0331; KK66; and KK98G.

In another embodiment of the method of the invention, the detecting comprises detecting the polymorphism in at least one marker selected from AG0093; AG0304; AG0378; AG0391; AG0482; BG1149; BG1230; BG1241; BG1453; BG1513; CA0120; CA0221; CA0546; CA0739; CA1027; PE0063; PE0203; UB0163; and UB0315.

In other embodiments, the method comprises detecting two or more markers located in two or more different linkage groups, three or more markers located in three or more different linkage groups, four or more markers located in four or more different linkage groups, five or more markers located in five or more different linkage groups, six or more markers located in six or more different linkage groups, seven or more markers located in seven or more different linkage groups, eight or more markers located in eight or more different linkage groups, nine or more markers located in nine or more different linkage groups, ten or more markers located in ten or more different linkage groups, eleven or more markers located in eleven or more different linkage groups, or twelve or more markers located in twelve or more different linkage groups.

In other embodiments, in the method, the detecting comprises amplifying the marker from genomic DNA of the plant or germplasm and determining if the marker comprises the polymorphism associated with the whole plant field resistance or improved whole plant field resistance to Sclerotinia. In other embodiments, the plant is Brassica napus; Brassica juncea; Brassica rapa; Brassica oleracea; or Brassica carinata. In other embodiments, the plant is spring canola, winter canola, or semi-winter canola. In another embodiment, the whole plant field resistance or improved whole plant field resistance results from decreased disease incidence compared to a plant lacking the allele of the QTL associated with the whole plant field resistance or improved whole plant field resistance. In another embodiment, the whole plant field resistance or improved whole plant field resistance results from decreased disease severity compared to a plant lacking the allele of the QTL associated with the whole plant field resistance or improved whole plant field resistance. In another embodiment, the plant has whole plant field resistance or improved whole plant field resistance to Sclerotinia sclerotiorum.

Another aspect of the invention features a method of introgressing Sclerotinia resistance in a second plant by cross pollinating the plant or a progeny identified according to the methods described above 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.

In another aspect, the invention features a method of producing an F1 hybrid seed, wherein the F1 hybrid plant derived from the F1 hybrid seed is resistant to Sclerotinia, the method comprising cross pollinating the plant or progeny identified according to the methods described above 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.

In another aspect, the invention features a method of positional cloning of a nucleic acid comprising a quantitative trait locus (QTL) associated with Sclerotinia whole plant field resistance or improved whole plant field resistance, the method comprising: providing a nucleic acid from a plant comprising a marker that is associated with Sclerotinia whole plant field resistance or improved whole plant field resistance with a statistical significance of p≦0.01, wherein the QTL is localized to a linkage group selected from N1, N3, N4, N7, N8, N9, N10, N11, N 12, N13, N15, N18 or N19, and wherein the linkage group comprises the marker; and cloning the nucleic acid comprising a quantitative trait locus (QTL) associated with Sclerotinia whole plant field resistance or improved whole plant field resistance. (a) an interval flanked by and including (i) markers CA0614 and PE0177 or (ii) markers AG0093 and AG0482 on linkage group N1; (b) an interval flanked by and including markers CA0410 and AG0023 on linkage group N3; (c) an interval flanked by and including markers BG1442 and BG0106 on linkage group N4; (d) an interval flanked by and including markers AG0510 and CA0105 on linkage group N7; (e) an interval flanked by and including markers CA0837 and BG1286 on linkage group N8; (f) an interval flanked by and including (i) markers CA1034 and AG0441 or (ii) markers AG0378 and KK66 on linkage group N9; (g) an interval flanked by and including markers BG0228 and PE0131 on linkage group N10; (h) an interval flanked by and including (i) markers CA0120 and CA0163 or (ii) markers CA0120 and CA1097 on linkage group N11; (i) an interval flanked by and including (i) markers BG1321 and CA0991 or (ii) markers CA0753 and PE0250 on linkage group N12; (j) an interval flanked by and including markers CA0603 and CA0736 on linkage group N13; (k) an interval flanked by and including markers PE0286 and AG0369 on linkage group N15; (1) an interval flanked by and including (i) markers BG0278 and CA0636 or (ii) markers UB0315 and CA0739 on linkage group N18; and (m) an interval flanked by and including (i) markers CA1107 and CA0221 or (ii) markers UB0307 and KK98G on linkage group N19.

In a particular embodiment, the QTL is localized to a chromosomal interval on linkage group N1, N9, N11, N12, N18 or N19.

In other embodiments, 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 whole plant field resistance or improved whole plant field resistance to Sclerotinia, and the detecting comprises identifying the polymorphism. The polymorphism may be, for example, a single nucleotide polymorphism (SNP) or a simple sequence repeat (SSR). In another embodiment of the method of the invention, the detecting comprises detecting at least one marker selected from AG0023; AG0045; AG0047; AG0070; AG0086; AG0093; AG0125; AG0148; AG0171; AG0203; AG0239; AG0243; AG0272; AG0304; AG0323; AG0324; AG0328; AG0359; AG0369; AG0370; AG0378; AG0391; AG0410; AG0441; AG0477; AG0482; AG0504; AG0510; BG0031; BG0106; BG0111; BG0119; BG0181; BG0228; BG0255; BG0278; BG0295; BG0452; BG0516; BG0647; BG0651; BG0713; BG0864; BG0869; BG0988; BG1062; BG1090; BG1101; BG1123; BG1127; BG1149; BG1182; BG1197; BG1230; BG1241; BG1244; BG1286; BG1288; BG1321; BG1368; BG1392; BG1442; BG1449; BG1453; BG1513; CA0105; CA0120; CA0163; CA0221; CA0226; CA0233; CA0328; CA0410; CA0423; CA0456; CA0488; CA0546; CA0552; CA0603; CA0614; CA0636; CA0681; CA0719; CA0736; CA0739; CA0753; CA0834; CA0837; CA0896; CA0991; CA1027; CA1032; CA1034; CA1035; CA1066; CA1080; CA1090; CA1097; CA1107; PE0012; PE0017; PE0063; PE0091; PE0131; PE0133; PE0177; PE0187; PE0203; PE0250; PE0281; PE0283; PE0286; PE0324; PE0340; PE0355; UB0015; UB0126; UB0163; UB0181; UB0196; UB0307; UB0315; UB0331; KK66; and KK98G.

In another embodiment of the method of the invention, the detecting comprises detecting at least one marker selected from AG0093; AG0304; AG0378; AG0391; AG0482; BG1149; BG1230; BG1241; BG1453; BG1513; CA0120; CA0221; CA0546; CA0739; CA1027; PE0063; PE0203; UB0163; and UB0315.

In other embodiments, the plant is a whole plant, a plant organ, a plant seed or a plant cell. In other embodiments, the plant is canola. The plant may be, for example, Brassica napus, Brassica juncea, Brassica rapa, Brassica oleracea; or Brassica carinata. The plant may be, for example, spring canola, winter canola, or semi-winter canola. In another embodiment, the Sclerotinia whole plant field resistant plant is resistant to Sclerotinia sclerotiorum.

In another aspect, the invention features a method of making a transgenic dicot comprising a quantitative trait locus (QTL) associated with Sclerotinia whole plant field resistance or improved whole plant field resistance, the method comprising the steps of: introducing a nucleic acid cloned according to the method described above into a dicot cell; and growing the cell under cell growth conditions. In one embodiment, the QTL is localized to a chromosomal interval selected from: (a) an interval flanked by and including (i) markers CA0614 and PE0177 or (ii) markers AG0093 and AG0482 on linkage group N1; (b) an interval flanked by and including markers CA0410 and AG0023 on linkage group N3; (c) an interval flanked by and including markers BG1442 and BG0106 on linkage group N4; (d) an interval flanked by and including markers AG0510 and CA0105 on linkage group N7; (e) an interval flanked by and including markers CA0837 and BG1286 on linkage group N8; (f) an interval flanked by and including (i) markers CA1034 and AG0441 or (ii) markers AG0378 and KK66 on linkage group N9; (g) an interval flanked by and including markers BG0228 and PE0131 on linkage group N10; (h) an interval flanked by and including (i) markers CA0120 and CA0163 or (ii) markers CA0120 and CA1097 on linkage group N11; (i) an interval flanked by and including (i) markers BG1321 and CA0991 or (ii) markers CA0753 and PE0250 on linkage group N12; (j) an interval flanked by and including markers CA0603 and CA0736 on linkage group N13; (k) an interval flanked by and including markers PE0286 and AG0369 on linkage group N15; (1) an interval flanked by and including (i) markers BG0278 and CA0636 or (ii) markers UB0315 and CA0739 on linkage group N18; and (m) an interval flanked by and including (i) markers CA1107 and CA0221 or (ii) markers UB0307 and KK98G on linkage group N19.

In a particular embodiment, the QTL is localized to a chromosomal interval on linkage group N1, N9, N11, N12, N18 or N19.

In other embodiments, 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 whole plant field resistance or improved whole plant field resistance to Sclerotinia, and the detecting comprises identifying the polymorphism. The polymorphism may be, for example, a single nucleotide polymorphism (SNP) or a simple sequence repeat (SSR). In another embodiment of the method of the invention, the detecting comprises detecting at least one marker selected from AG0023; AG0045; AG0047; AG0070; AG0086; AG0093; AG0125; AG0148; AG0171; AG0203; AG0239; AG0243; AG0272; AG0304; AG0323; AG0324; AG0328; AG0359; AG0369; AG0370; AG0378; AG0391; AG0410; AG0441; AG0477; AG0482; AG0504; AG0510; BG0031; BG0106; BG0111; BG0119; BG0181; BG0228; BG0255; BG0278; BG0295; BG0452; BG0516; BG0647; BG0651; BG0713; BG0864; BG0869; BG0988; BG1062; BG1090; BG1101; BG1123; BG1127; BG1149; BG1182; BG1197; BG1230; BG1241; BG1244; BG1286; BG1288; BG1321; BG1368; BG1392; BG1442; BG1449; BG1453; BG1513; CA0105; CA0120; CA0163; CA0221; CA0226; CA0233; CA0328; CA0410; CA0423; CA0456; CA0488; CA0546; CA0552; CA0603; CA0614; CA0636; CA0681; CA0719; CA0736; CA0739; CA0753; CA0834; CA0837; CA0896; CA0991; CA1027; CA1032; CA1034; CA1035; CA1066; CA1080; CA1090; CA1097; CA1107; PE0012; PE0017; PE0063; PE0091; PE0131; PE0133; PE0177; PE0187; PE0203; PE0250; PE0281; PE0283; PE0286; PE0324; PE0340; PE0355; UB0015; UB0126; UB0163; UB0181; UB0196; UB0307; UB0315; UB0331; KK66; and KK98G.

In another embodiment, the detecting comprises detecting at least one marker selected from AG0093; AG0304; AG0378; AG0391; AG0482; BG1149; BG1230; BG1241; BG1453; BG1513; CA0120; CA0221; CA0546; CA0739; CA1027; PE0063; PE0203; UB0163; and UB0315.

In another embodiment, the dicot cell is regenerated to form a first plant. In another embodiment, the first plant is crossed with a second plant of the same species. In another embodiment, the dicot is a soybean, sunflower, canola, or alfalfa. In another embodiment, the dicot is canola, for example, spring canola, winter canola, or semi-winter canola. In another embodiment, the dicot is Brassica napus, Brassica juncea, Brassica rapa, or Brassica oleracea. In another embodiment, the Sclerotinia whole plant field resistant plant is resistant to Sclerotinia sclerotiorum. In other embodiments the whole plant field resistance results from decreased disease incidence or from decreased disease severity compared to a dicot lacking the QTL.

Another aspect of the invention features a method of identifying a candidate nucleic acid comprising a QTL associated with Sclerotinia whole plant field resistance from a dicot, the method comprising: providing a nucleic acid cloned according to the methods described above; and, identifying a homolog of the nucleic acid in a dicot.

Another aspect of the invention features a method of marker assisted selection comprising (MAS) of a quantitative trait locus (QTL) associated with whole plant field resistance to Sclerotinia, the method comprising the steps of: obtaining a first Brassica plant having at least one allele of a marker locus, wherein the marker locus is associated with the whole plant field resistance or improved whole plant field resistance to Sclerotinia with a statistical significance of p≦0.01; crossing the first Brassica plant to a second Brassica plant; evaluating the progeny for at least the allele; and selecting progeny plants that possess at least the allele. In one embodiment, the plant is a member of a segregating population. In another embodiment, the marker assisted selection is done via high throughput screening.

Another aspect of the invention features a Brassica plant identified by the above method, and progeny thereof, including F1, F2 and F3 progeny.

Another aspect of the invention features an isolated or recombinant nucleic acid comprising a polynucleotide selected from the group consisting of: a sequence selected from any one of marker sequences AG0023 (SEQ ID NO:1); AG0045 (SEQ ID NO:2); AG0047 (SEQ ID NO:3); AG0070 (SEQ ID NO:4); AG0086 (SEQ ID NO:5); AG0093 (SEQ ID NO:6); AG0125 (SEQ ID NO:7); AG0148 (SEQ ID NO:8); AG0171 (SEQ ID NO:9); AG0203 (SEQ ID NO:10); AG0239 (SEQ ID NO:11); AG0243 (SEQ ID NO:12); AG0272 (SEQ ID NO:13); AG0304 (SEQ ID NO:14); AG0323 (SEQ ID NO:15); AG0324 (SEQ ID NO:16); AG0328 (SEQ ID NO:17); AG0359 (SEQ ID NO:18); AG0369 (SEQ ID NO:19); AG0370 (SEQ ID NO:20); AG0378 (SEQ ID NO:21); AG0391 (SEQ ID NO:22); AG0410 (SEQ ID NO:23); AG0441 (SEQ ID NO:24); AG0477 (SEQ ID NO:25); AG0482 (SEQ ID NO:26); AG0504 (SEQ ID NO:27); AG0510 (SEQ ID NO:28); BG0031 (SEQ ID NO:29); BG0106 (SEQ ID NO:30); BG0111 (SEQ ID NO:31); BG0119 (SEQ ID NO:32); BG0181 (SEQ ID NO:33); BG0228 (SEQ ID NO:34); BG0255 (SEQ ID NO:35); BG0278 (SEQ ID NO:36); BG0295 (SEQ ID NO:37); BG0452 (SEQ ID NO:38); BG0516 (SEQ ID NO:39); BG0647 (SEQ ID NO:40); BG0651 (SEQ ID NO:41); BG0713 (SEQ ID NO:42); BG0864 (SEQ ID NO:43); BG0869 (SEQ ID NO:44); BG0988 (SEQ ID NO:45); BG1062 (SEQ ID NO:46); BG1090 (SEQ ID NO:47); BG1101 (SEQ ID NO:48); BG1123 (SEQ ID NO:49); BG1127 (SEQ ID NO:50); BG1149 (SEQ ID NO:51); BG1182 (SEQ ID NO:52); BG1197 (SEQ ID NO:53); BG1230 (SEQ ID NO:54); BG1241 (SEQ ID NO:55); BG1244 (SEQ ID NO:56); BG1286 (SEQ ID NO:57); BG1288 (SEQ ID NO:58); BG1321 (SEQ ID NO:59); BG1368 (SEQ ID NO:60); BG1392 (SEQ ID NO:61); BG1442 (SEQ ID NO:62); BG1449 (SEQ ID NO:63); BG1453 (SEQ ID NO:64); BG1513 (SEQ ID NO:65); CA0105 (SEQ ID NO:66); CA0120 (SEQ ID NO:67); CA0163 (SEQ ID NO:68); CA0221 (SEQ ID NO:69); CA0226 (SEQ ID NO:70); CA0233 (SEQ ID NO:71); CA0328 (SEQ ID NO:72); CA0410 (SEQ ID NO:73); CA0423 (SEQ ID NO:74); CA0456 (SEQ ID NO:75); CA0488 (SEQ ID NO:76); CA0546 (SEQ ID NO:77); CA0552 (SEQ ID NO:78); CA0603 (SEQ ID NO:79); CA0614 (SEQ ID NO:80); CA0636 (SEQ ID NO:81); CA0681 (SEQ ID NO:82); CA0719 (SEQ ID NO:83); CA0736 (SEQ ID NO:84); CA0739 (SEQ ID NO:85); CA0753 (SEQ ID NO:86); CA0834 (SEQ ID NO:87); CA0837 (SEQ ID NO:88); CA0896 (SEQ ID NO:89); CA0991 (SEQ ID NO:90); CA1027 (SEQ ID NO:91); CA1032 (SEQ ID NO:92); CA1034 (SEQ ID NO:93); CA1035 (SEQ ID NO:94); CA1066 (SEQ ID NO:95); CA1080 (SEQ ID NO:96); CA1090 (SEQ ID NO:97); CA1097 (SEQ ID NO:98); or CA1107 (SEQ ID NO:99); (b) a polynucleotide sequence with 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 to a polynucleotide of (a); and (c) a polynucleotide sequence complementary to the polynucleotide sequence of (a) or (b). In one embodiment, the isolated or recombinant nucleic acid is associated with whole plant field resistance to Sclerotinia.

In another embodiment, the isolated or recombinant nucleic acid comprising a polynucleotide is selected from the group consisting of: (a) a sequence selected from any one of marker sequences AG0093 (SEQ ID NO:6); AG0304 (SEQ ID NO:14); AG0378 (SEQ ID NO:21); AG0391 (SEQ ID NO:22); AG0482 (SEQ ID NO:26); BG1149 (SEQ ID NO:51); BG1230 (SEQ ID NO:54); BG1241 (SEQ ID NO:55); BG1453 (SEQ ID NO:64); BG1513 (SEQ ID NO:65); (SEQ ID NO:67); CA0221 (SEQ ID NO:69); CA0546 (SEQ ID NO:77); CA0739 (SEQ ID NO:85); or CA1027 (SEQ ID NO:91); (b) a polynucleotide sequence with 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 to a polynucleotide of (a); and (c) a polynucleotide sequence complementary to the polynucleotide sequence of (a) or (b).

Another aspect of the invention features an isolated nucleic acid molecule for detecting a polymorphism in plant DNA associated whole plant field resistance or improved whole plant field resistance to Sclerotinia, wherein the nucleic acid molecule comprises at least 15 nucleotides and is identical to a sequence of the same number of consecutive nucleotides in either strand of the plant DNA in a region where the polymorphism is located, wherein the nucleic acid molecule comprises a sequence that is 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% identical to a marker sequence or a fragment of a marker sequence selected from the group consisting of marker AG0023 (SEQ ID NO:1); AG0045 (SEQ ID NO:2); AG0047 (SEQ ID NO:3); AG0070 (SEQ ID NO:4); AG0086 (SEQ ID NO:5); AG0093 (SEQ ID NO:6); AG0125 (SEQ ID NO:7); AG0148 (SEQ ID NO:8); AG0171 (SEQ ID NO:9); AG0203 (SEQ ID NO:10); AG0239 (SEQ ID NO:11); AG0243 (SEQ ID NO:12); AG0272 (SEQ ID NO:13); AG0304 (SEQ ID NO:14); AG0323 (SEQ ID NO:15); AG0324 (SEQ ID NO:16); AG0328 (SEQ ID NO:17); AG0359 (SEQ ID NO:18); AG0369 (SEQ ID NO:19); AG0370 (SEQ ID NO:20); AG0378 (SEQ ID NO:21); AG0391 (SEQ ID NO:22); AG0410 (SEQ ID NO:23); AG0441 (SEQ ID NO:24); AG0477 (SEQ ID NO:25); AG0482 (SEQ ID NO:26); AG0504 (SEQ ID NO:27); AG0510 (SEQ ID NO:28); BG0031 (SEQ ID NO:29); BG0106 (SEQ ID NO:30); BG0111 (SEQ ID NO:31); BG0119 (SEQ ID NO:32); BG0181 (SEQ ID NO:33); BG0228 (SEQ ID NO:34); BG0255 (SEQ ID NO:35); BG0278 (SEQ ID NO:36); BG0295 (SEQ ID NO:37); BG0452 (SEQ ID NO:38); BG0516 (SEQ ID NO:39); BG0647 (SEQ ID NO:40); BG0651 (SEQ ID NO:41); BG0713 (SEQ ID NO:42); BG0864 (SEQ ID NO:43); BG0869 (SEQ ID NO:44); BG0988 (SEQ ID NO:45); BG1062 (SEQ ID NO:46); BG1090 (SEQ ID NO:47); BG1101 (SEQ ID NO:48); BG1123 (SEQ ID NO:49); BG1127 (SEQ ID NO:50); BG1149 (SEQ ID NO:51); BG1182 (SEQ ID NO:52); BG1197 (SEQ ID NO:53); BG1230 (SEQ ID NO:54); BG1241 (SEQ ID NO:55); BG1244 (SEQ ID NO:56); BG1286 (SEQ ID NO:57); BG1288 (SEQ ID NO:58); BG1321 (SEQ ID NO:59); BG1368 (SEQ ID NO:60); BG1392 (SEQ ID NO:61); BG1442 (SEQ ID NO:62); BG1449 (SEQ ID NO:63); BG1453 (SEQ ID NO:64); BG1513 (SEQ ID NO:65); CA0105 (SEQ ID NO:66); CA0120 (SEQ ID NO:67); CA0163 (SEQ ID NO:68); CA0221 (SEQ ID NO:69); CA0226 (SEQ ID NO:70); CA0233 (SEQ ID NO:71); CA0328 (SEQ ID NO:72); CA0410 (SEQ ID NO:73); CA0423 (SEQ ID NO:74); CA0456 (SEQ ID NO:75); CA0488 (SEQ ID NO:76); CA0546 (SEQ ID NO:77); CA0552 (SEQ ID NO:78); CA0603 (SEQ ID NO:79); CA0614 (SEQ ID NO:80); CA0636 (SEQ ID NO:81); CA0681 (SEQ ID NO:82); CA0719 (SEQ ID NO:83); CA0736 (SEQ ID NO:84); CA0739 (SEQ ID NO:85); CA0753 (SEQ ID NO:86); CA0834 (SEQ ID NO:87); CA0837 (SEQ ID NO:88); CA0896 (SEQ ID NO:89); CA0991 (SEQ ID NO:90); CA1027 (SEQ ID NO:91); CA1032 (SEQ ID NO:92); CA1034 (SEQ ID NO:93); CA1035 (SEQ ID NO:94); CA1066 (SEQ ID NO:95); CA1080 (SEQ ID NO:96); CA1090 (SEQ ID NO:97); CA1097 (SEQ ID NO:98); or CA1107 (SEQ ID NO:99). In one embodiment, the nucleic acid is selected from any of SEQ ID NOs: 126-323.

In another embodiment, the marker sequence is AG0093 (SEQ ID NO:6); AG0304 (SEQ ID NO:14); AG0378 (SEQ ID NO:21); AG0391 (SEQ ID NO:22); AG0482 (SEQ ID NO:26); BG1149 (SEQ ID NO:51); BG1230 (SEQ ID NO:54); BG1241 (SEQ ID NO:55); BG1453 (SEQ ID NO:64); BG1513 (SEQ ID NO:65); (SEQ ID NO:67); CA0221 (SEQ ID NO:69); CA0546 (SEQ ID NO:77); CA0739 (SEQ ID NO:85); or CA1027 (SEQ ID NO:91). In one embodiment, the nucleic acid is selected from any of SEQ ID NOs: 136, 137, 152, 153, 166, 167, 168, 169, 176, 177, 226, 227, 232, 233, 234, 235, 252, 253, 254, 255, 258, 259, 262, 263, 278, 279, 294, 295, 306, or 307.

Another aspect of the invention features a kit for screening a plant or germplasm for a QTL associated with whole plant field resistance to Sclerotinia, comprising a container in which is contained one or more of the isolated nucleic acid molecules of claim as described above; and instructions for screening a plant for the QTL associated with whole plant field resistance or improved whole plant field resistance to Sclerotinia.

In one embodiment, the further comprises a buffer. In another embodiment, the kit is for use in high throughput screening and comprises at least one component for such use. In another embodiment the kit is for use in high throughput screening in an integrated system.

In another aspect, the invention features a Brassica plant that exhibits whole plant field resistance or improved whole plant field resistance to Sclerotinia, comprising alleles favorable to Sclerotinia whole plant field resistance in at least 1 QTL localized to a chromosomal interval selected from: (a) an interval flanked by and including (i) markers CA0614 and PE0177 or (ii) markers AG0093 and AG0482 on linkage group N1; (b) an interval flanked by and including markers CA0410 and AG0023 on linkage group N3; (c) an interval flanked by and including markers BG1442 and BG0106 on linkage group N4; (d) an interval flanked by and including markers AG0510 and CA0105 on linkage group N7; (e) an interval flanked by and including markers CA0837 and BG1286 on linkage group N8; (f) an interval flanked by and including (i) markers CA1034 and AG0441 or (ii) markers AG0378 and KK66 on linkage group N9; (g) an interval flanked by and including markers BG0228 and PE0131 on linkage group N10; (h) an interval flanked by and including (i) markers CA0120 and CA0163 or (ii) markers CA0120 and CA1097 on linkage group N11; (i) an interval flanked by and including (i) markers BG1321 and CA0991 or (ii) markers CA0753 and PE0250 on linkage group N12; (j) an interval flanked by and including markers CA0603 and CA0736 on linkage group N13; (k) an interval flanked by and including markers PE0286 and AG0369 on linkage group N15; (1) an interval flanked by and including (i) markers BG0278 and CA0636 or (ii) markers UB0315 and CA0739 on linkage group N18; and (m) an interval flanked by and including (i) markers CA1107 and CA0221 or (ii) markers UB0307 and KK98G on linkage group N19.

In a particular embodiment, the QTL is localized to a chromosomal interval on linkage group N1, N9, N11, N12, N18 or N19.

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

DETAILED DISCUSSION
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 Sclerotinia whole plant field resistance. 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 Sclerotinia. Accordingly, these markers are useful for marker-assisted selection (MAS) and breeding of Sclerotinia 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 Sclerotinia whole plant field resistance. Quantitative Trait Loci (QTLs) associated with Sclerotinia whole plant field 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 improved resistance to Sclerotinia. In addition, QTL nucleic acids isolated from one organism, e.g., canola, can, in turn, serve to isolate homologs of QTLs for Sclerotinia whole plant field resistance from other susceptible organisms, including a variety of commercially important dicots, such as soybean, alfalfa, sunflower, flax, beans, (for example, white beans), potatoes, peas and peanuts.

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.

The term “Sclerotinia whole plant field resistance” or “whole plant field resistance to Sclerotinia” refers to the resistance of a plant against the plant pathogen Sclerotinia, under field conditions or under extreme disease pressure field research conditions (as described, for example, herein and in WO 2006/135717). It reflects the resistance of the entire plant when exposed to Sclerotinia under these conditions. In one embodiment, a plant with Sclerotinia whole plant field resistance has a rating of disease development of 5.0 or greater, based on the Sclerotinia Sclerotiorum Disease Incidence Severity (SSDIS) rating scale. In other embodiments, a plant with Sclerotinia whole plant field resistance has a rating of disease development of 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 or greater, based on the Sclerotinia Sclerotiorum Disease Incidence Severity (SSDIS) rating scale. Ratings of disease development 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 Sclerotinia whole plant field resistance has a rating of disease development in the range of at least 5-6, or 6-7, or 7-8, or 8-9, based on the Sclerotinia Sclerotiorum Disease Incidence Severity (SSDIS) rating scale.

It will be understood by the skilled artisan that the greater the number (or percentage) of favorable alleles for Sclerotinia whole plant field resistance a plant possesses, the greater will be the level of resistance exhibited. This concept can be appreciated by reference to Table 8 herein. In certain embodiments, a plant with Sclerotinia whole plant field resistance has a genome containing at least about 50% favorable alleles. In more particular embodiments, a plant with Sclerotinia whole plant field 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, as shown in Table 8, 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 Sclerotinia whole plant field 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 Sclerotinia whole plant field 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 Sclerotinia whole plant field resistance may contain at least 6 of the QTLs described herein. More particularly, the plant may contain at least 7, 8, 9 or 10 of the QTLs described herein. Yet more particularly, the plant may contain 11, 12 or all of the QTLs described herein.

In the present invention, the evaluation for whole plant field resistance, using, for example, extreme disease pressure field research conditions, mimicked growers' field conditions and the worst-case field scenario in canola, requiring two fungicide applications to protect the crop from infection from Sclerotinia.

The term “Sclerotinia improved whole plant field resistance” or “improved whole plant field resistance to Sclerotinia” refers to the increase in resistance of a plant against the plant pathogen Sclerotinia, under field conditions or under extreme disease pressure field research conditions (as described, for example, herein and in WO 2006/135717). In one embodiment, a plant with improved whole plant field resistance to Sclerotinia is a plant having at least one allele of a QTL associated with whole plant field resistance to Sclerotinia and having rating of disease development of 5.0 or higher based on the SSDIS scale compared to a plant that does not have the at least one allele. Other embodiments of Sclerotinia improved whole plant field resistance parallel those outlined in the description of Sclerotinia whole plant field resistance set forth above.

Sclerotinia affects many different tissues of a plant. Natural infection by Sclerotinia begins with infection of the flower petal. The disease spreads to the leaves once the infected petals fall onto them. Lesions then develop simultaneously on a number of leaves per plant. These lesions further expand to colonize and wilt the leaf with infection proceeding further towards the stem via leaf petioles. The infection then reaches the stem to develop further in the stem causing premature ripening.

A plant with whole plant field resistance to Sclerotinia is a plant that is resistant to the pathway of Sclerotinia disease development in all tissues of the plant. Accordingly, a plant with whole plant field resistance to Sclerotinia can also be termed a plant with “pathway resistance” or “field pathway resistance” to Sclerotinia. The screening methods as described herein are used to identify plants with whole plant field resistance to Sclerotinia. These methods are unique compared to other screening methods known in the art for assessing resistance to Sclerotinia. Other screening methods known in the art to assess resistance to Sclerotinia examine only part of the plant, and these methods take place at growth stages not associated with natural disease development. For example, Zhou et al. (2003, TAG 106: 759-764) performed phenotyping by inoculating the leaf at the seedling stage and by inoculating the stem at the mature plant stage. Zhou et al. (2006, TAG 112:509-5160) phenotyped based on petiole inoculation on a single plant per line, while Bela et al. (17th Crucifer Genetics Workshop (Brassica 2010), September 2010, Saskatoon, Canada) phenotyped based on petiole inoculation on 12 plants per line. Yin et al. (2010, Euphytica, online version: DOI 10.1007/s10681-009-0095-1) utilized three inoculation methods: mycelial toothpick inoculation, mycelial plug inoculation and infected petal inoculation onto cauline leaves. While it may be possible to identify one or more QTLs associated with Sclerotinia resistance using such screening methods, these screening methods are not as comprehensive as the screening methods to identify whole plant field resistance to Sclerotinia as described herein. This means that while these other screening methods may be used to uncover one or a few QTLs associated with resistance to Sclerotinia in a particular tissue of the plant, they cannot be used to identify all of the QTLs associated with Sclerotinia resistance throughout the entire plant. In addition, screening methods involving a single tissue, rather than the whole plant, will not be able to detect epistatic effects resulting from genes in different tissues working together to influence Sclerotinia resistance.

There are a number of advantages to using a whole plant approach to detecting resistance to Sclerotinia. First, this methodology most closely resembles the natural interaction between Sclerotinia and plants in the field, and should, therefore, be a superior system in which to identify QTLs associated with resistance to Sclerotinia. Second, the whole plant approach allows for a larger number of QTLs to be identified relative to other screening methodologies that only examine one plant tissue. Third, this approach permits analyses of epistatic effects, unlike other screening methods. Fourth, this approach allows actual field performance to be predicted from the data.

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, whole plant field resistance to Sclerotinia or improved whole plant field resistance to Sclerotinia. For example, the QTL may have a favorable allele that confers, or contributes to, whole plant field resistance to Sclerotinia or improved whole plant field resistance to Sclerotinia.

The term “favorable allele” is an allele at a particular locus that confers, or contributes to, a desirable phenotype, e.g., whole plant field resistance to Sclerotinia or improved whole plant field resistance to Sclerotinia, or alternatively is an allele that allows the identification of plants with decreased whole plant field 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 whole plant field resistance to Sclerotinia or improved whole plant field resistance to Sclerotinia are provided, for example, in Tables 7 and 13.

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 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 physically) to any other marker that is in close proximity, e.g., at or less than 10 cM distant. 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 also refer 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 are provided, in SEQ ID NOS: 1-125. 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: 1-125, or a fragment of such a sequence. The fragment can be, for example, the SSR (as set out, for example, in Table 14, or a sequence that flanks (e.g., those as set out as SEQ ID NOS: 126-325) and includes the SSR. 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: 1-125 or a fragment of such a sequence will have some variation from line to line. 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: 1-125 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., SSR technology is 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.

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 and a tri-nucleotide repeat would be ATGATGATGATG. 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: GAGAGAGA (4 repeats of the GA sequence) and Allele B: GAGAGAGAGAGA (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.

The term “extreme disease pressure field research conditions” means controlled disease research conditions as described, for Example, in WO 2006/135717. For example, extreme disease pressure can be generated with the application of Niger seed carrier mimicking Sclerotinia-colonized petals. Natural inoculum may be present in the field as a backup inoculum. The percent disease incidence of the test plants are adjusted to running checks, and given an SSDIS score of 1 to 9. However, under these extreme conditions plants are more susceptible to Sclerotinia for at least the following reasons: (1) under extreme disease pressure field research conditions, the plants are subjected to wetness provided by misting irrigation, which is favorable for Sclerotinia development, (2) under extreme disease pressure field research conditions the plants are in a semi-enclosed environment due to the artificial canopy, which ensures continuous moist conditions favorable for Sclerotinia development, and (3) under extreme disease pressure field research conditions there are six rows of different test plants in each plot, therefore any one row of test plants having a particular morphological phenotype may be surrounded by two different rows of plants with different morphological phenotypes. Accordingly, any benefits from a morphological phenotype that is less conducive to Sclerotinia infection (for example high branching) are decreased because any one row may be surrounded by plants having a different morphological phenotype (for example low branching). In contrast, a plant growing under natural field conditions is (1) not enclosed in an artificial canopy, which ensures continuous moisture and (2) is grown in plots surrounded by plants with the same morphological phenotype, which allows all benefits from the morphology to be expressed. Accordingly, selections having a morphology that is less conducive to Sclerotinia infection, for example high branching, perform significantly better under natural field conditions compared to extreme disease pressure field research conditions.

It is well established that generating reliable field data on an annual basis is not common. Sclerotinia is a potent disease but it only develops during wet summers with moderate temperatures. A number of issues become critical in screening for Sclerotinia resistance in the field in years when the conditions of Sclerotinia are sub-optimal. Duration of wetness, water quality, availability of inoculum, and presence of moist or humid microenvironments affect disease development in the crop. Although the methods described are directed to Brassica, it is to be understood that the methods may be applied to any plant susceptible to Sclerotinia infection via ascospores. This includes sunflower (head rot), safflower (head rot), dry bean (pod rot), dry pea (pod rot), soybean (stem and pod rot), alfalfa (blossom blight), and lettuce (lettuce drop). Bardin and Huang 2001. See also U.S. Patent application publication 2003/0150016 for Sclerotinia effects in soybean.

The critical issues in the field have been resolved as follows:

(a) Appropriate artificial inoculum for continuum of data collection: Since natural inoculum is not always triggered in the field, an inoculum that mimics infection via petals has been developed. The carrier for the fungus can be Niger seed (Guizotia abyssinica-Nyer seed) colonized with Sclerotinia and distributed at the time of full petal drop.

(b) Water quality and Sclerotinia: Initially, ground water was used to irrigate the Sclerotinia colonized fields. However, a lack of infection transfer in years with low rainfall and either high or low temperatures was observed. In vitro tests have confirmed that ground water inhibits Sclerotinia growth. Through lab and field testing, it was determined that deionizing (Dl) water treatment alters the ground water quality sufficiently to prevent inhibition of Sclerotinia development. Henceforth, Dl water was used to irrigate extreme disease pressure field research plots. In theory, the treated deionized water differs from the original ground water in that the minerals, for example magnesium and calcium (lime), are reduced while the pH is not affected. Sclerotinia produces oxalic acid, a diffusible toxin, to aid in the infection process (U.S. Pat. No. 6,380,461). Calcium can bind with oxalic acid to create calcium oxalate. Removal of calcium is very likely the qualitative change in the deionized water that enables growth of Sclerotinia. Accordingly, a water source low in minerals or having no minerals, for example reduced or eliminated magnesium and calcium, can be used.

(c) Irrigation operated by leaf-wetness sensors: To enable continuous wetness in the field, leaf wetness sensors (Campbell Scientific) that trigger irrigation only if moisture is lower than a set threshold are used. Optimized irrigation enables disease development and enhances screening for disease resistance. However, excess irrigation may interfere with meaningful evaluation. In particular, in a research setting with rows of unique genotypes in close proximity, lodging of one entry can lead to transfer of the pathogen by plant-to-plant contact and increased disease incidence on a second genotype. Thus the Sclerotinia resistance score for the second genotype may underestimate its potential performance in a more homogeneous population. In natural field data trials, excessive irrigation can create a more conducive environment for Sclerotinia through an increase in lodging over what is usual for a given genotype. Thus, the performance of the trial entries may be distorted due to excessive irrigation.

(d) Providing an enclosure to help maintain a microenvironment necessary for disease development: To enable development of disease in dry, hot and/or windy seasons, a netting enclosure may be used.

The new methodologies enable controlled disease development, reliable expression of phenotype, and characterization of many different lines under optimal Sclerotinia conditions.

Markers

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

Genetic mapping of several hundred molecular markers has developed a genetic linkage map covering approximately 1400 cM (centiMorgans) corresponding to the 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.”

Exemplary marker loci associated with whole plant field resistance to Sclerotinia are localized to thirteen linkage groups in Brassica napus: N1, N3, N4, N7, N8, N9, N10, N11, N12, N13, N15, N18 and N19. These exemplary marker loci delineate chromosomal intervals including Quantitative Trait Loci (QTLs) associated with phenotypic measures of whole plant field resistance or improved whole plant field resistance to Sclerotinia. For example, Tables 5 and 11 herein list markers that localize to linkage groups N1, N3, N4, N7, N8, N9, N10, N11, N12, N13, N15, N18 and N19. Additional primers and probes corresponding to these markers or fragments of these markers can be designed based on the sequence information provided herein.

AG0023 (SEQ ID NO:1); AG0045 (SEQ ID NO:2); AG0047 (SEQ ID NO:3); AG0070 (SEQ ID NO:4); AG0086 (SEQ ID NO:5); AG0093 (SEQ ID NO:6); AG0125 (SEQ ID NO:7); AG0148 (SEQ ID NO:8); AG0171 (SEQ ID NO:9); AG0203 (SEQ ID NO:10); AG0239 (SEQ ID NO:11); AG0243 (SEQ ID NO:12); AG0272 (SEQ ID NO:13); AG0304 (SEQ ID NO:14); AG0323 (SEQ ID NO:15); AG0324 (SEQ ID NO:16); AG0328 (SEQ ID NO:17); AG0359 (SEQ ID NO:18); AG0369 (SEQ ID NO:19); AG0370 (SEQ ID NO:20); AG0378 (SEQ ID NO:21); AG0391 (SEQ ID NO:22); AG0410 (SEQ ID NO:23); AG0441 (SEQ ID NO:24); AG0477 (SEQ ID NO:25); AG0482 (SEQ ID NO:26); AG0504 (SEQ ID NO:27); AG0510 (SEQ ID NO:28); BG0031 (SEQ ID NO:29); BG0106 (SEQ ID NO:30); BG0111 (SEQ ID NO:31); BG0119 (SEQ ID NO:32); BG0181 (SEQ ID NO:33); BG0228 (SEQ ID NO:34); BG0255 (SEQ ID NO:35); BG0278 (SEQ ID NO:36); BG0295 (SEQ ID NO:37); BG0452 (SEQ ID NO:38); BG0516 (SEQ ID NO:39); BG0647 (SEQ ID NO:40); BG0651 (SEQ ID NO:41); BG0713 (SEQ ID NO:42); BG0864 (SEQ ID NO:43); BG0869 (SEQ ID NO:44); BG0988 (SEQ ID NO:45); BG1062 (SEQ ID NO:46); BG1090 (SEQ ID NO:47); BG1101 (SEQ ID NO:48); BG1123 (SEQ ID NO:49); BG1127 (SEQ ID NO:50); BG1149 (SEQ ID NO:51); BG1182 (SEQ ID NO:52); BG1197 (SEQ ID NO:53); BG1230 (SEQ ID NO:54); BG1241 (SEQ ID NO:55); BG1244 (SEQ ID NO:56); BG1286 (SEQ ID NO:57); BG1288 (SEQ ID NO:58); BG1321 (SEQ ID NO:59); BG1368 (SEQ ID NO:60); BG1392 (SEQ ID NO:61); BG1442 (SEQ ID NO:62); BG1449 (SEQ ID NO:63); BG1453 (SEQ ID NO:64); BG1513 (SEQ ID NO:65); CA0105 (SEQ ID NO:66); CA0120 (SEQ ID NO:67); CA0163 (SEQ ID NO:68); CA0221 (SEQ ID NO:69); CA0226 (SEQ ID NO:70); CA0233 (SEQ ID NO:71); CA0328 (SEQ ID NO:72); CA0410 (SEQ ID NO:73); CA0423 (SEQ ID NO:74); CA0456 (SEQ ID NO:75); CA0488 (SEQ ID NO:76); CA0546 (SEQ ID NO:77); CA0552 (SEQ ID NO:78); CA0603 (SEQ ID NO:79); CA0614 (SEQ ID NO:80); CA0636 (SEQ ID NO:81); CA0681 (SEQ ID NO:82); CA0719 (SEQ ID NO:83); CA0736 (SEQ ID NO:84); CA0739 (SEQ ID NO:85); CA0753 (SEQ ID NO:86); CA0834 (SEQ ID NO:87); CA0837 (SEQ ID NO:88); CA0896 (SEQ ID NO:89); CA0991 (SEQ ID NO:90); CA1027 (SEQ ID NO:91); CA1032 (SEQ ID NO:92); CA1034 (SEQ ID NO:93); CA1035 (SEQ ID NO:94); CA1066 (SEQ ID NO:95); CA1080 (SEQ ID NO:96); CA1090 (SEQ ID NO:97); CA1097 (SEQ ID NO:98); CA1107 (SEQ ID NO:99); PE0012 (SEQ ID NO:100); PE0017 (SEQ ID NO:101); PE0063 (SEQ ID NO:102); PE0091 (SEQ ID NO:103); PE0131 (SEQ ID NO:104); PE0133 (SEQ ID NO:105); PE0177 (SEQ ID NO:106); PE0187 (SEQ ID NO:107); PE0203 (SEQ ID NO:108); PE0250 (SEQ ID NO:109); PE0281 (SEQ ID NO:110); PE0283 (SEQ ID NO:111); PE0286 (SEQ ID NO:112); PE0324 (SEQ ID NO:113); PE0340 (SEQ ID NO:114); PE0355 (SEQ ID NO:115); UB0015 (SEQ ID NO:116); UB0126 (SEQ ID NO:117); UB0163 (SEQ ID NO:118); UB0181 (SEQ ID NO:119); UB0196 (SEQ ID NO:120); UB0307 (SEQ ID NO:121); UB0315 (SEQ ID NO:122); UB0331 (SEQ ID NO:123); KK66 (SEQ ID NO:124); and KK98G (SEQ ID NO:125) (sometimes referred to as “the markers exemplified by SEQ ID NOs: 1-125”). contain simple sequence repeat (SSR) polymorphisms or single nucleotide polymorphisms (SNPs) that identify QTLs contributing to Sclerotinia whole plant field resistance or improved whole plant field resistance and can be used as markers thereof. It will be appreciated that the number of repeats in the SSR can vary. Favorable alleles that contribute to whole plant field resistance to Sclerotinia or improved whole plant field resistance to Sclerotinia are provided, for example, in Tables 7 and 13.

It will be noted that, regardless of their molecular nature, e.g., whether the marker is an 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 Sclerotinia whole plant field 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 resistance to Sclerotinia 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, AG0023; AG0045; AG0047; AG0070; AG0086; AG0093; AG0125; AG0148; AG0171; AG0203; AG0239; AG0243; AG0272; AG0304; AG0323; AG0324; AG0328; AG0359; AG0369; AG0370; AG0378; AG0391; AG0410; AG0441; AG0477; AG0482; AG0504; AG0510; BG0031; BG0106; BG0111; BG0119; BG0181; BG0228; BG0255; BG0278; BG0295; BG0452; BG0516; BG0647; BG0651; BG0713; BG0864; BG0869; BG0988; BG1062; BG1090; BG1101; BG1123; BG1127; BG1149; BG1182; BG1197; BG1230; BG1241; BG1244; BG1286; BG1288; BG1321; BG1368; BG1392; BG1442; BG1449; BG1453; BG1513; CA0105; CA0120; CA0163; CA0221; CA0226; CA0233; CA0328; CA0410; CA0423; CA0456; CA0488; CA0546; CA0552; CA0603; CA0614; CA0636; CA0681; CA0719; CA0736; CA0739; CA0753; CA0834; CA0837; CA0896; CA0991; CA1027; CA1032; CA1034; CA1035; CA1066; CA1080; CA1090; CA1097; and CA1107; as well as PE0012; PE0017; PE0063; PE0091; PE0131; PE0133; PE0177; PE0187; PE0203; PE0250; PE0281; PE0283; PE0286; PE0324; PE0340; PE0355; UB0015; UB0126; UB0163; UB0181; UB0196; UB0307; UB0315; UB0331; KK66; and KK98G are useful for the identification of homologous nucleotide sequences with utility in identifying QTLs associated with Sclerotinia whole plant field resistance in different lines, varieties, or species of dicots. Such homologous markers are also 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_mis 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_mcan 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_mis 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_mcan 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_mof 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 (http://www.ncbi.nlm.nih.gov/). 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 (GCG 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), Qβ-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. Favorable alleles for whole plant field resistance to Sclerotinia or improved whole plant field resistance to Sclerotinia are provided, for example, in Tables 7 and 13.

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 whole plant field resistance, or improved whole plant field resistance to Sclerotinia. QTLs associated with Sclerotinia whole plant field 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 whole plant field resistance to Sclerotinia 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 applications provide additional details regarding alternative statistical methods applicable to complex breeding populations that can be used to identify and localize QTLs associated with Sclerotinia whole plant field 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 are resistant, or exhibit improved resistance, to Sclerotinia whole plant field resistance 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, e.g., eliminated from subsequent crosses. 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 are resistant or have improved resistance to Sclerotinia.

After a desired phenotype, e.g., Sclerotinia whole plant field 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 disease tolerant loci with genes for high yield and other desirable traits to develop improved plant varieties. Disease 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 disease 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 disease 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 disease, or multiple loci each involved in resistance to different diseases, 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 AG0023; AG0045; AG0047; AG0070; AG0086; AG0093; AG0125; AG0148; AG0171; AG0203; AG0239; AG0243; AG0272; AG0304; AG0323; AG0324; AG0328; AG0359; AG0369; AG0370; AG0378; AG0391; AG0410; AG0441; AG0477; AG0482; AG0504; AG0510; BG0031; BG0106; BG0111; BG0119; BG0181; BG0228; BG0255; BG0278; BG0295; BG0452; BG0516; BG0647; BG0651; BG0713; BG0864; BG0869; BG0988; BG1062; BG1090; BG1101; BG1123; BG1127; BG1149; BG1182; BG1197; BG1230; BG1241; BG1244; BG1286; BG1288; BG1321; BG1368; BG1392; BG1442; BG1449; BG1453; BG1513; CA0105; CA0120; CA0163; CA0221; CA0226; CA0233; CA0328; CA0410; CA0423; CA0456; CA0488; CA0546; CA0552; CA0603; CA0614; CA0636; CA0681; CA0719; CA0736; CA0739; CA0753; CA0834; CA0837; CA0896; CA0991; CA1027; CA1032; CA1034; CA1035; CA1066; CA1080; CA1090; CA1097; CA1107; PE0012; PE0017; PE0063; PE0091; PE0131; PE0133; PE0177; PE0187; PE0203; PE0250; PE0281; PE0283; PE0286; PE0324; PE0340; PE0355; UB0015; UB0126; UB0163; UB0181; UB0196; UB0307; UB0315; UB0331; KK66; and KK98G 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 Sclerotinia whole plant field 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 pathogen (e.g., Sclerotinia whole plant field resistance).

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 Sclerotinia whole plant field 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 Sclerotinia whole plant field resistance may contain at least 6 of the QTLs described herein. More particularly, the plant may contain at least 7, 8, 9 or 10 of the QTLs described herein. Yet more particularly, the plant may contain 11, 12 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 Sclerotinia whole plant field 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 Sclerotinia whole plant field resistance. 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 having whole plant field resistance to Sclerotinia 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, QIAprep™ 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 Bacteriophage (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, P W J 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 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 whole plant filed resistance to Sclerotinia associated QTLs 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, Solanum, 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 computer, 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 Sclerotinia whole plant field resistance or improved whole plant field resistance to Sclerotinia. 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.

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. Examples 1-6 describe analyses of one Brassica napus mapping population. Examples 7-10 describe analyses of a different Brassica napus mapping population.

Example 1: Mapping Population #1

The parents used for the mapping population were 04DHS11418 (a Sclerotinia resistant double haploid deposited as ATCC Accession No. PTA-6778) and PHI2004HS1 (a non-resistant spring canola DH line polymorphic to the resistant line, selected for having a similar agronomic phenotype with consistent high susceptibility in the same testing environment where the 04DHS11418 line was selected for resistance) (see Table 2). These lines were used to develop a double haploid mapping population consisting of 186 progeny.

TABLE 2

Measuring field performance under

extreme disease pressure (research trials)

Disease

Rating

incidence
Spring
Mapping

SSDIS**
Category
SSDI %*
Checks
Parents

1.0
Highly susceptible
≧80
44A89

1.1-2.0
Susceptible
79-70
46A65 = 2
PHI2004HS1

2.1-3.0
Moderately
69-60
46A76 = 3

3.1-4.0
susceptible
59-50

4.1-5.0
Moderately
49-40

5.1-6.0
resistant
39-30

6.1-7.0
Resistant
29-20

04DHS11418

7.1-8.0

19-10

8.1-9.0
Highly resistant
9-0

*SSDI % Sclerotinia sclerotiorum Disease Incidence %. SSDI % is UNSSDI (where UNSSDI is the percentage of plants in a population infected with Sclerotinia) rating adjusted for a deviation from the expected mean of checks 04DHS11418 (25%) and PHI2004HS1 (75%) as described on Table 4. This rating is used only under controlled extreme disease pressure field research conditions. It is calculated by multiplying the observed SSDI % by Factor X, where Factor X is the factor that brings the average SSDI % of the appropriate checks to 50%. Adjustment for severity is not done.

**SSDIS Sclerotinia sclerotiorum . SSDIS is the UNSSDI rating adjusted for a deviation from the expected mean of checks 46A65/46A76 for spring canola under extreme disease pressure This rating is used only under controlled extreme disease pressure field research conditions. It is calculated by multiplying the observed UNSSDI by Factor X, where Factor X is the factor that brings the average SSDI % of the appropriate checks to 50%. Adjustment for severity is done after incidence adjustment.

UNSSDS is a rating of the extent of disease development on an affected plant. Two scales are used in the invention. The Pioneer SSDS scale ranges from 1 (dead) to 9 (no disease) and the Public scale ranges from 0 (no disease) to 5 (dead) plant. For details of the Pioneer SSDS scale, see Table 15 of WO 2006/135717. The Public scale is provided as follows: 0 = no disease; 1 = superficial lesions or small branch affected; 2 = large branch dead; 3 = main stem at least 50% girdled; 4 = main stem girdled but plant produced good seed; 5 = main stem girdled, much reduced yield.

Both parents have good standability. Therefore, standability or lodging resistance is fixed, thus eliminating this variable in the mapping process. The choice of a highly susceptible line resulted in a population without transgressive segregation (i.e., all resistance came from 04DHS11418 and no DH progeny lines were more resistant than the resistant parent). Over a period of four years, the population was phenotyped in the field and genotyped with SSR molecular markers. Phenotyping was carried out as described in WO 2006/135717, the entire teachings of which are hereby incorporated by reference.

Example 2: RNA Expression Profiling

RNA expression profiling experiments were carried using 04DHS11418, PHI2004HS1, resistant and susceptible bulks (comprised of susceptible and resistant double haploid (DH) progenies). Intact leaf tissues (not inoculated) were used, as well as leaf tissues sampled 6, 24 and 48 hours after leaf inoculation with mycelium. RNA expression profiling was performed by probing the different bulks with a chip consisting of Brassica (85,820) and Arabidopsis stress-related (17,617) nucleic acid sequences. A number of genes related to Sclerotinia disease resistance or to pectin (these genes are associated with cell wall integrity) were upregulated when inoculated with mycelium at 6, 24 and 48 hours. A summary of the results can be found in Table 3. Some of these genes were sequenced in the resistant and susceptible lines to find any SNPs. These SNPs were then added to the genetic map (termed KK).

TABLE 3

Number of disease related genes upregulated at different time treatments.

Pectin-Related
Disease Resistance-Related

Gene Sorting
Gene Sorting

Treatment
Total
1 Set
2 Set
3 Set
4 Set
Total
1 Set
2 Set
3 Set
4 Set

6 Hour
25
13
4
7
1
19
11
4
3
1

Treatment

24 Hour
65
21
38
6
0
48
38
9
0
1

Treatment

48 Hour
60
53
5
2
0
52
43
6
3
0

Treatment

Total
150
87
47
15
1
119
92
19
6
2

Example 3: Sclerotinia Screening

Disease Scoring

The plants of the double haploid mapping population created as described in Example 1, were rated for disease as described in Table 4. The unadjusted parameters (e.g., UNSSDI and UNSSDS) showed year to year variation due to environmental variation such as positional variation in the field and weather conditions. Such variation would be expected by one skilled in the art.

TABLE 4

Field-collected Sclerotinia parameters UNSSDI and UNSSDS and their relationship to

derived parameters SSDI %, SSDIS (research data) and SSFS (natural/research data).

SSFS

Field

severity

SSDIS
based on both

SSDI %
Based on adjusted
UNSSDI and

Based on adjusted
UNSSDI and
UNSSDS

UNSSDS
UNSSDI under
UNSSDS under
used in

UNSSDI
Disease
extreme disease
extreme disease
natural field

Disease
severity of
pressure field research
pressure field
conditions or

Trait
Incidence
affected plants
conditions
research conditions
research

Scale
0-100%
Pioneer SSDS
0-100%
1-9
0-100%

scale
Conversion of
Conversion of
% field impact -

1 = dead
UNSSDI and
UNSSDI and
quantifies

9 = no disease
adjustment for checks'
UNSSDS
damage in the

Public scale
UNSSDI
adjustment for
field

0 = no disease

checks' UNSSDI
irrespective of

5 = dead plant

and UNSSDS
disease

pressure

Usage
General
General
Pioneer only
Pioneer only
General

Adjustments
N/A
N/A
Adjusted to checks
Adjusted to checks
Unadjusted

Hypothetical

Pioneer
Public
*assuming checks do
**assuming checks

Examples (HE):

SSDS
scale
not deviate for
to do not deviate

Different

scale

UNSSDI
for UNSSDI and

combinations of

UNSSDS

disease

incidence and

disease severity

HE 1
80
1
5.0
*80
**1.0 (80)
80

HE 2
80
5
2.0
80
2.6 (64)
32

HE 3
50
5
2.0
50
5.0 (40)
20

HE 4
30
7
1.0
30
7.3 (17)
6

HE 5
10
8
1.0
10
8.5 (5)
2

UNSSDI is the percentage of plants in a population infected with Sclerotinia.

UNSSDS is a rating of the extent of disease development on an affected plant. Two scales are used in the invention. The Pioneer SSDS scale ranges from 1 (dead) to 9 (no disease) and the Public scale ranges from 0 (no disease) to 5 (dead) plant. For details of the Pioneer SSDS scale, see Table 15 of WO 2006/135717. The Public scale is provided as follows: 0 = no disease; 1 = superficial lesions or small branch affected; 2 = large branch dead; 3 = main stem at least 50% girdled; 4 = main stem girdled but plant produced good seed; 5 = main stem girdled, much reduced yield.

SSDI % is UNSSDI rating adjusted for a deviation from the expected mean of checks 04DHS11418 (25%) and PHI2004HS1 (75%) as described on Table 4. This rating is used only under controlled extreme disease pressure field research conditions. It is calculated by multiplying the observed SSDI % by Factor X, where Factor X is the factor that brings the average SSDI % of the appropriate checks to 50%. Adjustment for severity is not done.

SSDIS is UNSSDI rating adjusted for a deviation from the expected mean of checks 04DHS11418 (25%) and PHI2004HS1 (75%) as described on Table 4. This rating is used only under controlled extreme disease pressure field research conditions. It is calculated by multiplying the observed UNSSDI by Factor X, where Factor X is the factor that brings the average SSDI % of the appropriate checks to 50%. Adjustment for severity is done after incidence adjustment.

SSFS is a measure of both disease incidence and severity under natural disease pressure in the field. It is calculated as follows: SSFS = [SSDI % × SSDS(0-5 scale)] ÷ 5

Example 4: 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.

Genetic Mapping Genetic mapping has placed 351 molecular markers to 19 linkage groups (Lg) that correspond to 19 canola chromosomes and public linkage group nomenclature. The linkage map covers ˜1400 cM.

QTL Analysis

QTL analysis using simple interval mapping and composite interval mapping (CIM) (Zeng (1994), Genetics 136:1457) identified 7 linkage groups (N1, N7, N9, N11, N12, N18 and N19) contributing to whole plant field resistance to Sclerotinia. In addition, regions identified by interval mapping as being associated with Sclerotinia resistance were confirmed by single-factor analysis of variance (PROC GLM, SAS Enterprise Guide 4.2) on Sclerotinia parameters at the P≦0.01 significance level. These QTLs are identified in Tables 5 and 6 below. As shown by the “Phenotypic Variation Explained” values in Table 6, some QTLs had a larger effect on Sclerotinia resistance than others.

TABLE 5

Markers significantly associated with Sclerotinia resistance at P ≦ 0.01.

Map

Linkage

Position

Group
Marker
(cM)
Parameter
Year

N1
AG0093
48.1
UNSSDS
2008

AG0243
52.2
UNSSDS, SSFS, SSDIS
2008

BG1453
52.8
UNSSDS, SSDIS
2008

UB0163
54.3
SSFS, SSDIS
2008

AG0391
55.4
UNSSDS, SSFS, SSDIS, SSDI %
2008

AG0045
55.7
SSFS, SSDIS
2008

AG0304
56.4
SSFS, SSDIS
2008

PE0203
57.2
SSDIS
2008

BG0119
58.6
SSFS, SSDIS
2008

BG0988
60.3
SSDI %
2005

UNSSDS, UNSSDI, SSFS, SSDI %
2008

AG0482
66.1
UNSSDS, SSFS, SSDIS, SSDI %
2005

SSFS, SSDIS
2006

UNSSDS, UNSSDI, SSFS, SSDIS, SSDI %
2008

UNSSDS, SSFS, SSDIS
Across years

N7
AG0510
0.0
UNSSDS, UNSSDI, SSFS, SSDIS, SSDI %
2007

UNSSDS, UNSSDI, SSFS, SSDIS, SSDI %
Across years

CA0105
0.5
UNSSDS
2006

UNSSDS, UNSSDI, SSFS, SSDIS, SSDI %
2007

UNSSDS, UNSSDI, SSFS, SSDIS, SSDI %
Across years

N9
AG0378
0.0
UNSSDI, SSFS
2006

UNSSDI
Across years

KK66
4.0
UNSSDI
2006

UNSSDI
Across years

N11
CA0120
21.9
UNSSDS
Across years

CA0233
28.8
UNSSDS
2006, Across years

SSFS
2007

CA0226
30.7
UNSSDS
2006, Across years

CA0546
30.7
UNSSDS
2006

SSFS
2007

UNSSDS, SSDIS, SSFS
Across years

BG1149
30.8
UNSSDS
2006, 2007,

Across years

SSFS
2007, Across years

CA1080
30.8
UNSSDS, SSFS
2006

SSFS, SSDIS
2007

UNSSDS, SSFS, SSDIS
Across years

BG1230
31.3
UNSSDS
2006

UNSSDS, SSFS
Across years

AG0370
31.3
UNSSDS
2006

UNSSDS, SSFS
Across years

BG0713
31.7
UNSSDS
2006, Across years

SSFS
2007, Across years

BG0869
32.9
UNSSDS
2006, Across years

BG1513
33.3
UNSSDS
2006, Across years

BG0181
33.4
UNSSDS
2006, Across years

CA1097
36.2
UNSSDS, SSFS
2006

UNSSDI, SSFS
2007

UNSSDI, SSFS
2008

UNSSDS, SSFS, SSDIS
Across years

N12
CA0753
60.5
UNSSDS
2005, Across years

CA1027
78.9
UNSSDS, SSFS
2005

UNSSDS, SSFS, SSDIS, SSDI %
2006

UNSSDS
Across years

PE0063
79.3
UNSSDS, SSFS
2005

UNSSDS, SSFS, SSDIS, SSDI %
2006

UNSSDS, SSFS
Across years

UB0331
83.1
UNSSDS, SSFS
2005

UNSSDS, SSFS, SSDIS, SSDI %
2006

UNSSDS
Across years

CA0681
84.7
UNSSDS
2005

UNSSDS, SSFS, SSDIS, SSDI %
2006

UNSSDS, SSFS
Across years

AG0359
93.0
UNSSDS, UNSSDI, SSFS, SSDIS, SSDI %
2006

UNSSDS, SSFS, SSDIS, SSDI %
Across years

CA0423
95.6
UNSSDS
2005

UNSSDS, UNSSDI, SSFS, SSDIS, SSDI %
2006

UNSSDS, SSFS, SSDI %
Across years

AG0086
96.1
SSFS
2005

UNSSDS, UNSSDI, SSFS, SSDIS, SSDI %
2006

UNSSDS, SSFS, SSDIS, SSDI %
Across years

CA0896
96.4
UNSSDS, SSFS, SSDIS, SSDI %
2006, Across years

PE0250
96.6
SSFS
2005

UNSSDS, UNSSDI, SSFS, SSDIS, SSDI %
2006

UNSSDS, SSFS, SSDIS, SSDI %
Across years

N18
UB0315
34.8
UNSSDI, SSDI %
2008

CA0739
42.2
UNSSDI, SSDI %
2008

N19
UB0307
30.1
UNSSDS, SSFS, SSDIS, SSDI %
2005

UNSSDI, SSFS, SSDIS, SSDI %
2006

UNSSDI, SSFS, SSDIS, SSDI %
Across years

CA0221
31.6
UNSSDS, SSFS, SSDIS, SSDI %
2005

SSDIS
Across years

BG1241
32.3
UNSSDS, SSFS, SSDIS, SSDI %
2005

UNSSDI, SSFS, SSDIS
Across years

KK98G
41.8
UNSSDS, UNSSDI, SSFS, SSDIS, SSDI %
2005

UNSSDI
2006

UNSSDI, SSFS, SSDIS, SSDI %
Across years

TABLE 6

QTLs associated with Sclerotinia whole plant field tolerance.

Phenotypic

variation

LOD
explained

Linkage
Parameter
Year
QTL interval
score
(%)

N1
SSDI %, SSDIS, SSFS, UNSSDS
2005
BG0988-AG0482
3.2
7.9

SSFS, SSDIS
2008
AG0243-AG0482
2.4
16.5

UNSSDS
2008
AG0243-BG1453
2.4
16.6

SSDI %
2008
BG0988-AG0482
2.6
17.8

SSDIS
across years
BG0988-AG0482
2.2
5.5

N7
SSDI %, SSDIS, UNSSDS
2007
AG0510-CA0105
3.4
8.7

SSDIS
across years
AG0510-CA0105
2.3
5.7

N9
UNSSDI
2006, across years
AG0378-KK66
3.4
8.3

N11
UNSSDS
2006
CA0226-BG0713
3.4
7.7

SSFS
2007
CA0233-CA1080
2.2
5.5

SSFS, UNSSDS
across years
CA0233-AG0370
3.3
7.9

N12
SSFS
2005
CA1027-PE0063
2.3
5.7

UNSSDS
2005
CA1027-UB0331
3.0
7.1

SSDI %, SSDIS, SSFS, UNSSDS,
2006
CA0423-PE0250
4.4
10.3

UNSSDI

SSDI %
across years
CA0423-PE0250
2.5
6.0

SSDIS, SSFS
across years
AG0359-PE0250
2.7
6.4

UNSSDS
across years
AG0359-CA0896
3.9
9.2

N18
SSDI %, UNSSDI
2008
UB0315-CA0739
3.2
22.2

SSDI %, UNSSDI
across years
UB0315-CA0739
2.4
17.2

N19
SSDIS, SSFS
2005
CA0221-KK98G
2.6
6.3

SSDIS
2006
UB0307-BG1241
2.1
5.0

SSDI %, SSFS
across years
BG1241-KK98G
2.2
5.4

SSDIS, UNSSDI
across years
CA0221-BG1241
2.4
5.7

Additional information about the SSR markers flanking the seven QTLs associated with whole field plant resistance to Sclerotinia are shown in Table 14 in Example 12. The forward and reverse primer sequences for each marker are also provided. “Repeat” indicates the SSRs or SNPs associated with each marker. The positions of the SSRs and SNPS are shown in the sequence information located at the end of the specification. Additional information about the alleles and allele size of each SSR marker flanking the 7 QTLs associated with whole plant field resistance to Sclerotinia is provided in Table 7.

TABLE 7

The alleles and allele size of each SSR marker

flanking the seven Sclerotinia (SCL) QTLs.

Favorable

Linkage

allele for

Group
Marker
Allele
Allele Size
SCL

N1
AG0093
a
221

AG0093
b
223
yes

AG0243
a
177

AG0243
b
182

AG0243
c
193

AG0243
d
189

AG0243
e
null
yes

BG1453
a
120

BG1453
b
122

BG1453
c
130

BG1453
d
132
yes

BG1453
e
134

BG1453
f
146

BG1453
g
148

BG1453
h
152

BG1453
i
154

BG1453
j
156

BG1453
k
172

BG1453
l
142

BG1453
m
138

BG1453
n
158

BG1453
o
162

BG1453
p
144

BG1453
q
136

BG1453
r
176

BG1453
s
114

BG1453
t
160

UB0163
b
111

UB0163
c
129
yes

UB0163
e
107

UB0163
f
117

AG0391
c
139

AG0391
a
127
yes

AG0045
a
168

AG0045
b
170
yes

AG0304
a
163

AG0304
b
226

AG0304
c
229
yes

PE0203
a
205

PE0203
b
209
yes

PE0203
c
211

PE0203
d
203

PE0203
e
207

BG0119
a
270

BG0119
b
252
yes

BG0988
a
184

BG0988
b
208

BG0988
c
200
yes

BG0988
d
190

BG0988
e
208

BG0988
f
186

AG0482
a
277
yes

AG0482
b
280

AG0482
c
283

AG0482
d
286

AG0482
e
271

N7
AG0510
a
272

AG0510
b
278

AG0510
c
282
yes

CA0105
a
152
yes

CA0105
b
170

N9
AG0378
a
275

AG0378
b
281
yes

AG0378
c
290

AG0378
d
293

AG0378
e
284

AG0378
f
312

AG0378
g
299

AG0378
h
296

N11
CA0120
a
138

CA0120
b
160

CA0120
c
172
yes

CA0120
d
163

CA0120
e
169

CA0233
a
298
yes

CA0226
a
229

CA0226
b
250
yes

CA0226
c
252

CA0226
d
221

CA0226
e
344

CA0546
a
110

CA0546
b
120

CA0546
c
123

CA0546
d
146
yes

CA0546
e
149

CA0546
f
126

CA0546
g
144

CA0546
h
112

BG1149
a
260
yes

BG1149
b
266

BG1149
c
263

CA1080
a
300

CA1080
b
303

CA1080
c
306

CA1080
d
325

CA1080
e
336

CA1080
f
339
yes

CA1080
g
342

CA1080
h
345

CA1080
i
348

CA1080
j
351

CA1080
k
354

CA1080
l
357

CA1080
m
333

CA1080
n
330

BG1230
a
252

BG1230
b
288
yes

AG0370
a
283
yes

AG0370
b
290

AG0370
c
298

AG0370
d
316

AG0370
e
287

BG0713
a
222

BG0713
b
226
yes

BG0713
c
228

BG0713
d
216

BG0869
a
212

BG0869
b
216
yes

BG1513
a
164
yes

BG1513
b
214

BG1513
c
216

BG0181
a
210

BG0181
b
216
yes

CA1097
a
245

CA1097
b
248
yes

CA1097
c
251

CA1097
d
260

CA1097
e
239

N12
CA0753
a
214

CA0753
b
216

CA0753
c
281
yes

CA0753
d
291

CA0753
e
218

CA1027
a
297
yes

CA1027
b
300

CA1027
c
303

CA1027
d
306

PE0063
a
114

PE0063
b
126
yes

UB0331
a
123

UB0331
b
126
yes

CA0681
a
264

CA0681
b
266
yes

CA0681
c
268

CA0681
d
276

AG0359
a
306

AG0359
b
330
yes

AG0359
d
315

AG0359
f
312

CA0423
a
195

CA0423
b
201

CA0423
c
204

CA0423
d
207
yes

CA0423
e
210

AG0086
a
238

AG0086
b
226
yes

AG0086
c
232

CA0896
a
254
yes

CA0896
b
266

CA0896
c
260

PE0250
a
239

PE0250
b
245
yes

PE0250
c
269

N18
UB0315
a
127

UB0315
b
131
yes

UB0315
c
133

CA0739
a
220

CA0739
b
222

CA0739
c
232

CA0739
d
234
yes

CA0739
e
240

CA0739
f
224

CA0739
g
236

CA0739
h
242

N19
UB0307
a
120
yes

UB0307
b
126

UB0307
c
134

UB0307
e
108

CA0221
a
271

CA0221
b
253
yes

CA0221
c
265

BG1241
a
370

BG1241
b
329
yes

Example 5: Validation of the 7 QTLs Associated with Whole Plant Field Resistance to Sclerotinia

Simulated validation of the 7 QTLs associated with whole plant field resistance to Sclerotinia was performed on 16 Sclerotinia-resistant and 10 Sclerotinia susceptible breeding lines from Pioneer Hi-Bred's spring canola program under extreme disease pressure field research conditions. This allowed the development of extreme disease conditions every year, regardless of the natural environment.

Referring to Table 8 below, each resistant line was genotyped for SSR markers flanking the seven QTLs identified. In Table 9, the first digit in the QTL group refers to the linkage group and the second digit refers to the QTL number on that linkage group. The number above the marker names represent the positions (in centiMorgans) of the marker on the linkage group. Each allele was denoted as i) present only in resistant lines (light gray), ii) present only in susceptible lines (dark gray), or iii) present in both resistant and susceptible lines (white). The total number of favorable alleles were added for each breeding line by assigning either 1) (allele present only in resistant line), 0.5 (allele is present in both resistant and susceptible) or 0 (allele present only in susceptible line). The percentage of favorable alleles in the resistant lines ranged from 63-90% and only 13-47% in susceptible breeding lines. This correlation indicates that the markers flanking the seven QTLs identified as being associated with Sclerotinia in breeding populations can be used to select for individuals that had the highest number of favorable alleles in breeding populations. Those individuals with the highest percentage of favorable alleles would be selected as good candidates for Sclerotinia resistance.

Example 6: Introgressing Sclerotinia Resistance from Spring Brassica napus to Winter Brassica napus

The Sclerotinia resistant source 04DHS11418 was crossed to winter canola lines in bi-parental, 3-way or complex crosses (as shown, for example, in Table 9). The F1 cross was then backcrossed to an elite susceptible parent. At the BC1F1 generation, approximately 500 progeny (minimum number required to identify at least one individual with all favorable alleles present) were generated for each cross and submitted for marker analysis using the markers identified as being associated with Sclerotinia in spring canola. Each individual sample was examined for presence of the favorable alleles from the Sclerotinia resistant line 04DHS11418. The percentage of favorable alleles present in each sample was calculated and the top three from each population were used to cross back to the recurrent parent. This process was repeated again at the BC2 stage. In addition, selections were intermated to develop populations in which individuals could be identified with homozygous desirable Sclerotinia alleles.

TABLE 9

Example of introgression of Sclerotinia resistance into winter B. napus using

marker-assisted selection

N9
N7
N7
N18
N18
N1
N1
N11
N19
N12
N12
Number of
Percent

Sample
AG
AG
BG
CA
UB
AG
BG
AG
BG
AG
CA
favorable
favorable

Name
0378
0510
1439
0739
0315
0482
0988
0370
1241
0359
0423
alleles
alleles

Example Set 1 MAS Results

Fe-
04DHS11418
b
a, c
a, b
a, d
c
a, b
c
a
d
b
b, d

male
WC-058
b
a, b
a
g
a
d
a
d
e
a
a, e

Male
WC-022
b
a, b
a
g
a
d
e
a
e
a
e

WC-663
b
a, b
a
c, g
a
d
a, e
d
e
a
d, e

09-
b, d
b
a, b
a, d, g
a, c
b, d
c, e
a
d, e
a, b
b, d, e
9.5
86.4

CMAS-

01-1641

09-
b, d
a, b
a, b
a, d, g
a, c
a, d
c, e
a
d, e
a, b
b, d, e
9.5
86.4

CMAS-

01-1737

Example Set 2 MAS Results

Male
04DHS11418
B
a, c
a, b
a, d
c
a, b
c
a
d
b
b, d

WC-058
D
a, b
a
g
a
d
a
d
e
a
a, e

Fe-
WC-227
D
a, b
a
d
a
d
b
a
d
a
a, e

male
09-
b, d
a, b, c
a, b
a, d
a, c
a, b, d
c, e
a
d
a
a, b, d, e
10.0
90.9

CMAS-

01-715

09-
b, d
a, b
a, b
a, d
a, c
a, b, d
c, e
a
d
a, b
a, b, d, e
10.0
90.9

CMAS-

01-929

Example Set 3 MAS Results

Male
04DHS11418
B
a, c
a, b
a, d
c
a, b
c
a
d
b
b, d

WC-058
D
a, b
a
g
a
d
a
d
e
a
a, e

Fe-
WC-063
D
a, b
a
d
a
d
a
c, d
d
a, c
d, e

male
WC-457
D
a, b
a, b
c
a
b, d
a
c, d
e
a
d, e

WC-227
D
a, b
A
d
a
d
e
a
c, d
a
a, e

09-
b, d
a, b, c
a, b
a, d
a, c
a, b, d
a, c
c, d
d
a, b
a, b, d, e
9.5
90.5

CMAS-

01-1205

09-
b, d
a, b
a, b
a, d
a, c
a, d
c, e
a
d, e
a, b
b, d, e
9.0
85.7

CMAS-

01-1234

09-
b, d
a, b, c
a, b
a, d
a, c
a, d
c, e
a
d, e
a, b
d, e
9.0
85.7

CMAS-

01-1241

Example 7: Mapping Population #2

The parents used for the mapping population were 06DSB13911 (a Sclerotinia resistant double haploid) and PHI2008HS1 (a susceptible spring canola DH line polymorphic to the resistant line, selected for having a similar agronomic phenotype with consistent high susceptibility in the same testing environment where the 06DSB13911 line was selected for resistance) (see Table 10). These lines were used to develop a double haploid mapping population consisting of 187 progeny.

TABLE 10

Measuring field performance under extreme

disease pressure (research trials)

Disease

Rating

incidence
Spring
Mapping

SSDIS**
Category
SSDI %*
Checks
Parents

1.0
Highly susceptible
≧80
44A89 = 1

1.1-2.0
Susceptible
79-70
46A65 = 2
PHI2008HS1

2.1-3.0
Moderately
69-60
46A76 = 3

3.1-4.0
susceptible
59-50

4.1-5.0
Moderately
49-40

5.1-6.0
resistant
39-30

6.1-7.0
Resistant
29-20

7.1-8.0

19-10

06DSB13911

8.1-9.0
Highly resistant
9-0

*SSDI % Sclerotinia sclerotiorum Disease Incidence %. SSDI % is UNSSDI (where UNSSDI is the percentage of plants in a population infected with Sclerotinia) rating adjusted for a deviation from the expected mean of checks 06DSB13911 (15%) and PHI2008HS1 (75%). This rating is used only under controlled extreme disease pressure field research conditions. It is calculated by multiplying the observed SSDI % by Factor X, where Factor X is the factor that brings the average SSDI % of the appropriate checks to 45%.

**SSDIS Sclerotinia sclerotiorum . SSDIS is the UNSSDI rating adjusted for a deviation from the expected mean of check parents for spring canola under extreme disease pressure. This rating is used only under controlled extreme disease pressure field research conditions. It is calculated by multiplying the observed UNSSDI by Factor X, where Factor X is the factor that brings the average SSDI % of the appropriate checks to 45%. Adjustment for severity is done after incidence adjustment.

UNSSDS is a rating of the extent of disease development on an affected plant. Two scales are used in the invention. The Pioneer SSDS scale ranges from 1 (dead) to 9 (no disease) and the Public scale ranges from 0 (no disease) to 5 (dead) plant. For details of the Pioneer SSDS scale, see Table 15 of WO 2006/135717. The Public scale is provided as follows: 0 = no disease; 1 = superficial lesions or small branch affected; 2 = large branch dead; 3 = main stem at least 50% girdled; 4 = main stem girdled but plant produced good seed; 5 = main stem girdled, much reduced yield.

Both parents have good standability. Therefore, standability or lodging resistance is fixed, thus eliminating this variable in the mapping process. The choice of a highly susceptible line resulted in a population without transgressive segregation (i.e., all resistance came from 06DSB13911 and no DH progeny lines were more resistant than the resistant parent). Over a period of three years, the population was phenotyped in the field and genotyped with SSR molecular markers. Phenotyping was carried out as described in WO 2006/135717, the entire teachings of which are hereby incorporated by reference.

Example 8: Sclerotinia Screening

Disease Scoring

The plants of the double haploid mapping population created, as described in Example 7, were rated for disease as described in Table 4 of Example 3. The unadjusted parameters (e.g., UNSSDI and UNSSDS) showed year to year variation due to environmental variation such as positional variation in the field and weather conditions. Such variation would be expected by one skilled in the art.

Example 9: Genetic Mapping and QTL Analysis

Genetic Mapping

Genetic mapping has placed 278 molecular markers to 19 linkage groups (Lg) that correspond to 19 canola chromosomes and public linkage group nomenclature. The linkage map covers ˜1100 cM.

QTL Analysis

QTL analysis using simple interval mapping and composite interval mapping (CIM) (Zeng (1994), Genetics 136:1457) identified 12 linkage groups (N1, N3, N4, N8, N9, N10, N11, N12, N13, N15, N18 and N19) contributing to whole plant field resistance to Sclerotinia. In addition, regions identified by interval mapping as being associated with Sclerotinia resistance were confirmed by single-factor analysis of variance (PROC GLM, SAS Enterprise Guide 4.2) on Sclerotinia parameters at the P≦0.01 significance level. These QTLs are identified in Tables 11 and 12 below. As shown by the “Phenotypic Variation Explained” values in Table 12, some QTLs had a larger effect on Sclerotinia resistance than others.

TABLE 11

Markers significantly associated with Sclerotinia resistance at P ≦ 0.01.

Map

Linkage

Position

Group
Marker
(cM)
Parameter
Year

N1
CA0614
8.2
UNSSDI
2010

UNSSDS, SSFS, SSDIS
2009

BG0111
10.9
UNSSDI, SSDI %
2010

BG1392
22.7
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSDI %, SSDIS
2010

BG1182
35.6
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDI, SSDI %
2010

BG1090
41.9
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDI, SSDI %
2010

AG0093
46.3
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

BG1453
53.1
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

PE0017
53.5
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

AG0391
56.9
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

AG0304
56.9
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

UB0163
57.0
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

PE0203
57.5
UNSSDS, SSFS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

AG0482
68.5
UNSSDI
2010

PE0177
73.6
UNSSDS, SSFS, SSDIS
2009

UNSSDI
2010

N3
CA0410
0.0
UNSSDS, SSDIS
2009

UNSSDI, SSFS
2011

BG1368
2.2
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSFS
2011

BG1197
37.3
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSFS
2011

UNSSDI, SSDI %
2010

AG0272
40.1
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSDI %
2010

AG0023
40.1
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSDI %
2010

N4
BG1442
0.0
UNSSDS, SSFS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDI, SSFS, SSDI %, SSDIS
2011

UB0181
3.9
UNSSDS, SSFS, SSDIS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

UB0126
10.3
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2011

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

AG0477
10.8
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDI, SSFS, SSDI %, SSDIS
2011

BG1127
11.0
UNSSDS, SSFS, SSDIS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDI, SSFS, SSDI %, SSDIS
2011

AG0125
14.2
UNSSDS, SSFS, SSDIS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDI, SSFS, SSDI %, SSDIS
2011

BG1244
15.0
UNSSDS, SSFS, SSDIS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

SSFS
2011

AG0239
15.2
UNSSDS, SSFS, SSDIS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

SSFS
2011

AG0203
16.1
UNSSDS, SSFS, SSDIS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

SSFS
2011

BG0106
18.5
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

SSFS
2011

N8
CA0837
0.0
UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2009

BG0647
3.1
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDI
2011

AG0070
3.1
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDI
2010

UNSSDI
2011

PE0281
3.2
UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2009

UNSSDI
2011

AG0324
4.6
UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2009

BG1101
7.3
UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

AG0328
15.6
UNSSDS, UNSSDI, SSFS, SSDIS
2009

BG1449
25.9
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

BG1062
25.9
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

AG0410
30.3
UNSSDS, SSFS, SSDIS
2009

BG1286
33.2
UNSSDS
2009

N9
CA1034
0.0
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

CA0834
0.0
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

AG0378
0.1
UNSSDI
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

BG1123
33.4
UNSSDI, UNSSDS, SSFS, SSDIS
2011

AG0323
45.8
UNSSDI, UNSSDS, SSFS, SSDIS
2011

BG0295
49.5
UNSSDI, UNSSDS, SSFS, SSDIS
2011

AG0441
66.6
UNSSDI, UNSSDS, SSFS, SSDIS
2011

N10
BG0228
0.0
UNSSDS, UNSSDI, SSFS, SSDIS
2009

PE0355
5.1
SSFS
2009

AG0171
7.1
UNSSDS, SSFS
2009

BG0651
13.6
UNSSDS, SSFS
2009

BG0255
17.9
UNSSDS, SSFS
2009

SSDI %
2011

AG0047
19.9
UNSSDS, SSFS
2009

SSDI %
2011

UB0196
29.4
SSDI %
2011

UB0015
29.5
SSDI %
2011

PE0131
36.8
SSDI %
2011

N11
CA0120
20.6
UNSSDS
2010

BG0452
32.8
UNSSDS
2010

UNSSDS, UNSSDI, SSFS, SSDIS
2011

BG0031
34.1
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDS, UNSSDI, SSFS, SSDIS
2011

CA1035
37.5
UNSSDS, SSFS, SSDIS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDS, UNSSDI, SSFS, SSDIS
2011

CA0546
37.5
UNSSDS, SSFS, SSDIS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDS, UNSSDI, SSFS, SSDIS
2011

CA1032
37.5
UNSSDS, SSFS, SSDIS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDS, UNSSDI, SSFS, SSDIS
2011

BG1149
37.8
UNSSDS, SSFS, SSDIS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDS, UNSSDI, SSFS, SSDIS
2011

BG1230
39.1
UNSSDS, SSFS, SSDIS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDS, UNSSDI, SSFS, SSDIS
2011

BG1513
42.0
UNSSDS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDS, UNSSDI, SSFS, SSDIS
2011

CA0328
42.3
UNSSDS
2010

UNSSDS, UNSSDI, SSFS, SSDIS
2011

PE0324
49.1
UNSSDS
2010

UNSSDS, UNSSDI, SSFS, SSDIS
2011

PE0283
53.1
UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2009, 2010

CA0163
56.6
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

N12
BG1321
0.0
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDI, SSDI %
2010

UNSSDI, SSDI %
2011

PE0133
19.9
UNSSDS, SSFS, SSDI %, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDI, SSDI %
2011

CA0456
21.2
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDI, SSDI %
2011

PE0063
27.2
UNSSDS, SSFS, SSDI %, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDI, SSDI %
2011

CA1027
27.2
UNSSDS, SSFS, SSDI %, SSDIS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDI, SSDI %
2011

BG0864
28.9
SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDI, SSDI %
2011

CA1090
28.9
SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDI, SSDI %
2011

CA0991
28.9
SSFS, SSDI %, SSDIS
2009

UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDI, SSDI %
2011

N13
CA0603
0.0
UNSSDI, SSFS, SSDI %, SSDIS
2010

BG1288
2.9
UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDS, SSFS, SSDIS
2011

CA0488
11.9
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDS, SSFS, SSDIS
2011

PE0012
13.1
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDS, SSFS, SSDIS
2011

PE0340
13.5
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDS, SSFS, SSDIS
2011

BG0516
22.1
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDS, SSFS, SSDIS
2011

AG0504
42.8
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDI, SSDI %, SSDIS
2010

UNSSDS, SSFS, SSDIS
2011

AG0148
55.5
UNSSDS, UNSSDI, SSFS, SSDIS
2009

CA0736
65.8
UNSSDS, SSFS
2009

N15
PE0286
0.0
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSDI %, SSDIS
2010

UNSSDI, SSFS, SSDI %, SSDIS
2011

PE0091
6.7
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDI, SSFS, SSDI %, SSDIS
2011

PE0187
15.2
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDI, SSFS, SSDI %, SSDIS
2011

CA0719
24.8
UNSSDI, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

UNSSDI, SSFS, SSDI %, SSDIS
2011

AG0369
43.9
UNSSDI, SSFS, SSDI %, SSDIS
2009, 2010

N18
BG0278
12.9
UNSSDI, SSFS, SSDIS
2009

CA0739
21.6
UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2009

UNSSDI, SSDI %, SSDIS
2010

UB0315
27.0
UNSSDS, UNSSDI, SSFS, SSDI %, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

SSDI %
2011

CA0636
32.4
UNSSDS, UNSSDI, SSFS, SSDIS
2009

UNSSDI, SSDI %
2010

SSDI %
2011

N19
CA1107
0.0
UNSSDI, SSFS, SSDI %, SSDIS
2010

CA0552
16.1
UNSSDS, SSFS, SSDIS
2009

UNSSDI
2010

CA1066
19.7
UNSSDS, SSFS
2009

BG1241
27.6
UNSSDS, SSFS, SSDIS
2009

UNSSDI, SSFS, SSDI %, SSDIS
2010

CA0221
29.7
UNSSDS, SSFS, SSDIS
2009

TABLE 12

QTL interval, LOD score and explained phenotypic variation of QTLs

associated with Sclerotinia whole plant field resistance or improved whole plant field

resistance

Phenotypic

Variation

Linkage

LOD
Explained

Group
Parameter
Year
QTL Interval
Score
(%)

N1
SSFS, SSDIS
2010
AG0093-PE0203
2.9
7.8

SSFS, SSDIS
2009
BG0111-BG1392
3.5
8.4

SSDI %
2010
BG0111-BG1392
2.8
7.4

UNSSDI
2010
BG1090-AG0482
5.0
11.5

UNSSDI, UNSSDS, SSFS, SSDIS
2009
BG1090-PE0203
3.9
10.5

SSDI %
2010
BG1090-PE0203
4.7
11.1

UNSSDS
2009
CA0614-BG1392
5.5
13.0

UNSSDI
2010
CA0614-BG1392
3.2
12.5

N3
UNSSDS, SSFS, SSDIS
2009
BG1197-AG0023
4.0
9.6

UNSSDI, SSDI %
2010
BG1197-AG0023
2.2
5.4

SSFS, SSDIS
2009
CA0410-BG1368
2.8
6.7

UNSSDI, SSFS
2011
CA0410-BG1197
2.9
3.9

N4
UNSSDS, SSFS
2009, 2010
BG1442-BG0106
6.3
14.7

UNSSDI, SSDI %, SSDIS
2010
BG1442-BG0106
8.1
18.5

UNSSDI, SSFS, SSDI %, SSDIS
2011
BG1442-BG0106
7.6
11.1

SSDIS
2009
UB0181-BG0106
3.4
8.1

N8
UNSSDI
2010
BG1449-BG1062
2.3
6.0

SSDI %
2009
CA0837-AG0328
2.3
6.5

SSDIS
2009
CA0837-BG1062
6.6
15.1

UNSSDI
2010
CA0837-BG1101
2.3
5.6

UNSSDS, SSFS
2009
CA0837-BG1286
7.5
17.2

UNSSDI
2009
CA0837-BG1449
3.7
10.5

UNSSDI
2011
BG0647-PE0281
2.3
2.6

N9
UNSSDS, SSFS, SSDIS
2009
AG0323-BG0295
4.5
10.6

UNSSDI, SSDI %
2010
AG0323-BG0295
2.9
6.9

UNSSDI, SSFS, SSDI %, SSDIS
2010
CA1034-AG0378
5.3
12.2

UNSSDI, UNSSDS, SSFS, SSDIS
2011
BG1123-AG0441
3.2
5.7

N10
UNSSDS, SSFS
2009
BG0228-AG0047
3.3
7.9

SSDI %
2011
BG0255-PE0131
3.0
4.3

N11
SSFS, SSDIS
2009
BG0031-BG1149
4.4
10.5

UNSSDS
2009
BG0031-BG1230
4.1
10.5

UNSSDI, SSDI %
2010
BG0031-BG1230
3.4
8.5

SSFS, SSDIS
2010
BG0031-BG1513
5.3
12.2

UNSSDS
2010
CA0120-CA0328
5.4
13.9

UNSSDI, SSFS, SSDIS
2009, 2010
PE0283-CA0163
5.2
12.2

UNSSDS
2009
PE0283-CA0163
5.2
12.3

SSDI %
2010
PE0283-CA0163
2.4
5.6

SSDI %
2009
PE0324-PE0283
2.2
5.4

UNSSDS, UNSSDI, SSFS, SSDIS
2011
CA0328-PE0324
5.8
7.2

N12
SSFS, SSDIS
2009
BG1321-CA0991
5.1
13.8

UNSSDI, SSDI %
2010
BG1321-CA0991
4.6
11.6

UNSSDS
2009
BG1321-CA1027
3.7
10.2

UNSSDI
2009
BG1321-PE0133
2.7
6.5

UNSSDI, SSDI %
2011
BG1231-PE0133
4.1
6.6

SSDI %
2009
PE0063-CA0991
2.0
5.0

SSFS, SSDIS
2010
PE0133-CA0991
4.9
12.2

N13
UNSSDI
2009
BG0516-AG0148
2.9
9.7

SSFS, SSDIS
2009
CA0488-AG0148
7.3
21.7

UNSSDS
2009
CA0488-CA0736
9.8
27.3

SSDI %, SSDIS
2010
CA0603-AG0504
7.3
17.3

UNSSDS, SSFS, SSDIS
2011
BG1288-AG0504
4.6
6.8

N15
SSDI %
2009
CA0719-AG0369
2.7
8.5

SSFS
2010
PE0091-PE0187
4.2
10.0

UNSSDI, UNSSDS, SSFS, SSDIS
2009
PE0286-AG0369
7.1
17.8

UNSSDI, SSDI %, SSDIS
2010
PE0286-PE0187
7.4
17.2

UNSSDI, SSFS, SSDI %, SSDIS
2011
PE0286-CA0719
8.7
12.1

N18
UNSSDS, SSFS, SSDIS
2009
AG0285-CA0636
8.4
20.0

UNSSDI
2009
BG0278-CA0779
6.6
16.4

SSDI %
2009
CA0739-CA0636
2.9
7.0

UNSSDI
2010
CA0739-CA0636
3.3
8.0

SSDI %
2011
CA0739-CA0636
2.7
4.1

SSDI %
2010
UB0315-CA0636
2.6
6.4

N19
SSDIS
2009
CA0552-CA0221
2.9
9.8

UNSSDI, SSFS, SSDI %, SSDIS
2010
CA1107-CA0552
2.8
8.0

UNSSDS, SSFS
2009
CA1107-CA0221
4.3
13.5

Additional information about the SSR markers flanking the twelve QTLs associated with whole field plant resistance to Sclerotinia is shown in Table 14 of Example 12, where exemplary sets of forward and reverse primer sequences for each SSR are also provided. “Repeat” indicates the SSRs or SNPs associated with each marker. The positions of the SSRs are shown in the sequence information located in Example 12. Additional information about the alleles and allele size of each SSR marker flanking the 12 QTLs associated with whole plant field resistance to Sclerotinia is provided in Table 13.

TABLE 13

The alleles and allele size of each SSR marker

flanking the twelve Sclerotinia QTLs. as well as

favorable allele for Sclerotinia (SCL) resistance.

Favorable

Allele for

Linkage
SSR
Allele
Allele
SCL

Group
Marker
Name
Size (bp)
Resistance

N1
CA0614
a
160

CA0614
b
178
yes

CA0614
c
188

CA0614
d
196

BG0111
a
141
yes

BG0111
b
146

BG0111
c
147

BG0111
d
144

BG1392
a
251
yes

BG1392
b
257

BG1182
a
299

BG1182
b
301

BG1182
c
303
yes

BG1182
d
347

BG1182
e
345

BG1090
a
268
yes

BG1090
b
272

AG0093
a
221

AG0093
b
223
yes

BG1453
a
120

BG1453
b
122

BG1453
c
130

BG1453
d
132
yes

BG1453
e
134

BG1453
f
146

BG1453
g
148

BG1453
h
152

BG1453
i
154

BG1453
j
156

BG1453
k
172

BG1453
l
142

BG1453
m
138

BG1453
n
158

BG1453
o
162

BG1453
p
144

BG1453
q
136

BG1453
r
176

BG1453
s
114

BG1453
t
160

PE0017
a
81
yes

PE0017
b
84

PE0017
c
78

PE0017
d
87

AG0391
a
127
yes

AG0391
b
130

AG0391
c
139

AG0304
a
163

AG0304
b
226

AG0304
c
229
yes

UB0163
b
111

UB0163
c
129
yes

UB0163
e
107

UB0163
f
117

PE0203
a
205

PE0203
b
209

PE0203
c
211
yes

PE0203
d
203

PE0203
e
207

AG0482
a
278

AG0482
b
281

AG0482
c
284

AG0482
d
287
yes

AG0482
e
272

PE0177
a
197

PE0177
b
199
yes

PE0177
c
201

PE0177
d
205

N3
CA0410
a
140

CA0410
b
142
yes

CA0410
c
148

CA0410
d
154

BG1368
a
128
yes

BG1368
b
131

BG1197
a
262

BG1197
b
267
yes

BG1197
c
272

BG1197
d
286

AG0272
a
151
yes

AG0272
b
157

AG0272
c
163

AG0023
a
128

AG0023
b
131
yes

AG0023
c
139

AG0023
d
145

AG0023
e
148

N4
BG1442
a
232
yes

BG1442
b
238

BG1442
c
250

BG1442
d
258

BG1442
e
266

BG1442
f
242

BG1442
g
254

BG1442
h
230

UB0181
a
147

UB0181
b
283

UB0181
c
369

UB0181
d
379
yes

UB0181
e
275

UB0181
f
285

UB0181
g
289

UB0126
a
216
yes

UB0126
b
220

UB0126
c
230

AG0477
a
268

AG0477
b
276

AG0477
c
278

AG0477
d
289
yes

AG0477
e
292

AG0477
f
285

BG1127
a
284

BG1127
b
290

BG1127
c
304
yes

BG1127
d
306

BG1127
e
292

BG1127
f
294

BG1127
g
302

AG0125
a
212

AG0125
b
238

AG0125
c
255

AG0125
d
264
yes

AG0125
e
267

AG0125
f
225

BG1244
a
247

BG1244
b
284
yes

BG1244
c
275

AG0239
a
295

AG0239
b
311

AG0239
c
314
yes

AG0203
a
206

AG0203
b
209

AG0203
c
215

AG0203
d
221
yes

AG0203
j
213

AG0203
k
203

AG0203
l
227

BG0106
a
216

BG0106
b
286

BG0106
c
310
yes

BG0106
d
288

BG0106
e
276

BG0106
f
284

BG0106
g
280

N8
CA0837
a
257

CA0837
b
269

CA0837
c
253
yes

CA0837
d
279

CA0837
e
283

BG0647
a
247
yes

BG0647
b
250

BG0647
c
259

BG0647
d
272

BG0647
e
275

BG0647
f
244

AG0070
a
270
yes

AG0070
b
274

AG0070
c
272

PE0281
a
179

PE0281
b
209
yes

PE0281
c
211

PE0281
d
213

PE0281
e
233

PE0281
f
221

PE0281
g
215

PE0281
h
236

PE0281
i
203

PE0281
j
194

PE0281
k
206

PE0281
l
249

AG0324
a
226
yes

AG0324
b
229

AG0324
c
244

BG1101
a
210

BG1101
b
219
yes

AG0328
a
222

AG0328
b
228
yes

AG0328
c
255

AG0328
d
258

AG0328
e
267

AG0328
f
270

AG0328
g
279

AG0328
h
276

AG0328
i
281

AG0328
j
273

AG0328
k
287

BG1449
a
132

BG1449
b
134

BG1449
c
158

BG1449
d
160
yes

BG1449
e
164

BG1449
f
156

BG1449
g
170

BG1062
a
188

BG1062
b
210

BG1062
c
212
yes

BG1062
d
186

BG1062
e
214

BG1062
f
216

BG1062
g
224

BG1062
h
208

BG1062
i
196

BG1062
j
176

BG1062
k
206

BG1062
l
218

BG1062
m
202

BG1062
o
182

BG1062
p
222

AG0410
a
322

AG0410
b
325

AG0410
c
328
yes

AG0410
d
334

AG0410
e
337

AG0410
f
331

BG1286
a
156

BG1286
b
159

BG1286
c
162
yes

BG1286
d
165

N9
CA1034
a
275
yes

CA1034
b
290

CA1034
c
293

CA1034
d
306

CA1034
e
309

CA1034
f
321

CA1034
g
299

CA1034
h
284

CA1034
i
278

CA1034
j
315

CA1034
k
296

CA1034
l
324

CA1034
m
327

CA1034
n
287

CA0834
a
274
yes

CA0834
b
289

CA0834
c
292

CA0834
d
301

CA0834
e
304

CA0834
f
307

CA0834
g
298

CA0834
h
319

CA0834
i
283

CA0834
j
313

CA0834
k
295

CA0834
l
277

CA0834
m
286

CA0834
n
325

CA0834
o
322

AG0378
a
275

AG0378
b
281

AG0378
c
290

AG0378
d
293
yes

AG0378
e
284

AG0378
f
312

AG0378
g
300

AG0378
h
295

BG1123
a
202

BG1123
b
216

BG1123
c
220
yes

BG1123
d
252

BG1123
e
254

BG1123
f
256

BG1123
g
222

BG1123
h
226

BG1123
i
234

BG1123
j
258

BG1123
k
218

BG1123
l
224

BG1123
m
264

BG1123
n
248

AG0323
a
210

AG0323
b
216
yes

AG0323
c
219

AG0323
d
231

AG0323
e
213

AG0323
f
222

AG0323
g
201

AG0323
h
228

AG0323
i
195

BG0295
a
286
yes

BG0295
b
288

BG0295
c
283

AG0441
a
292
yes

AG0441
b
301

AG0441
c
283

N10
BG0228
a
137
yes

BG0228
b
143

BG0228
c
146

PE0355
a
217

PE0355
b
223
yes

PE0355
c
225

PE0355
d
237

PE0355
e
229

AG0171
a
241

AG0171
b
245
yes

AG0171
c
247

BG0651
a
230

BG0651
b
245

BG0651
c
253

BG0651
d
256
yes

BG0651
e
269

BG0651
f
248

BG0651
g
251

BG0651
h
275

BG0651
i
272

BG0255
a
180

BG0255
b
183

BG0255
c
186
yes

AG0047
a
293
yes

AG0047
b
311

AG0047
c
319

UB0196
a
281

UB0196
b
287

UB0196
c
293

UB0196
d
295
yes

UB0196
e
283

UB0196
f
275

UB0196
g
271

UB0196
h
277

UB0196
i
265

UB0196
j
289

UB0015
a
241

UB0015
b
243

UB0015
c
253
yes

UB0015
e
251

UB0015
f
255

PE0131
b
144

PE0131
c
148
yes

PE0131
d
152

PE0131
e
150

PE0131
f
154

N11
CA0120
a
138

CA0120
b
160

CA0120
c
172
yes

CA0120
d
163

CA0120
e
169

BG0452
a
197

BG0452
b
209
yes

BG0452
c
212

BG0452
d
215

BG0452
e
221

BG0452
f
194

BG0452
g
191

BG0031
a
225

BG0031
b
228

BG0031
c
237
yes

CA1035
a
255

CA1035
b
258

CA1035
c
282

CA1035
d
294

CA1035
e
297
yes

CA1035
f
285

CA1035
g
306

CA1035
h
300

CA0546
a
110

CA0546
b
120

CA0546
c
123

CA0546
d
146
yes

CA0546
e
149

CA0546
f
126

CA0546
g
144

CA0546
h
112

CA1032
a
203
yes

CA1032
b
211

BG1149
a
260
yes

BG1149
b
266

BG1149
c
263

BG1230
a
252

BG1230
b
288
yes

BG1513
a
164
yes

BG1513
b
214

BG1513
c
216

CA0328
a
237

CA0328
b
240

CA0328
c
252
yes

CA0328
d
255

CA0328
e
258

CA0328
f
234

CA0328
g
264

PE0324
a
258

PE0324
b
270
yes

PE0283
a
149
yes

PE0283
b
167

PE0283
c
173

PE0283
d
176

CA0163
a
311

CA0163
b
317
yes

N12
BG1321
a
197
yes

BG1321
b
200

BG1321
c
330

PE0133
a
131

PE0133
b
141

PE0133
c
147
yes

PE0133
d
153

PE0133
e
133

CA0456
a
179

CA0456
b
185
yes

PE0063
a
114
yes

PE0063
b
126

CA1027
a
297

CA1027
b
300
yes

CA1027
c
303

CA1027
d
306

BG0864
a
169
yes

BG0864
b
175

BG0864
c
185

BG0864
d
191

BG0864
e
193

BG0864
f
197

BG0864
g
195

BG0864
h
187

CA1090
a
282

CA1090
b
288
yes

CA1090
c
292

CA1090
d
295

CA1090
e
279

CA0991
a
162

CA0991
b
165
yes

N13
CA0603
a
179

CA0603
b
182
yes

BG1288
a
199

BG1288
b
205
yes

CA0488
a
202

CA0488
b
217

CA0488
c
237

CA0488
d
231
yes

CA0488
e
234

CA0488
f
240

PE0012
a
115

PE0012
b
134
yes

PE0340
c
279
yes

PE0340
d
281

PE0340
e
277

PE0340
f
255

PE0340
g
257

BG0516
a
165
yes

BG0516
b
170

BG0516
c
179

BG0516
d
154

BG0516
e
182

BG0516
f
173

BG0516
g
158

AG0504
a
320

AG0504
b
332
yes

AG0504
c
338

AG0148
a
268

AG0148
b
272

AG0148
c
280
yes

AG0148
d
286

CA0736
a
324
yes

CA0736
b
474

N15
PE0286
a
186

PE0286
b
194
yes

PE0091
a
152

PE0091
b
164

PE0091
c
176

PE0091
d
180
yes

PE0187
a
176

PE0187
b
178

PE0187
c
180

PE0187
f
182
yes

PE0187
i
184

CA0719
a
300

CA0719
b
304
yes

AG0369
a
180

AG0369
b
184
yes

AG0369
c
186

N18
BG0278
a
239

BG0278
b
241
yes

CA0739
a
220

CA0739
b
222

CA0739
c
232

CA0739
d
234
yes

CA0739
e
240

CA0739
f
224

CA0739
g
236

CA0739
h
242

UB0315
a
127

UB0315
b
133

UB0315
c
131
yes

BG0278
a
239

BG0278
b
341
yes

CA0636
a
257

CA0636
b
263
yes

N19
CA1107
a
225

CA1107
b
228
yes

CA1107
c
328

CA1107
d
216

CA0552
a
192

CA0552
b
195
yes

CA0552
c
204

CA0552
d
207

CA1066
a
196

CA1066
b
217

CA1066
c
232

CA1066
d
235

CA1066
e
238

CA1066
f
241
yes

CA1066
g
244

BG1241
d
329
yes

BG1241
e
370

CA0221
a
253
yes

CA0221
b
265

CA0221
c
271

Example 10: Introgressing Sclerotinia Resistance from Spring Brassica napus to Winter Brassica napus

The Sclerotinia resistant source 06DSB13911 is crossed to winter canola lines in bi-parental, 3-way or complex crosses. The F1 cross is then backcrossed to an elite susceptible parent. At the BC1F1 generation, approximately 800-1000 progeny (minimum number required to identify at least one individual with all favorable alleles present) are generated for each cross and submitted for marker analysis using the markers identified as being associated with Sclerotinia in spring canola. Each individual sample is examined for the presence of the favorable alleles from the Sclerotinia resistant line 06DSB13911. The percentage of favorable alleles present in each sample is calculated and the top three from each population are used to cross back to the recurrent parent. This process is repeated again at the BC2 stage. In addition, selections are intermated to develop populations in which individuals can be identified with homozygous desirable Sclerotinia alleles.

Example 11: Use of Sclerotinia Resistant Lines for Hybrid Seed Production

Sclerotinia resistant lines with scores of 5 and higher for SSDIS are selected for use in hybrid seed production and hybrid testing. Production of these seeds can be done according to methods known to the skilled person and are described, for example, in WO 2006/135717.

Example 12: Marker Sequences Containing Polymorphisms, and Exemplary Primers

Set forth below is sequence information for markers of QTLs significantly associated with Sclerotinia whole plant field 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 14 below and sequences in brackets indicate polymorphic regions (SSRs, SNPs).

AG0023

(SEQ ID NO: 1)

CGAATTCGCCCTTCTCTTGCTTAGATCTGGACTAACTACTTCnnAAAGAAAACATTnnnTTAATGTTTAT

GTCGAATGTCATTTATGCTGAACAAAATAACCTTGAAAATATGTTCTGTAGGCTAAAGTTGGGAGAGAGA

AGGAGGTTGAAGAGATTTTGTCAAGATTGCGAGGAGAAAATTCTGATGTATCAGATGAGGCAGGAGAGAT

ATTAGTAAGCATATATATGCATGAATAATCATATGATCAATGTATATATTTTTTACTTCACAATATTTTG

ATGATCATCAGGCATATACAGAACATGTTAAACAACAAGGAGATGATCGCGGTTTCCTCAAGTTGTTTCA

GCGAAAATACGCGTTCTCACTTACTGTAATT[CTTCTTCTTCTTCTTCTTCTTCTTTTTCTTCTTCTTCT

TCTTCTT]TAATAACCCGTTTGGTTTACACAGATTGGAGTTGTTCTTATAGCTTTGCCTCAACTTGGAGG

TCTTAGTGGTTATTCTTTTTACACTGAGTCCATTTTCATATCTACAGGTAnnnTAACTCTTACTTCTTCA

ACAAAATCTTGATTTTTATATATTTATTTACCGTAACGATAATTGTTGATAATTACGnnnATCAGGTGTA

TCGAGTGATGTTGGATTCATATCGACATCTATAGTTC

AG0045

(SEQ ID NO: 2)

ACGAATTCGCCCTTCTCTTGCTTAGATCTGGACTAnnnnnTGATTTGCCCGCTATGTTCGACGGGTGGAG

ATTTTAGTTTTACTTCCTCGATCTGATTGTATGGGTTGGGAGTAGGGTCTAATATATCAACTGCGAGTGT

ATGTTCGTTTCCTCCTCAGTTTCGAAGTTGGGTTCTTATGTGTTTAGCCTAAGnnnCTGTGAnnnGnTAG

TTTTTTTTTAATCAGTTCCAACAGGATTCATTTCAGGnnnTTGGAACTTGTGTATATGTGTTAGCCTGAG

ATCTCTGTAGTGTCCGGAAATGATATTTnnnnATTATCATTAATTTAGTTCGAAGnATGAAGCTCAGTGT

TGTTGGACTTGTGTATATGGAGCTCGAAGAGTGAAGCTCAGTGCGTTTTCATCTGAGGATGATGATGATG

GAGCTAATGTGCTGAGCAATGAGAACTCGAGATGATAAGGCTTGAGGGACATGCCAGTGAGT[GAAGAAA]

CCGTCGGGCTATAGCTTAGT[GAAGAAGAAGAAGAA]GAGCTCGTGGAGTGATCAAATTTGCAGGTATG

CCCAAACTTGCCAATCCCACATTGTGGAGAATGGCTGCATTTTTACCACAAAGCTGTTTCTGTGGAGCCA

AAAATGAATGGAGGATAGTAAAACAGAACGTCATAATCAAATCAAGAAATTTTAACTTTTTTTGTCAGCA

CAAATTTnnnCTTTATCTTTAATTATTTAC

AG0047

(SEQ ID NO: 3)

CGAATTCGCCCTTCTCTTGCTTAnnnnCTGGACTAACAACAATTCCAAAATACTAATTCACAAACTTTGT

TTACAATCCAAAGAAAATCCGCTCTTTTGAAGCGCGGATCAAGATCTAGTGTTATAATATATCTAGAACA

TGGGAGTTTGGTCCAATGAACTACTGTATAGTTCTATCGAAATTTTTGAGTGATAAGATTGAAGCTCCAG

CACTCACTTATCTATTTGAGAAGCAAATAATAGAAAAAGAAGTAGATTTGAGGAAGAGATGATGGAGTTG

AACAAGGAGCTTTAAGATTTGAGTTCTGACAGTGTAGAAGCTGCAATACTGAGGCACCAAGGAAGAAAT

[CATCATCATCATCATCATCATCATCAT]CAAGAATTAGTTTCAGTTCATATCCACAACCATTTTTTCTTT

CAAAGAAATTTGCTGGTAGTAATTTTGAAGTTGTAAATTTTACATTTTCAGTGTTTCATTTTTCTCACGT

TTTCTTAATAATTGTTTACTTGCCAAATGATTCCATCACTTGGAAACTCACTATTGTTTGACATTTTGGT

GTGCTTAAGTGACTCTTTTCGAGTATTCATACATTATAGAAATTGTTTGGGACAACAGGTAAGAATTGCT

TGGCACAAGTAATGGCATCCCTCCCTGCAAATATATATAAATATTACAGTTGTCCTGGAACTTTTnnnnT

CTATCCTCTGCTGACAGGATGAGATATATGCATATAGAATATTAACTTCnnTCnGCCCGTATGTTCATGG

ATGnnnAGCTCCAT

AG0070

(SEQ ID NO: 4)

CGAGGAGTTGAATGACCCTGACTGTACTTTGGCCTCGAGACAGTCCCATCAAGAATAATTTACTGGGTCG

ATTTTTATTTTTAATTCTGGTCGAGCCAACTCCGAACTGGTCGAGCGGGATTTTTTAATTCTGGTCGACC

AAAATCATATCCGCTCGTGAGGGTCTTTACAACCACCATCACCACACTCGGACGATCACCCCACCACCAC

TTGGACGA[CCACCACCACCACCA]CCGGCGGCTCGGCTAGCTCTCGGGGGGCTCGCGGCGAG[GAGAGA]

GGAAGATATCCNACGGAAAGAGAAAAGAGAGG[GAGAGAGAGAGAGA]GGCGTGAGAGAAGAAGAGAGA

AAAGGAAAAGAGAAGCTTGACGGCTAGGGTTTCCTAGTCTCTATAAATTCCTGCAGAGCTTCACTCAAGT

TTCAGAATGAGAGAAGAGTAAGAGGAGGCAGCTTCATTTATAGAAACAGGAGGAAACCCTAGGTCATTTA

CCCTAATGGGCTGCAGTCTTAATGGGCTCTCCTTAAGAAAATTTTGGGCTAGGAACCGGGACGTTACAAT

AATGCTTCTTATGAATATGTCTGAGTAGTTCTTTTGTTAGATTTAGGGTTCTTCAAGGGGTGAATTATGG

TTTGCTANATTTATATTGTTGTTTGTGTGATTT

AG0086

(SEQ ID NO: 5)

GCTCGCCGACTTCGGAGTCGCCTCGCCGCTCGATATCCCTTTCAGCCTCGCCTCCATCTCTTTTCCCCAA

ACTCTAGGCTGTTGCTGTTGCGTCGCCGCCGCCGCCGCCGTGGCTGTTATCGAGCTATTTGATCTACCGT

ACAGCATTTTAAACCGTTGATCAGATTCGGGATCAGACTTTGTCGTCACCGGAGGGCTCTTGATCGGCGG

TTGCACTTCCCCCTCCGTACACGGCGTACAATGTCGGTAAGCACCGGAAGCTTTCAGAGCCATATCTTTG

AGCTGAGAATGAATTTACGAAAATACCCTTGATCAGTATAGAGAATGACAAGAGGTGGAGGATGAGCAAA

[GAGAGAGAGAGAGAGAGAGA]AGTCTACCTGAGATGTTAGAGATTTGGCTTGCTTGGAATCCGGATCGT

CGGGTTGACCCGAGGTTTCATCGCCGGCTCGCTTCGAACGAGCTATACAAGTCAGCATTTTCCGGCAGCT

GCTGTTTCTTGGTAATGTGATTTTGTTTCTTCTCTTTTTGGATACG[GAGAGA]CAGTAGATGCTGTCAG

TTTCTAACTTTGGTTTGTGTTGTGTGTTTGGTCATGGTGCTCTTTTTATGTTTTATACTCACTTTACCAN

NGAAAACGGTTCCATTTTTTTAA

AG0093

(SEQ ID NO: 6

TAGGAGATGAGATGTACTGTTGCTTAGGGCTCTTATTTCTCTTGAAACTAGAATAAGCTGCCATCGGGTC

GGTGTAATAATCAAACCTTGGCTTATCATACGATTCCTGCTGATGTATGGATGCTTCAGCCAAGGGATTT

GAGAGGTGACTTGTGTTCATAGAGGTTCCAAGCTCTGTAGAACCATCATTCTCTG[CAGCAGCAGCAGCA

GCAG]CTTCCATCCGCATTGCTTTTAGCATTT[CTTTTCTTTT]CTCTGAATCTTCCATTACTGCTCAGC

TTCAAAGCTAATCAACTACAAAAATATAAACTTTTTTTCGAAATTATCAATCGAATCGCACCAAAAGAGC

TAAGATCTCCACGCGAGAACAATCTAACTAACCCTAAACCCCCAAATTATCCCAAACTCTGTACGGATAC

TCAAATTGGAAAAGCGAAATTGAGAGGATGCTAACCTTGGTTTACTCAACTTCTTCACTTCCTGGTCGCC

AGAGGTAGAGGATGAATGACAAGTGAAAACCCAGAACACGATGATGACGACAACNAAGCCTCCACAAATA

AATATAANACCCGGTTCGTGTTCGACCGTGTTTTTCCNATTAAAACCGGTTTACGGCGATNAGAATCATA

AACCAAATACGATNATCACGAAGGGTGACGATTAANACGAGACTTCCCAAAACCGGTTCGT

AG0125

(SEQ ID NO: 7)

CTGTTGAGGGGAGGAAACAAGAGCCTGGGAGGAGAACTCCTTGCTGGGGAAGACGAGATCATTCTCCTCA

GAGGCAATGGATTCACCTAAGACCACAGCGTTTAACTGAGAGATCTTGCCGGCACCTGAGGGTAGCAGAG

ACATGGACTCCACATGCCTTAGGTGATTGGATGACATTGTCTTACACCGGAGAAGTTTGTCCAACGGAGA

TGATCTGCCACACCCTTACAAGTCAGATGTCATTTGAATAAAATTTAAAACAAAACCACAAATGTCTTTT

TGACTTATTTATCAAAACTGCCTAAACCCCAAACCCAAT[CATCATCATCATCATCATCAT]AACCATAT

TCATCAATCATTCTATCATTATTGTCATCATTGGATCAGATTATTCATTCACCTTTGAGAAGCCGGAAGA

ATCCGAGATCCAAGTGATCCGCTTGTTTTCAGATCCTGAAGAAACAAAAACAGATCANAGGCGAATATTC

TTTTTTGATTACNATCAGATCATAAGAAGAAGAANAGATTGAAACTTTCGTANACCCAAAACATATCATT

ATGACNAAAGATCACATCTTTAACTCCNATGATCCCTAAGATTCGACTTACAGGTCGAGAACGAAGAGAG

GAAATTTTTTGAAAAATTGTAAGAAGGGGCG

AG0148

(SEQ ID NO: 8)

CTTGAGAGAGAGATTGAAAGTGAGCTGCGCCAAGAACCAAAACGGACATATCACAAGGCTTACTTTAGCA

ACACGCATCCTTGTCCAACGTCCCTTCTCATCTAAACCTTTAGCACAAATCTCACCACTAACATCAAGCT

CTACACTTTCCACACCAGATTCAATCCCACTACCATCTCCCTCCTCTTCTCTAACAACAACCCCTGATCC

TTCAGACACATCTGACA[CCACCACCACACCACCTCCTCCT]AGCAAAACATCCTTCTGTGACTCAGCCG

ACTCCTGCAAATGAGACCTTTTGCTTCTCCGAAACAGTATACTCCCATACGCATCTGCTAAATACCGACC

GATCTCGAAGAGAGGAAGGTTAGTCCAGATCTAAGCAAGAGAAGGGCGAATTCGCGGCCGCTAAATTCAA

TTCGCCCTATAGTGAGTCGTATTACAATTCACTGGACCGTCGTTTTACAACGACATGACTGGGAAAACCC

TGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCATCTGGCGTAATAGCTAACAGGCC

CGGACCGATCGCCCTTCCCAACAGTTGCGCAGCCTATACGTACGGCGGATTAAGGTTTACACCTATCAGA

GAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCGGGG

AG0171

(SEQ ID NO: 9)

CACTTTGCATAAATACTTTTACGAGCAATTTTAAAAAAAAATTCTAAAAATGTCTATTATTTGTGGACTT

GGAATAGCCGTTTATCTGCTTTGATTTTGTCGTTTCTTAAATCAAAGTCCTAATCAGGGTCCTTATATAA

GATCAGTCCTCTAGAATCTGAATAGCTTTTTAAGACAAAAAAAAAACAAAACAGATTAGAGTCCGAATCG

GACTCGAACATCTCCAATTTAACTTCTATCTTTTTTTTTTCTAAAATAAAATGTAAAATAAAAATATTTT

AATTGTATGAAAAATTGCATTCAATAGCTAAAAAAAAATAAAAATTCAATACATAACTAAAATCCCACTT

T[CTCTCCTCTTTTCTCTTCCTATCTCTCTCTTCTCTCTCTCT]AAAAATCTAATTTTTCTTTTTTTTTC

TGGTTATTCCCTAAATAAGCCCTAATTGTATTCTATTTTCACTCTAAAAAATAGCTTGATTTTATAAATA

GAATAATTCATTTGTTTTTTTAAAAATAAATTATCATTAGAATATAATTTAACTTTATTATAAAATTATT

CTCTTTTAGAGCAAAAAAATAAAATAAACCATTAGAAATTGTTTTAGAGAAATCATCAGTCAAAATCTCA

CNNNTTCCACTATTTAGTTTCATCTCTNNNGCAATCAGACGTAAGAACACAAAAACATATGTTAT

AG0203

(SEQ ID NO: 10)

CAGCCATTCTCTATGGCCTTGGTGACCATGGAGATCAAGTGCCTTAACAGCAACCGGTTGGGCTTCTAAA

CCCGGTTTAACCTTGTCATCAATGAACCCTTTGTAAACTGGCCCAAACCCTCCTTCTCCCAGCATGTTAC

TTCTTGAGAAATTATGCGTAATAACTCTCAGCTCAGACAAGGTGAACATACGAAGCTTTTGAGATGTGGA

GGAGTTTGAGAGGTCATCCATGACCGACATGGGCGAGCTTGGATCACTTATGTCCGATAACGACAGCCTC

TTGATCACCGGACAAGTTCTTATTTTCATTGCGTTGCCCCTCTCCTCTACTTCGTATCTACTCGCGTTCT

TTGTCCTGTAACATCCTAAAAACAGAGATGTCAATGATGT[CTTCTTGTTCTT]GGTTACTGCCATTTT

[CTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTT]CAACTTTTTGGAGAA

AATGGAAGATAGATAGTGTTTTACTCTTTTTGATGTATCTTTTGTAGATTGCGTNNNNNTNNNAATTAAG

GGTGTTTTAGTTAACCTACTTGTCTTCAAAGTCCTGTATTTATAGNGGTTGTTGTGTCTTATTCGTAGTG

ACTACCTTGGAACTTTCTNNNGACATATACTGTN

AG0239

(SEQ ID NO: 11)

AAGCAACCTACGGTGTTCTCTTATTACCGAGTGCTATTAGGCTTGTCTTAATGTCCTAAACAATTACGTT

TTTACTTTGAATGTGAACCTGCTCTTGAACGAAATATCTTCATGACTTCATATCATTTGTTTAATTCTAC

TACTGTCTGGTTAAAAATGGTTGTATGTTGCATATATTGCTATTTTAACCACATGTAAGAAAAGAGAAAC

GATCCCACGTTTATACTCAAATTCAAGAATCCAACACAAATCCTCTCACAATTCTATTAGATTGGAAGAA

AGAAACTCATAAAATAATTATAAATAACAAAAGGACAAGTAAATAGAGTCTATCTGGAACATAAAAACAC

TAACGGGTCTTGTGGGGTTTACAGA[TTTTCTTCTTTCTCTTCTTGACAGATTTGGGTGTCTTGTTCTTC

CTAGCCTCTCTCTTCTCTTCTTTTACTTTCTTCTTGAGCTCTTTCCTTGCTGCTTTCTTGTCTT]CANGA

CCCAACTCCTCTTCACTCTCACTTCCACTTTCCTCTTCTTCNNNNTCACTTTCTTCTTCTCCTTCTCCTT

TTTCTTCCTCTTTTCCCTCTTCAACATCAACCTTTACTNNCTGCTCATCTACTTTAGGANNNCATCCTTA

NNN

AG0243

(SEQ ID NO: 12)

TTTAGAATCAACACAAGACATAGNCAANTTGATTTTGGCTACTGGGTATTAATAATCCCCAAAATCACAT

CCTTCAAACCGAACGAACAATCAACAAGAAAATAAGAGCTACGATTCAGAAAAAGCCTATGATCAAAACG

CCTAAAATAACNNNAAAAAAANNNGGAGCAATTTTAAACAAATGGAATTGAATAGTATGAGATGAGATGA

GAAACAAAAGAGAAAGCAGTGTGCATAGATCATCAGGGAAGCTAACCTGAAATGATTTGTTGATTGGGGG

ATGAGATTGTTGGAAGGGGACAGAGA[GAAGAAGAA]GGTTTGCTTGAAGACTCGAAAATTAAAGCTTGT

TAAG[GAAGAAGAA]GAG[GAAGAAGAA]GAGGATATAAATTGACATGGACCTATTAAATGCCCATTTTG

TTCTGNTTATTTACTTAAGATTGCCACTATGACCTTTGACTTTTGGACGGCGNNTGTAGCTAAGCTACTG

TTTCTTCATTAATCACGCTTGCCATGATTAGTTTTTTTTTTCCTCCTATAGNNTTCATANNTAGCCCGAA

ATTACTGACTTTTATGAGATAAAGATCGTATTTTTTTATTTCTTANNGTTTAATACCCT

AG0272

(SEQ ID NO: 13)

CTACTTCCAAAAGAAAACATTAAATTAATGTTTATGTCGAATGTCATTTATACTGAACAAAATAACCTTG

AAAATATGTTCTGTACGCTAAAGTTGGGAGAGAGAAGGAGGTTGAAGAGATTTTGTCAAGATTGCGAGGA

GAAAATTCTGATGTATCAGATGAAGCAGGAGAGATATTAGTAAGCATATATATGCATGAATAATCATATG

ATCAATGTATATATTTTTTACTTCATAATATTTTGATGATCATCACGCATATACAGAACATGTTAAACAA

CAACGAGATGATCGCGGTTTCCTCAAGTTGTTTCAGCGAAAATACACGTTCTCACTTACTGTAATT[CTT

CTTCTTCTTCTTCTTTTTCTTCTTCTTCTTCTTCTTCTTCTTCTT]TAATAACCCNNNTGGTTTACACAG

ATTGGAGTTGTTCTTATAGCTTTGCCTCAACTTGGAGGTCTTAGTGNGTATTCTTTTTACACTGAGTCCA

TTTTCATATCTACAGGTAAAATAATTCTTCTTCTTNNNCAAAATATTGATTTTTATATATTTATTTACCT

TAACGATAATTGTTGATAATTACNNNNATCACGTGTATCGAGTGATGTTGGATTCATATCGACATCTATA

GTTCNNNNNTTACCGATTTCGAGTGACCTTGTTTAGAGTTC

AG0304

(SEQ ID NO: 14)

ACAAACATAATTGCAATTAAACGGATAGTAAGGGTCACAGATCACACAATACTGCATCGAAGTTTTGTCA

TCAACACAAGTGCGCATCGTTTCATTCTTTTCTTTCTTCCGGCTTACCTGAGCCCGGCCGTGGCACAATC

TTCTTCAACAGACAGCGTTTAAATAAAACTTAACTTGGTAGGGCTGAGGATTCAAGAATCATTTCTTGTA

ATTCACTGGCACATCGTCGTCATCTTCTTCAAGATCACTAAACGTTACATCTTCATCCTCATCCACAACA

TGTTTGGTTGCTACGGTGGAGCTAACAGTTCCTGCATTATCTTCATCCTTCAACCAATCATCAA[CATCA

TCATCATCATCATCAT]CGACTTGAACGTCAATAACTCTAGGTGAAGATCCAGTGACTGGCTTGTCATGG

GGCGCTGGTGCTGGTTTTTCTTCAATCACAGGCTTGTCAACAACTTGTATCTCCTTGCTCTCGATTGGGT

GCTTCTCCGTCTCAACCTCNNNTGTATCACTCAAATGTATTGTTTCCANNNAGATGNANTTGACTGNNNC

TGGTGAAGACACAGTAAGTGGTTCCTCATTGGCA

AG0323

(SEQ ID NO: 15)

AATCATCCTAAATTTACCTAACCCATGGATTCAAAAGGTAACTAACTACTCGCTAATAGACATGATAAAC

CCAAAACCAATAGTGATTGAAGGTTAATCATGATTAGTGATCAAGATAATCCAATAAACACAAGACAAAG

ATGAAAAGAGGCTAAAGATTGAATCTTTCACCAAAATATGTTCATGTCTAGAGAAAACAAGATAGATCCT

AAGAATCTAACAATACTAAAAGCATGATAGTAAGCCCTCTAAGCGTGTCCACGTAAGTTAATATATTCAG

CTAATCAGAGATTACTAGCTATTTTGCCATGTCATAACAATTTTAAGTCGACCAATACAAAAACCGGGCA

AGGCGTCTGGGCCATTAGTAATATCCAGTGGCCAATACGAAACCCATCTCATTAATATCAAATCTCCAAT

GAAAGCCATTATCGTGGCGACTCTTCTTTTT[CATCATCATCATCATCCTCATCATCATCATCATCATCA

TCAT]CGCTTGGGATCACAACAATTTCCTGTTAGCACAACCCACTCTCCATCAATCAATCAGGGTCTTTA

CTACTCTTTTCATGCTTTCGTNTCAACTCCCTTTGTTTATCCTCCTATATAAATCATTGAATATCNNNAT

TTTGATCCAAG

AG0324

(SEQ ID NO: 16)

TAGTCTCACCAACTCCAAACTTGTTAACATCTGAAGGAGTCTGAATATTCTCCTCCT[CATCATCATCAT

CATCATCATCATCATCATCAT]CACAATCAACAACTTCAACCTTCTTCTTATTGTTATCATCATCCTTTA

CTAATTTCTCCTCATCACAATCAATAACTTCAATGACTGAATCTTGAGAAACTGCTTCTTCTTCTTCTTC

CTCCAAATCGATAAACTCTTTATCTTTGGTAAGGAACCTGAAGGCATTCAAAGCCGATCTCTTGGCGTTA

TCATACTCGCGAGTGAAAAGCTCCCCCTTCTCGCAGCAATCGTTAGGCTTAGCCGTGGGTGATCCTCCGT

ATATAAGGTTCGCTCTGCTANNAGCGTGGATTGCTCGTCTAAGTGGAGTTTTGGCGTCGGGATACCTCGA

AATCCTCGAAGATGGCTTGTTGGGATCCTGAGACATGGTTCCGAAGGAGAATCTGCGTTTCTTCGGAGCT

TGGAAGTAGGGCGAATGTCAGCGGAATTGGGCGGTGGAAGGGTGGTTGGAGTATAAGGAATCTTCGCTGC

GCTTGCGATTGATGGCGACNGCGCTCATTTNNNGAGTCGATCACTGAACCCTANNGATTGGGAGATCGAC

GNNNGGAGAGGAACCATAAGAGTNGAGA

AG0328

(SEQ ID NO: 17)

TTGCCAAAACATTTAACCAGGTGAGCACTTAAACTCTTGTCTGGCCCAAAAAAAAAAGAGTGAGACTGTA

TAGAGGATCAAGCCAACAGTAGATGAGGAAGAGGAAGGCCATGTAATCTCTAATCCACAACGATCTTGAT

TACCCATATAGGTCCATTGACTTTGAATCTTAATTTCAGAACATCAACAAATCTTCATCTTTACTAAAAT

TACAAAAAATCTTTTAACTTTTTAATTTTTGAAAAAAATACATATACACACATACAGCTAGTCTCTTACG

AAACACTACACAACTAGATAACTCCAAACATTTACAACTGAAAGTTTATCAGCTTGGAAAATCATCACTC

AGATTTCTTGTGGAACTTCACGGAGTCTATCAAGTGTATTAACAATCTCACTCAGACAAGCGATGAGCTC

GTCTCCTTGCTGTCTCACAAACTTTAACTTGTCCTCAATGCCAACATCATCCTCTGTTTCACTTCCATTT

GCTAGATTTCTCCCGACTATTGCATGTATCATGTCAGCTTTCTCACCTAGCTTTGCTTCCAACGCTTTAA

CCTCTTCTTCTATGCTCATCGTTT[CTTCCTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTT

CTT]CTACATCANNNGAAATTTCTACCACCGGCTTCTTCTTCACTTCCATGGTTTTATTCTTCTGATGGN

NNGCTTTTGCACG

AG0359

(SEQ ID NO: 18)

CTCTTGCTTAGNTCTGGACTAACCCATATCCGAAAAGNTTCGAGGACCGACTCGAAAACCCAACCCGAAG

ATCTGCACGCCTAGGCCTAGTTTACAAGCAAGCCTACCAAAATATGTCAACTCGTTAAAAGCCTTTTAAC

CTGTCTGGTTCGGTGCACGGTTCAATTCCCGGTTTAGTTGTAACCGGTTTGTGATTGCTCAAAACCCTAG

TCGTCACCCTTTTTTATCATTATTGTGAACAAGTAGTCACCTCTACAAGTAAAACCTTAAACCCTATTGA

GCGAGTAGCAGAGCGCAGCAAGAAGAAACAAAACCAAAATATGAGACCACCACGTGGCGGCGGAAGCTTC

AGAGGAAGAGGAGGAAGAGATGGCAGCGGACGCGGAGGTGGCGGACGTTTTAATCGTGGAGGTGGCCGCT

TT[GGTGGTGGTGGTGGTGGT]GGCTGGCGTGACGAAGGACCTCCCGACCAAGTCGTNNNTTCGTTTTCT

CTCCTCTCTTGGTTTTCGCTCTCACTTTACAGCTCAAGCAGAAGTCTTTATTAACAAAAGTTGTCACCTT

TGACAAATTAGTCTTATCCCTTTGTTAGAGTCATCTTTAAGTTAAAGGTATAAACTTTGTGAAGTTATTC

GTTATGACAAAGTTTCTTTCTTTCGTTGGGTTATAACAGAAGTTGCAACGTTTGTTCATGCTNN

AG0369

(SEQ ID NO: 19)

TTTTGAAGGAGGTTGCTTGGGTGATTTTTGATGAGATTCATTACATGAAGGATAGGGAGAGAGGGGTTGT

TTGGGAGGAGAGTATTATTTTCTTGCCGCCTGCTATTAAGATGGTTTTTCTTTCGGCCACGATGTCTAAT

GCTACTGAGTTTGCGGAGTGGATTTGCTATCTGCATAAGCAGCCGTGTCACGTGGTGTATACGGACTTTA

GGCCCACGCCTCTGCAGCATTATGCTTTTCCTATGGGTGGGAGTGGGCTGTACCTTGTAGTTGATGAGAA

TGAGCAGTTTAGAGAGGCTAATTTCATTAAGATGCATGATACTTTCCCAAAACCAAAATCTGAGGGGAAA

AAGAGTGCAAATGGCAAATCA[GGTGGTAGGGGCGGCGCTAAAGGTGGTGGCGGCGGC]GGTGGTGATTC

TGATGTTTACAAAATTGTAAA

AG0370

(SEQ ID NO: 20)

TAAAAAATACCTTAAATATATAAAAAATCTATCTTTGTCGAACAAGTAAAAAATTTAAAACATCTTACTT

TTGGAAACGAGGGAATATGATATTTTGGAATGAATCAAAATTGACAATCACCTTGTATAGAACCCATAGG

TTCGTGGATTCGCGGTCCTCACCACTAATAGGTTGGACCTGGTTAAGAATCCTTGCACACAGAAATAAAC

TTAACCATTGCCGCCTTACATTGTATTCCAAATTTGTTAATTACCGGCCAACAACACAATTATGTTATCT

CCATTATTACAACCACCCGCCGCAATAATTATCTCAATCA[GTTTTGTTTTGTTTT]TATTTATATTCAA

ACGGAATATCGTTATTTAATTATTAGAGCTTTAAATAATCTATATAAAGTCCATACAATTTTTGTTTATG

GAAATAGACACTACAAACGCGTATTTTCAGTTTTTTTTTCATATGGAGAGAACTACCAATCAGTTGAAAG

AAAAAAGAGAACTACCAATCTACCATATAATATAAAAACAATAATAGTATTAAAAAAAGGAGAGGGCAAC

GGAACGGGACGGAAGAGAATGGAAATGGTTACGTTTATAATAGCAATGATCTGTTGAACAGCTTATGACA

CCTCACTCTGCGCTTGCTTCCATTCCTCATCTCTCTCTCTCTCTNNNAACTCTCTACGAAACCCTACCTT

CTTCN

AG0378

(SEQ ID NO: 21)

TACAAGACAGACGGACATAATAAGAAGAAATGCAAATAGACAAGTCCTGAGAGAGAGTATCCAAAATAGA

GATTTTAAAATCTGTCACTAACTTTTAGGCATAGCTTGTTTAGTTCGCTTAGTCCCTCCTTTCTTCACAA

AACCAAAACCAAAAAAAAAAAGATGAGAGAGAGCAGTTTGTTGATAAAGAAGCAAATGAAATGTAACTTA

CTTTTCTACCGGCGGTGGTGGTTGCTGGTGGAGTTGCTGCTGCAATTGAGTAACGTCACAACAAAAAGGA

AGATGGAAATGAAAAAAAGGAGGATTCAATGGATATCAACAAAAACGTGTTAGAAAGACTCACCACTCTG

TTGAGCTTCACCACCTTGGCTGCCTCCTCTTCTCGTCGGGGTCTACACATTTCAATAGATTCGTCAACAA

CAGTAACGAGATTGCGTCGAAACCTAACTCAGAAAAAAAAAGAGATGACGTACCGCACTCGGACCTG[CC

GCCGCCGCCGCCG]AAGATGAAGCAGTCGCATCTACAAATTTCAGATGCCAAATTAGGGTTTAACCTAGA

AAATAAAAATATCAATAAGGCAAAGAGAGAGAGAGAGAGTACTAGTTGGATTGCGATCT

AG0391

(SEQ ID NO: 22)

GTCTTCTCACGGCCGTACGGATAACGCCGTGAAAAACCACTGGAACTCGACGCTCAAGAGGAAATGCTTA

GGCGGCGGTGGAGATGGTAATCTCATTGTGATGAGGAC[GGAGGAGGAGGAGGA]TCAGGATCGGCGGAA

GAAGAGGAGATCGGTGAGCTCTGAGTCTGCTACTCCGGTGGACACTGGGTTGTACATGAGCCCGGAGAGT

CCCACCGGAATCCCATCTCCGCCGTCTCCGGTTGATGCTCAGCTTTTAAAACCAATGGCGATGCCGTCAC

CGGTGGAAATGTCTTCGGTGGAGGAGGATCCGACAGCGTCATTGAGCCTGTCACTGTCACTTCCTGGTCC

TGATGTCAGACAGGAGTTGAAGAACGCGGGTTCGAAACACAACTCGTTGCTGTTTCCCCGGTTTGGGAGT

CAAATGAAAATTAATGTTGAGGAGAGAGGAGAAGCACGTGTTGGACATAAAGCTGAGTTTTTGACGGTGG

TGCAAGAGATGATTAAGGTGGAAGTGAGGAGTTATATGGCGGAGATGCAAAAAAATAGCGGTGGTGGCGG

TGGTGAATTCATCGTCAGTGGTTTTTATGATGCCGGCAACGGCGGTTTCAGGGATAGTGGG

AG0410

(SEQ ID NO: 23)

AAAAGGAAAGTTTAAGACTTTAAGCTTTACCTGATGATCCATATCGGGGAAAATCGCGGCGAGGTGATCG

AGGAGAAGC[GAGGAGGAGGCAGGAGGAGGAGGA]GGGGGAAAACGCGGCGGAGAAGAGGATGAGAAGTA

ACGGAGTTTCTTGGAGACGGGAGGGGAAGAAGCGGCGGACAAATCCTCGAACAGAGATCTCTTGCTACCG

CAAACAATCGCAGACATGTTATCTGCTTCCACCTTCTTTTTCTTCTTCTTTCTTCCTTCCTTCAGATCTC

AACCTTTCCTTTTTGTTTGGTTTTTTTTTTTCCTTTTT]CCTCTAATCCATCTCTGATCTGTTTCTGTCG

GAAACCAAGCAAAAAAAAGTCAAAACACATCGGATCTTCTTCCGCATCTAAATAGATCCAACAACCCGGA

CTCGGATTCAAAT

AG0441

(SEQ ID NO: 24)

AAAAGACGTTGACTTGATTTGATATGCCATAGAGCTAAACCCTAATTGAATTTCAATCAATTAGGGGTAA

AGATCTCTCAATCTACGAACAAAAGATCTAATATTTACAGTCAAAATCTACGGAAAACACAAAGAAAAGG

CTTACGGCGACTGGGCTGCGAGGAGCGCGATTCTGATTACGGATCGGCATCTTCCGATTCTGGATCCGAG

CTAGGCGATACGAAAGATGATTTCTTCCGGAAACCTCCGATTGAGTAACAAGAATCTCGACAGAAGTTGT

TTCTTCTCAGTTAGAGAGAAGAGATTAGGCTTCGGCCCTTTTTGTGATTTTGAGAAGGAT[GAGAGAGAG

AGAGA]GT[GGAGGAGGAAGGAGGAGGAGGAGGAGGAGGA]GCCTTTGTTATTTTGAAAGTTTGAAAATA

GATCTTGAGAATAATTGTAACGTTACTCTTGGTCCTCTATATGCTTATTTATTTATTCCACTAATACTTT

ATAAGGTATATGGGCTTTATATGGACTATAATCTCGGCCCATCTATGTTAAACTAATCCGTAATTTTCTT

GGTTTTTTTTAAACTTGCGCGCTCCTTAATTTGAAT

AG0477

(SEQ ID NO: 25)

ACTTTTTATAACCGACACTTAAATCAAAACTTGAAAAATAGCATCAATTAGATTTGTAACGGAGTATCAT

CAATCATCAAGAAACAACAATCTTGTAGGTGAGTAAATAAAAGATACCGTGAATAATGTCAACAATCGTA

ATCTCATACCACTAATACGTAATTAAAGAAAATAATCATATAATTAGGGAGATAATGTTGGGAATCTTAA

TCGTATAATCAGAAGCGTATTCATTTCATTACAAATTGATTCTCTTGTCATTTGTTATAT[AATAATAAT]

AAAAAAAACGTTAAATCAATTCAAACTAAACCTT[CTCTCTCTCTCTCTCTCT]TTCTATTTCGCTCAT

CATCATTTTATCTGATGAATACGCCCAATTGAAATCCTTTCCTTATCAACTCAAATTGAGTTTTCAAAAT

TATTCAATTTTCGGATCTCCGTAGATTTGCTCGGCGGAGGAGGAGGAAGGATGGCTCAGTTGGCGGCGGC

GGCGGGGAGGAGAATAGGGGATTACGCGGTGGGAAGACAAATCGGGTCGGGTTCGTTTTCGGTGGTGTGG

GAAGGGAGGCATCTGGGAGATGGAAACGTGGTTGTAATCAAGGAGATAGCCATGGCGAGGCTTAGTAAGA

AGTTGCAAGATAGTCTCATGTCCGAGATTATCATCTTGAGGAA

AG0482

(SEQ ID NO: 26)

ACCCAAACGAATTGCTCTGTCCGTAGAAAGAACAGGCTCGGGAGCTGAGT[GGTGGTGGTGGTGGT]GGA

GAAGCGACGGTGGACCATCCGGGAACGAGTGCAGCGAGAGACGGAGATCTTGACTCGGAGGAGCTTCCGT

CGAGGAGCCAACCACCGGGAAAAACGACTCCGAATCCATCGACGGCGGAAGAAAACTCGGAAGCTCCGCC

ACTCCGTCGAATCCACCGGACCGTGCACCACCGAACTGAAGCTGTTCCCGCTG[CGGCGGCGGCGG]CGA

CGGAGATATTTTAGTTTTGGCGGCGGTTCTTCTCGGTTTAGCGTTTGCGGCGGCGTTGCGAACAGCGTCG

GCGGGACTCCACGGAGGAAGCTGAGCGAGCTCGTCGATGGAAGTCTGAGCCTTTCTGATCAGCCAGTCAA

CGGCTTTGCTCGGTCGGTCGAAGCCAAGGCGGTCTTGAACGTCGTAGAACTGAATCGCCGTGTGAGCCGA

TAGCCTCACGCGCCGGTCACGTGGCCCTTTGGCCGTGCAGACTTTGCTGTGCCGGTCTTTTCTCCCCGTC

GACCGCACAATGTGACCTCCTTGCACCTCCACTATCTCGTCTGACGCAGCGCGGTGCCTCATTGAAGAAG

GCNGNNNGGGTTGAGGGGTGGAGGAAGTGGTGAGCTTCGTCGTGGTCGTCTGCCATTGGTTGAGCATAC

AG0504

(SEQ ID NO: 27)

ACAACTTTGAAGTGTGAATAGAGTAAAAGATTCAATCTTTCATATCAAAAGACTAACCTAGACTCGAACT

CACGGATCTCAGCAAGTTCTTTCCCCATCAAATCCACCCACTGCACCTTCTTCTTCTTCTCCCTTCTCTC

ACCATCCTCTGAATCTAAAGTTTCCTTTTTTAGACTACTCTTCAGGATCTCTCCATTACTTTGACCTTCC

TCTAAGGCACAATCTTCTTCCTTTCCCTCTTCTTCTGTTCCCTCATTCACCAAACTATCAACGTGATCAA

CAACTTCCTCTACTTGTGCATCAACATACTGAT[CATCATCATCACCGTCAT]CAACAACCGAAGAGACT

TGA[GGAGGAGGAGGAGGAGGAGGAGGAGGA]GTGTCCTCCAATTTCAGAGAACCAGATGCGTAGATGTG

AGGAGAAGGCTTACAGAAGCAGATGAAAGAAGGGCACTGGATCTTACAAAGCAAAACCCTCATCAAAACA

TATGTTCCAATCATCAACCAATTCAACAAGATCTCTTTTGTCTTTGGCAAAGTTAGAAACTTTGTGTGCC

CATTGATATGCCCAGATTGAGAAAAGGAAACACTTTTGATTTCTGAATAAAAAGTAGAAACAGAGCAGCA

AAGAAGTTAGTATATATCTCTCGTCTTGGATAGAATCCAAAGACCATAAATAACGAGTTGATCAGATGAN

NNAGCAGACAAA

AG0510

(SEQ ID NO: 28)

ACTGGTCTCAATGGAGGTGGTGGAGGAGGAGGAGGAAGAATACGACTGGAATCTCTTTTAGACCTTGTAG

AGAGTTAAAAGATAGTTTTAAGAAACAAATGTCAATCTCTCAAAATAGGAATACTATACTCTTTACCTGT

GAGATAGCTGAGCAAAATCATCATCTGATTCATCGTCATCATCATGATTGATGCTGACAAGTTGAGCT[G

GAGGAGGAGGAGGAGGAGGA]GCTGTTGCACCGCCTCCATTTGAAGGAACAGAGGCGATATCGTCATGAC

GCTGAAGAACACGCTGCAAGTTATCGTTCAATGCTAATCCCTGGCACAGAAGCTCCTCGTCTCTGAAGAA

AAGAAACATCATCAAAAGGGGATGAAACATCAGGTGAT[GAAGCAAGAAGAAGAAGAA]AACTCACGTGG

TGGTGTTGACAAGAGTCATCACACGTTTCTGATAGGT

BG0031

(SEQ ID NO: 29)

CGAGGAAGCAT[AGGAGGAGGAGGAGG]AAGCAGTTTGAGTGTTTGGAGGAGATGCCTGAGG[AGAGAGA

G]GAAGGGAGGGAGTCGGACGAAGGCGTGTGTTTTTGCACGTGCAGCGGAGGAGAATCCGGATCCGAAGA

CGGAGAGGGAGAGTGGGAGTCGGTGGAATGGACGGCTGAGATGGAGGCGGAGGCTGAGGGAATGGGATGG

GCCGTTGATTTGGGGATTTGGGTTATGTGTTTAGGTGTGGGCTACTTGGTGTCCAAAGCCTCAACTAAAA

CCTTGAGAGGTGGAGGAAGGAGAAGAAGATCAAAAAGTTTCTTTTAGAGTTCTCTGTAATCAGTCAGTCT

AGTTGTTCAATAACGTTCTAATGTAATAGTACAGATCAATAAACCATAAATGTAAAACAATCCATGATTT

TGAATACCAAGAGTCGCACGAGTTCCATTTTATTTGAGAGCATAGAACAATAAACTTTCTCCTCTGACCT

GATGAACTAAGGCAAGTTCATGCAAGAATCTAATGAATGCAAGCAATCAAGTACGTCAAATCATATTGCA

TTTACAAATTATACAAATACACAAAGGATCCAAAAAGTGCCTTCTCCCTTTTCTTACTAACAATAATAAT

AATGCAGCAAAAAGGAATAAAAGTTTATCAAAAACGTGTGATGATAATTCAATGTAAATAAGCAAATATG

TGGAGAGCT

BG0106

(SEQ ID NO: 30)

GTAGCCTTGTGTGAGTTGGAACCAGACTTTCCTGTTTCCCTGCCTTGTCTCAAGGTTATGCATTTAGAGA

GAGTTATAGCTAACCTTGAGAGGCTTATAACTAGCTGCCCTGTTCTTGAAAAGTTAACCATAATCAGGGA

TTCTTTTGAAGTTCTCGAAATTATGTGTGTGCGCTCCAAGTCTTTAAAAAGTTTGGCTCTACTGATTGAA

GCTTCTGATACTGATCTCTTAGAAGATCACGATTTGGAGATCGATGCCCCAAAGCTTGAGCGTATGAGTC

TCTGTGATCACTTATCCAGAAGCATCGTTATACACAGTATTGCTCCCTCTGCAGTGGTACAGATCGATGT

TAACTTTAAT[AGGGAGGG]TGGTGATACATTATTGGACCAA[GATGATGATGATGATGATGATGATGAT]

TCCAAGAGAACTATGATCCGTAATTTCCTAACCGGGATATCCACAGTCAGCCTCATGAAGATCTCCTCT

GATACTCTACAGGTAC

BG0111

(SEQ ID NO: 31)

GTAATCATTTCTTTGTTATCTCTCTTTCCATGATCGTCCGTCCAAGAGATATGTAATTGGCGTTGTTTGA

TTCTGCAATCCGTACAATCCATTTCTAGCTGTTAATCTGAATATAGCCATCTTATTAGACTGAAATCTAA

GCGCCTGGATGGGGTGGTTTTATTTTCATTTTGACTTTTGGCGTTTGGTTTTCAGATCTTTAAGATAT[G

ATGATGATGATGATGATGATGAT]GAAAATGATGAGATTTAGATTTTACTG[ACCACC]CTTTTTTTTTT

TTTGTCTTTACGTTTCTTTCAGCTCAATTCAGAGAAGAGCCCTTTTCAACGTACTTATGCAGCTCAGGTA

AATTTCATGTTTATCTGACACTTGTCTAGTAATGTGTGATACAATCTAAGAATGTAAATCTTACAATTGT

GATAAAAATATTCTCTCTCGTGTTTAGATAAAAAGATGTGGAGAGATGGCAC

BG0119

(SEQ ID NO: 32)

GTGCGGGTTCGAGCAGCTCTCAGCGCTCGCGGAGGGAGGCATGAACGTGGCCAGGCTCAACATGTGCCAC

GGCACTCGCGACTGGCACCGTGACGTCATCCGCAGCGTCAGGAGGCTCAATGAGGAGAAAGGATTCGCGG

TCGCGATCATGATGGATACCGAAGGTAGCGAGATTCACATGGGAGATCT[CGGCGG]CGAGGCCTCGGCT

AAAGCAGAGGTTCCTTCCTCTTCTTGAAATCTT[GATGATGATGATGATGATGATGAT]GCAT[GTTGTT]

AATCAGATTATTGGATATAATCCGGTTTAGTTAGAGACCGGTTTAGTTAG[ATTAATTA]TGGTTAAGT

TTCTTTTTGCTTAATCATGTATATAAAGAAATGTTAACACAGATGAGGTTTTTGTAGGATGGTGAGGTTT

GGACGTTTACCGTTAGAGCTTTTGATTCGTCTCGTCCTCAACGTACCATTAGTGTGAGTTATGATGGTTT

CGCTGAAGGTAATGTGTCTTTTTTTTTTGTGTTATGAAAGCATCAAGTGGATGTGAGTATGAGATGGGGA

TCGATTTTTTTTTTTTTTTTGTGATTTCAGATGTAAGAGTTGGTGATGAGCTTCTTGTTGATGGTGGAAT

GGTTAGATTTGATGTGATTGAGAAGATTGGTTCCGATGTGAAGTGTCTGTGTACTGACCCTGGGCTGTTG

CTTCCTCGAGCTAACTTGACTTTCTGGAGAGATGGGAGTCTTGTAC

BG0181

(SEQ ID NO: 33)

GTAACATATACAAATACTTCTAGGAATCAATCGAAATATATATTTCATATCGCAATTTCACAATACTGTT

GAACTTACAAACGTGTATAATTACACCATTTTTTTACACAAAATCTTTAACATGTCGATTTCTTATACCA

TTTGTAATTAACTCAACATATTTTTTTAACTAAATCAGCCTCGCCAATTTGTGTTGGTTTACGGAACCGG

TACAAATATTGTTGGCCTGGCCGTTATTAATTTCAAATGATTGATTCATAGGTAACATGAGAAGTTTGGA

GAGCTTACTAACGAAAGCAGGAGCGGAGACATTGCCATTGGCAGAGCAACGAAGTACGCCTTGAATATTG

ATGAGACCTAAAATGCCTCCAAGGAC[ACCACCACCACC]AAGAATCCCACCAAGGCCACCATTACCAAG

AAGCCCCCCTACTAGGCCACCATTACCAAGAAGGCCACCTAGGCC[ACCACCACC]AAGAAGGCCACCTA

GGCC[ACCACCACCACCACCACCACCACCACCACC]AAGAAGGCCACCTAGACCCCCAAGCTGAGCCTTA

ACCATTGGAGACACCATCACGAGACACACGAAGATCAGTGAGAAGGTTATGCGTTTGTTCTCAAGCATTG

TCATGTTCTTGG

BG0228

(SEQ ID NO: 34)

GTATCTATCTCCTCTTGCCTAAATCACACCATGACTGACTTTCCCAAAATAACCTAGAGATCCAGAAAGA

ACGGAGGAAAGAAAGAAAAAATGGAGGAGACGAAGCCATTGGTAGGGAACCATCCCCAGCAAC[AGCAGC]

AG[CAACAACAACAACAACAACAGCAG]CTCCTGTATCAACACCAATTACAACAGAGACAGCAACAGAT

GCTTCTATTACAGCAGTTGCAGAAACAGCAACAACAACAAGCCGCCATGTCTAGGTTCCCCTCCAACATC

GACGTTCATCTCCGACCTCCAGGGTCAATCCAGACCCGACCAATTGTTCCCCCTCAGCAGCAGAACCCTA

ATCCCAACCCTAGCTTGGGACAGCCTACACCGAATCTTCAGCAGCAGCAGCAGCAGCAGCAACAGCAGGT

TGTAGCGAGTCAGCAGATGCTGCAGCAGCAGCAACAACAGCAGCAGCAGAAGTTGATGCGTCCTTTGAAT

CACATCGAGCTTCAATTCGCTTATCAGGACGCTTGGCGTGTCTGCCACCCTGATTTCAAGCGACCTTTCT

CTTCTCTCGAAGACGCTTGCGAAAGGTTCAGTTCTAATTTTTATCTAATTACATTTGTCTTTTTGAGATA

TTTCCTTAAATAAAATCGGTTATAGACAATCTCATCCGTTCAATCTTATTTCAGGCTATCGTGTGATATA

TGCATACGGGTCTTGTGATCTTTGAAATGAAACATTGATCTGTTAATGACTTACTTACTGGTCATATCTG

CAACTTGTATGTTCTTCTTTAGTTCGTGTTTGGTATTATGGTGATGATATCTGTTAGCCTTTTCGTTAAT

TTCTATACTTCTTTTCATTGATATTGTTTGTGTTAGATCCAATAGATCCTGCTTCTTTTGGTGTTCGTGC

GAAACTTAAATCTCTTTCTGAGTTTAGTGTGGTTGATTTTATATTATTTTTGTCATCTAATGTGGTTGAT

TTAGAATTACAAAACTTTGTGATTGTTTCCTATTTTAGTATAACCACCTGATTCACTGATACTGATAATT

ATTCCCTGACTTTTATATTTATGCTAAAAGTTTACAACTTTACATTAGCATATTATTGGTTTTATTAGAT

ACATTTGTTGCCTTGATTGAACATTTCTGTATATTGTTTGTTTTATCTTACCTCATAC

BG0255

(SEQ ID NO: 35)

CTAGTGGCTACAAATCCAACTGTCGGTTCTCACTTGGGAGACCCAGGTTTGGATCTATCAAGTTTAAAAA

TCCAAACTCAGCACAGCCTTTCATTTCTGAAACAAGAAAAGAGATG[GAAGAAGAA]GATCAAGAAGCCA

AAAGCATCGGTTTCAGGGAG[GAAGAAGAAGAAGAAGAA]GATTAT[GATGAT]GGAGCTAAGGGTATTG

ATCTAGAAGGAGAAGAGAAGAAGCATATATGCTGTGAATGTGGCAAACGTTTCAAGTCAGGCAAGGCGTT

AGGTGGCCATAAAAGGATCCATGTGCTCGAAACTCGCAAATTCTCAATGGTGAGACCGAAGATGGTGGTG

ACGTCTGGTGCGGTTGCGGTTGCGGTTGGTAGATCTGATGAGCAGAGAGATGATTTCGAAGTTGATTGCT

GTGTTTGTCATAAGAAGTTTACATCGATGAAGGCTTTGTCTGGACACATGAGGTTTCATCCAGACAGAGG

ATGGAAAGGTGTTTTGCCTCCTCATCATCCACTTGATGATCATCATGGTGGGGAGTTTATAAGCTCCGAT

TACGATGATGATGCTGATTATGATTATCATGAGGATGATGATTATGAGAACTCGGAGTTATGGGATATTA

ATCGTTGGGAATTGGACAACGTGGTTGACCTTAAGGACTCGATCAAAGAAGGATGGACGGTGACAGGAAA

GAGAGGAAGGAGAAGTGCTTTGAAGATTGATGAACCTGATGATATTGATGCTAAGGATCTATTGTTCTTA

GCTACTACAGCAGAATCTGTCGATGCTGCAGAGACTTGTTGTGATTCGCTTTTGGGGGAAGAGATGATGA

TGAAGAAGAGGAAAAAGAAGAAGAAAAGATTGTCTGAGATGGAGAAAGAGTCATCATCTAGTCATGGTCA

TCATCAGCTTGAGGTTGGTGATGCTGCTGAGGGAGGTGGCGGTGCAC

BG0278

(SEQ ID NO: 36)

GTATATGTCTTTGGTTATTTTTTTTGGTATCCAAATAACCGTAAATAAAAATTAAAAATGGCCCGTTTTC

CCTCGGATAAAAAAATTGTAGAGTTTAAATCATGTCTTTTAAAACCATGGGAGCAAAATCAAAGGAAGAG

AGAAGATAAATTAAATGGTGGCTGTTCAGTTGTTTAGCTGGAAGACATTGATTCTTCTACCTTCACAAGC

TTCAAGACATAAGGGTTTCACTTCTTTTAACAGGTTTTTAATCTGT[CTTCTTCTTCTTCTTCTTCTTCT

TCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTATTATTATTATTATT]AT

AATAGTTTCAAGTTTCTGAAAAACAATTGATTCCATGGTGGTGCATGTGTTTTACAAGATATCTCACTGA

AAATTAACTTTGTTGCAGAACATTGAGTTTGCACTCTCTGCCTTCAAATGGGATTGATTCTTTTAGATCC

CGAGGTGAGGAGGCTCTGAAACACATTCCACGTCTTAATGTCCTTCCTCTCAACAAAGACTCATACTTTC

ATACTATCATATTTTCATAATTTCATTATTACAGGAACCTTCAGAGTCAAATCTCAAAAGACAGGAGACA

CAGAGTCATCTACTTCCAACTTGAATCAACCTAATGATTTAAAATCCAAATTCCATAAGGTGCGTGTGTG

TCATGCATGTCTTTACTTTTTTTATCTAATGATTTACTTAATGCTTTATGTTATAATCTTTCTTAATATA

CATATCTGCAGAGTCTCCAATATAAACTTGTACTAGGATGCATCCCACTGTATGCGGTATCGAGAATTGT

ACAAAAGATCATTCATGGGCTTCCACTCCACATTCAGAACTCAGTAGGGGCTGGCTTGCCTTTTGCTTGT

GCATCAGACTCTCTGAATAAACCATCTTTAAGTGGTATCAAATGGAGTCTTGCAAGGTTCTTTTTCCTGT

TCAATATTCGGCTCGAGAAGAACGTTGCTAC

BG0295

(SEQ ID NO: 37)

CTGTTCCGTTTAACTATGCTCGCACCTCCATTATCTCTCCTCTTTCATAACTCTCTCTCCTCCTTCTTTC

TTCTCCACATCTCTCCGATTTCATCGCTAGAATTCTCCACCGATTCTTAAGGTATGTTTTATCTTCACTT

CAACTCTTGTCGGAATTCACTCTCCTTGCCTGTCTGAAACTTTCCATTTGCAGATCTGTAAAACTTTCTA

TTTGTGTTTCCTCCTTTCCGTAGATCGAGAAGAAACGATGACTTCAACGG[AGGGAGGG]ATACGATCCC

TCTTGTCTC[TCCTCCTCCTCCTCCTCC]TTCTCTTATCCATAACCACTCTAATCTCAGCCGCTGACTAC

ACACCCACCGACAAAATCCTCTTAAACTGCGGCGGCTCCTCCGACCTAACCGACACAGATAACAGAACAT

GGATCCCCGATGTCAAATCCAAGTTCCTGTCTTCCTCCGGAGACTCCAAAACATCCCCCGCCGCAACACA

AGACCCCTCCGTCCCCACCGTCCCTTACATGTCCGCCAGAATCTTCAGATCTCCCTTCACTTACTCCTTC

CCGGTCGCCTCAGGTATTGGTTCAATCCTGGTTTAGTAATTGTACTTTGGTTTACTCATTTCCGGTTTAC

TAAACACTTTTCCCTATCACAGGTCGCAAGTTCGTGCGTCTCTACTTCTACCCCAACTCCTACGACAGCC

TCAACGCAACCAACTCCCTCTTCTCCCTCTCCTCAGGACCCTACACTCTTCTCAAAAACTTCAGCGCCGC

TCAAACCTCCCAGGCGTTGAACTACGCTCACATCATCAAAGAGTTCGTAGTCAACGTCGAAGGTGGGACC

TTAAACATAACCTTCACACCAGAGTCAACGCCTTCTAACGCCTACGCCTTCGTCAACGGTATCGAAGTAA

CTTCGATGCCTGATATCTACAGTAGCGCCGACGGGACGTTGACCGTTGTAGGGACTTCTAGTGGCGTCAC

GATCGATAACACCACCGCTCTCGAGAATGTCTACAGGCTCAACGTCGGCGGGAACGACATCTCTCCTTCT

GCTGACACCGGTTTGTTTAGGTCTTGGTACGATGATCAGGATTACATCTTCGCCGCGAGTCTCGGTATCC

CCGAGACA

BG0452

(SEQ ID NO: 38)

GGTCTGAGATATATCCTCGAGGGTTGTCCTAAACTAGAGAAGCTTGGGATCAGGGACAGTCCCTTTGGTG

ATGTTGGACTGCGCTCTGGGATGCATAGGTATAACGACATGAGGTTTGTTTGGATGTCGTCATGTCGGTT

ATCCCGGGGAGCCTGCAGGGACATTGCTCATACTCTGCCTAGTG[TGGTGGTGG]AGGCGTTTGGGTCA

[GATGATGATGATGATGATGAT]GACGAAGACGACAATGCAGATTATGTGGAGACGTTGTACATGTATCGG

TCCCTTGATGGCCCAAGGAAGGATGCTCCAAAGTTTGTAACAATTTTATGAAGACAAGCTTAGAGAAAGC

AGGAGCTGAAGTAGAAGAGAATGTGTGTTTGTATGATTGTTTGTACCATTTGATTTGATTGGCTCCCCTC

TGTTTTTGGATTTGTCTTGTACCAAGAAAGAGTGAAGAGTCAGTGAAGAAAGAGGTTGTTTGTGGAAGTC

AAAGAATGAAACTTTTATTATTTGTGTGTAATCAAGAATATGATTTTACAGCCATTTCACGATTATTTTT

GTCTACAAGAAGTATTGGTTATACATTACATTATAAGATCTTCACCAATCTTGACTTCGTCCTCCATCAG

CAGATGCTCTAAGGTGTCGATGAAAGCAGTAACTTTCTCCAAGCTCTTCTCATCAAGCCTTGGGACCGTG

TGGCCCTTGGGATGATGGACCACCACCGGATTCTTGAAGGAATCTATCAGCTCAGTTCCGTAAGGTTTCA

AAAAATCAGTCTCTCCTGCAAAGAAAAAACTCATTTTTCACATTGAAATTTGCAAACCAGATATACAATT

TAGTAGGTCATCAAATTACCTAGAAAGTGGAGGGAGGGAATGTCCATGGTAGACGAATACGCATCCTTCG

CCACCTTGGTGGATTTGAACATAGCTCCTCCAATAATTATGATAAACTTGATCTTTGGTACTTTCTGGAG

TGCAATTCCCTGCAATATAAAATATAATTCTAAGATAATGTAATGCGATTTCCCAACGCAAAAGCAACAC

TACTGACGTACCTTAGCTTGCAGTCCTGGTAATCCTCCAGACAATATTGCACCCTGCAAAATTAACATAG

AGATATATTATTAGATCTTATATAAGAAACTGTTAAATGAGAAATGAAGCAATTTTGTAATTAGAGTACC

TGAGAAAAGCCAATGAGACCATCAAAGGGACCAAGCTCGATCATACGATCCTCTAAATACTCCAAACATT

TCTCGAAATTCG

BG0516

(SEQ ID NO: 39)

GATGTGTTCTTCATTGTATCTAGCAGAAGCTTGGTCAACAGAAAATGGCCTGAAACAT[GATGATGATGA

TGATGATGATGATGATGAT]GAGACTATAAAACTTAGGACAAAGGTA[TAATAA]TC[TTGGTTTGGT]T

TCTCTTAGCTCACCTAGATGGTTAGTTGCGAATTGCAGCTCAATATTGTCCTTAGAGAGCATGAAAGGAC

ATGCCATTACCCCAGCGTTGTTGCTAACAAGATTTGAGAGATTACAAAACATTAAAACCGTCACAAAACA

CTAGACATGAACTACTGTGTTTCGAGAGCTTACATCAAGATGTTTAGTGGAAGACCAGTAGATTTGTAGT

CAGATGCAAATCTCCTGACAGATTCAATTGAGCTGAGATCTAACTCCATGACGTCGAGTTTAGCACCAGG

GACTTGATTGAGGATATCTTGCTTAACTTTAGCACCGGAGACAGTGTTCCTCACCGCCATAACC

BG0647

(SEQ ID NO: 40)

GCACATATGTCCGCACCTGTACAAAACCGCCTCGACCTGCGTTTCGTCGCAGACGCAGCATTTGCGTTTC

ATTGGGTTTTCTCTGTAAACCGATTGTTGCAAGCTCGCGTTAGCATCCAAACACGTTTTGACAGAATCTC

GTAGTAAGGACATTTCTTGTTGAAGCTGTTGGATCTGTGTTCTCATATCGGTTATCAGCTCCATTTCCTG

AAAACGTTTTAAAGCGGTTCAAAGATTTTACTATTCTACTAGTTGGGGTTTGCGAGTTTTCTATGCAATA

ACAAGAAATCGAAAATTACTTACATGTGAAGGAGGATTGTGAACAGACAAGACAGGAGTGGAAGTTACTT

CGGTGTCTTGACAACTCCATGATCCTGCAGGAGACGATGCAAAGATGGGCGAAGAAGACGATCTGCTTGA

GTCATCTCTATCGTTTTGTTCTTCACCTTCCGTTGATGGCTCTTCTTCAGTTTCCTCCGCAGTGTCATCT

CTATGTTCTTCCT[CTTCTTCTTCTTCTTCTTCTTCTT]GTTGCAATTCCCATGATTCAGAATGCTTTTT

CGAATGTGTCTGCAGACGAGACATCATGAGCCTATCGATCTGATCTCGTAACCCGCTCTCGAGAAAGTCT

GTCACTGTTCTTCTGTAATAGCAAGAAAATATTTATCTTCTTAGTTAATGGTTTAACAAATAAGAAAAGG

GATTTGTTGAATCGATGTTGCGTACCGCTCAAGGAGTCT

BG0651

(SEQ ID NO: 41)

AATGCATAACAAAAGATTTGAACCCGGGTCTTTGGTCAAACAATAATCATCCTAAATTTATGCTAATAGT

GATTCTTTTGTTAGCCACTGAACACAAACTCT[CTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTT]CCT

CTTTCCTCTGCACTCTCTCCGACACAAGACGGCGGTCAACGGAGTCCTTGTCGGTCAAATGATCCCTAAG

GACGAAGGAGGAGTTGTGGAGATTTCCGATTCTGTTCCGCTCTTTTGCTCCAACCTCGCTCTCCTTCCTC

CTCTCTAGATCTCGCTCATCATGGTCGCTCTCACTACATAAGTTTTTGAAATTGAATATTGAAAAACTTA

GGATCTGAGTGCACTGTTGCGAATTCTCAATATTGTTGTTCTGTAGCTGTGTTTGGGAGAGAGGCAGTGT

CTGTAATAC

BG0713

(SEQ ID NO: 42)

ACGGTTCAGCACAGTAAAAAAAAAGTTTTTTTGACTTTTTTTTCTTTGACCGCCAAAGAAGACGAAAATG

AGTCTTTGAGAAAATCACAAAAAAGAAGAAGAAGAAAAATGAATCCTTTTTGTTTCTTCTGCACAGAATC

TTCTT[CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT

CTCTCTCTCTCTCTCTCT]TTCTTGAGGTTTCTTTTCCTCCACGATTCCTCGTCCCTCTTGCTTCTGTGT

GATCGATTTTGGTGAAATTGAGCTGAGTGTATCTGTCCGCCGAGGCCTTTTGTTCACTGTTCAATTCAAC

ATCAGATCAATTTTAGGGGCTTTCAGTCAAAGATCGCTGCTTTGGTGTAAGTTTGAATTTGGGTAACTGA

ATGAATGTGATCTTTGGTTCCAGTTCATGTAATTATGTTTGATTGACTGGGAAAGTATCATCCTTTATTA

CGGATTGTAAACATTTAAGGTTGAATCTTAACATTAGCACCATTTGGATTCGAATTTGTTTGGTGGGTTT

GGCTTTAGATCCATAAGCAAGCTTATGAGCTCTTAAAGTTATGTTGTTTTTTTTTGCTTAAGCCATTCAA

ACTGATGAGATATACTCTCTTTGTCTTGCTTCCTAGGTTTGTGATTTTAGTATAGAATCCTGTTATCATG

GATGAACACAATAGGAATCCATTTGCAAGTGCAAGCGGAAGAGCAAGTGGAAGTACAAGTGTGAGTTCCA

ACTCCAGTTTTAGTAGCAGCGTGGCGGATACAGAGGATGATCAAACCATTGC

BG0864

(SEQ ID NO: 43)

TGCAAAGGAAGCAGGTGTAGCAGCTCAAGCTTATGAAGCTCTAAAGACACTGAGAGAAAAAAAAACATCT

GCAAAGTGGTAAACAAACTCTTCTTATTTCACACAACACATGGTAAAGAAAATACTTTTTCATGGAGAAT

AAGAAGAAGAAGAAGCTAAATGCGTTGCGTTGCAGGTG[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG

AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG

AGAGA]AGCGCATAAGTAGTGTTTGTGTGTTGGTGATTTTCTATTTGGAAACTCTTTTGTAAGCAATAAT

CTCAGATGCTAAAGCCATTGTATTTATTGCTCACTTCATTTTACAGCCAAACTAAGTTTTAAAAACTGAA

AATATAAAACGCTAAAATTTTCTTTGGTTGACATCAGCATAATATAAATTTAGCTTACTCCCTCGATTCA

ACAATACAAAAAAAACGACATAAGTTTGAGTTTACATGCTTTCAACCAATAAAATGGAACTCTTTATCAT

AAAATAACAGTCAACGTATTATTAAGTCCAAACCACCACAAACCAATATTTGCACAAATAAAAGTTTCCA

ACCTTAGCTGCCACTATAAAGTTATAAACCACCATCCAAAGTCCATTATTTTAAGATAGATTTCGTACGG

TAC

BG0869

(SEQ ID NO: 44)

TCGTATAAAATAAAATTCTGAACAAAAATAATTATATAATTTCAAATTGCGGCTCAAAATCTATTATTTT

TAAAAACTCAACAAAATTGTATATGGGCCGATGAAGCCCAAGTATTTTAATTACCGTAAAGGAGGGTTTG

AGTCGGCCACAAATCAAGGAATTATTTC[CTCTCTCTCTCTCT]CCTGTGACGAGTTG[CTCTCTCTCTC

TCTCTCT]CGTCTCGTCCGCGCTCCGAAGAAATTTCACAGATTCCTGTCATGTCTTCCGGCGGAAACTCT

ACCCTCTCCAACGTCGAAAAGATGTTCTTCTGTTACCAGTGCAATCGCACAGTCACCATCTCAATCTCCT

CCTCCTCCGACGATCCTTTCTGCCCTCGCTGCTCCGGTGGGTTTCTAGAAGAATACGACGAGCCAAACCC

TAATCCGCCCCCAAATCTCAACCCTCTCGGTTTCCTCCCCATGGCCGATCCTTTCTCCACCCTGCTCCCG

CTCCTATTCGGCTCCTCCTCCTCTCCTCCTTCCTCCACGAACCAGAGCTTCTTCGGCCAGAATCAGCACC

CTCCTCGCGGCGGAGCTTTCGATCCGGTGTCGTTTCTCCAGAACCATCTCCAGCACCTGCAATCCAGCGG

CACTCACGTCCAGTTCGTGGTGGAGGATCATCCCTCGGATCCGTTTGGCCGGATGCCGGGGAACATGGGG

GACTACTTCTTCGGCCCTGGCCTCGAGCA

BG0988

(SEQ ID NO: 45)

AACGGTTTTGTATAAATAGTATATTCTATATATGTATGCATATAATCTTTTTTCGTAACTTAAAAGGATT

AAACCGGATTTATTAAAGACACAAATCTAACTTCCAGATGAGAGGTGCAATACACATATGGATTATTTTC

CAGATATTTAAATGGACCATAAATATAGACCCATAACCGCGTGGCCACATATGGAACTAATGATTTCGCA

CTAGAAGGGAATCGATTCCTGACCTGAACCAACAGGACAATTCCTCCTCTAGCGGAAACCATTAAGCCAC

CACAACATGGTTTTAAACAAAAAATTGTACGCATCTGCGTGGCTTACTATTAAAACATCTCTATCTCTCT

CTTAAAATACATCAAGAGTATAATGAGAGATATCTCAGTTTCATGTAGTAAGACAAAACCCAAGACTCCA

ACCGGAAAATTCCAACCCTAAGAGGCAAACTAAATTTCATTGTACAATAAAATAATTAATGCTATTCAGT

TTTCTAAAAGCAGATTTAAGTCTCTAACTCCAATTTTCCAT[CTCTCTCTCTCTCTCTCTCTCTCTCTCT

CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT

CT]AAATCCCCACTAGGATTATGGGAACTCACGTCCTCGTTTAATGCGATTCATGACTCCTCAAAGCCCA

GCATTCCCACTCTGCAAATTACTAGTACCTCTTAGTCTTAATTACCATTTGACCAATCT

BG1062

(SEQ ID NO: 46)

GTAATGCGGCAGGACAGCCCTCCTCGGAGTCCACTTCATGGAGGAGCATACTATTCATCCAGTGATGATG

ATAACCACTCCACCTACCTCTTCCCAGAAATTGGCACCCCAACTCGTTCCATCCCAGTCTCCGCCAACAC

CACTGTATGAAT[CTCTCTCTCTCTCTCTCTATCTCTCT]TTCACCATTGTTTTTATGATCTTATGGACC

TTAATAAATAAACATATGCAGCCTGTTCACCACAACTACCAAATCATTGCGGTGGAAACCTACGAGCAAG

AGAAGCAGTACGAGCCACCGGAGCTAGCGGACGAGTCACAGAGCTTCTCGATCCAGGAGATCGCCAAAAT

GCGAGGACTCAAGGAAGAGAGCCAATCGATGATCTCCGAGTCCTAC

BG1090

(SEQ ID NO: 47)

GTGAAACCGGTTCTGGAGAACTCTAAGGTTGTTTTGAAAGATCGCAAAAGAGAGTGGAAGCGGAATTAGG

GTTCTGGTTATGGCAAGGAGATGAACCGGAAGGAGGTACGAATCCTTTGGGAAGCATAACAGAAAACGTA

TCCGGTCGGACCGGTCGGTTAAAACCGTTACTGTTTTTCTTGGTCATCATCTTGAAGTAGTTGGCACGGT

GACGAAGACCACGGCGACGAATGCTATGGCGGTAGTAGTAGTAGTGATGGTTGTAACGAAGACGACGGTG

CTGATCACGTTGCCGCGGGTTCAAGATAAACGGCGTCATTTTCTTGATGTAGACGAGTTGGTGCGGTGAT

TCGTCGCCGTTGAGGTAGGTTTCTGCCTTTTGTGTAGATACTCTTGTTTCTTGATTCGATAATGATGAGG

ATGATGATGATGGCGATGATGGTAAT[GATGATGATGAT]GGTGAGATAGGGAAGACGAGAATGAGAATG

AGAGAGATGATGAAGCATTTGACAAGGTTGTGTTTCATCAAAACATCCATTGCGATT[GAGAGAGAGA]G

GGAGTAGGACTTTTGGTTTAATAGAGAGAGGGAGAGTAAAGATGAAACAAAAAGATGTGAGCGAGGCAAC

TATAACAAATCTTGGTATGGCGTCTAAATAATTCGTTTAGTTATTCGAATTTTAATTAATTTTAGTATGA

TTTTTGATTGCGTATAATTTGGAAATTAGTTGGGCTTTTTGTTGGTCTGAGGC

BG1101

(SEQ ID NO: 48)

CACATGCTTGTGGATAAAT[CATCATCATCATCATCATCATCAACATCATCATCATCATCATCAT]CAAT

ATCAAATATATGGTAAGTCCATTTTCATTTAGCTTTCAGTAAAACTGTTAATCTATGCATTCGATAATTA

AGAGAATCAAACGAATTGTGTTTGCAACATTATAATTAATGGTTGAAATTCATTAAGAATATTTAGTTTG

GGTTTTCTCATTTTCATACAAACATTATCCATGCATACGGTTGGTCATTAGGTTTTGAAAATATATGAAA

TCAGAAACATTTTAATTTTTTTTAATGTAATTTGAAAGCATACAAGTTATGTATATTAACTTTGTGTAAT

TTGAAAGCATACAACTTATGTATATTAACTTTTCAAAATTTGGACTATAAATAAATATTTCTTTGATCTG

CCCAAAATCACAAAAGATTCTTTTACAAGATAAACTGTATCTTTTACTCTCTTTTTTGTCAATACTGTAT

GTTTCACTTGTCACGAATTTGCATTCAAATAACTATGTAGCAGCACATTATGATAAAGTTGGAAGTGTAT

GAATAAATTGATAATGTAGATTGTAGGGTGAGAAGTTAAAAAAAATGAGTAATTTTTAGGGGCCAAATGT

ATTTTCGTATAAATTAAGGGTGGAAACATGAAAATTAGATTTTTTATGTCCGAACTACCCACTGACTTGT

CCGAAGTCCGT

BG1123

(SEQ ID NO: 49)

GTTCAAAGGCATATTTATATTATATTTAAATTGGGACCAAGATTTGCTTTGGGACAAGCTGTGCCCCACG

ACTTTCTCGCTAGTGCTCTCTGGTCGCTTCTCCTTCTAGAGACCAACCATTTCCACCAACTCCGTTTTCA

GTTCACACCATGCCCACCACTGCATCAGTTAGTTGATATGAGCCCAACTTCTTTCTTCACTGTTTAACAA

AATGGACTGGTCAACACAGTCTCTGTCACACCCGAGAATTCTAATGTGGTGGACACAATCTTCACTAGGC

CACCTTTTGTCACCAGTCTCTCTCTCTCTTTTCCTGTTTTGATCCTTCCATAAGATTAAACCTTTATGGT

TACTACCATATTATAACGATCTCGGTGGTGGTAGCGTAGCCCAAAGATGATGATCCGAAACTGAATGTAA

ACTATGTACCAAA[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA]GG[GAGAGAGAGAGAGAGAGAGA

GAGAGAGAGAGAGA]GTAATAATTAAAACAAATGGGACAAATTAACCCCCC

BG1127

(SEQ ID NO: 50)

CCGCCATAACAAAAATCTTCCCACAAGCGTGTAGAGATCTG[GAGAGAGAGAGAGAGAGAGAGAGA]GCT

TTCAAGTGATTAAATCCAAAAGTAATAAAGAGAAGACGGAGAAACTAAAGTGACGCGCCCCCTTCCTAAC

GGATTATATTTATTTTATTCTTATATATTTATGGGCTTATTGCAGCAATAGCCATATTTGAAATGAAAAT

TAAGAGAGTAGCCATGATGTTGACATAATGTACTCACTGTCTTTTTACAATTTTACTAGCCGGTTATACC

TTTGTAGGAAACAGGTTCCCAGTTCCTTTAACTAAAGTAAACGATGTGGTGATTTACTGACCCATAGTAA

CAATGAGAGTATTTTAGCAACGCCTAAAATTAAAATGAAAGGAAGGAAAACATTCTATAGAGATGAAAAT

ATAAAAAAAACAGAAGTGTAAAAGAAAGAACGTTACAAACGGAGAATGCATGGATCGTAATGCTGATGCC

AAAATATGGAAATAGTTCCACTTCAAATAGAATACACATAGTATAACAATAGTTTAAAGTTTGTCACCGC

TATGTCATATGAGAATATTTTCCATTCTATCGGATATAGATCAGTTTATATTTACTAATATAATCAC

BG1149

(SEQ ID NO: 51)

GTAGAGTGATAAGAGAACATCTTGTCTGACTCATGCTTCTTTTCTGCTATGTTAGGCTATTGCTCTTAAT

GATCTGCCTTCCACTAACGTATCGAGACTGGGGCTTGCTCTTAACTTATCTCTTTTCTACTATGAGACTC

TCATATCAACTAAAGCTGCGCGTAAGATCGCAAAGGCGGTATGTTGTTGCTTTCTCTCATTTAGTATTTT

GGTTTATGTTATGCGATTATCATCTATTCTCCCAGATGCTCTGTTTGATAAAACTTAATGCTTTTCTTTC

TTTTTTTTTGTTCTGACAATCGTTACTGTTCAATTGTATTCATATTGTGGCATAAATATGTATATGTTGC

TACACTTCCCTGTGGGTGTGCAATCTTCATATGATATAGTAATGGTTTGCAGATTGCTTATCATTTGGAA

GATAGATATTGTAATTGATTATGATGATGATGTGCATAATTTGGAAAGTAGAGCCTATCGTTATTCCCTT

ACACTAATGGAATTATATATTGATGATGTTCCAAATTTTTTAAATATGATGAATT[GATGATGATGATGA

TGATGAT]GGTAGGCTTTCGAAGCGTCAATAACAGAAATGCACGCAGTGAGAGAGGAATCATACGAGCAA

ACTGCATTGATCACGAATCTTATCCTTGACCGTATCACCCCTCTGG

BG1182

(SEQ ID NO: 52)

GTGTTTCTAAGCACTTTTTTTTTATAATCAAATCACATACAGCAAACATAAACATGAGTCTCAGCTTCAA

GAGCCAATGAAATTAGCTTCCTTTATAATATTCAAGAACTAACCAGTTTCACTTCTACTAATCCTCGGCG

CACTGATGATGTTTCTAACTAAATTGGATACTAAAGAACGTAGCTTTTCACCTCGAATCAAACAACTTGA

AAACCAAAACAATCTAAACGAAATTTCATAACCTAAGGAGGATCGGAAACTAAAATTTCTACATCGGAAT

CGAATCGACGCGAAGTGAAACGAAGATCGAT[AGAGAGAGAGAGAGAGAGAG]GACTCACTCGCCAGGAG

AAGACATGTTCGTCGATTTCGAAGATCCGATTGATTCAGAAGCGGAGAACAATCCAAGTTTTTTATTGAG

AGAGCACCGAACAAACTCTCTCCCTAGAACGTTCCTTCCCAGCTTCTCTACAAATCACTTGTTCCGCGAC

TGCGTATCTTATCCAATCATGTCTTGCCACG

(SEQ ID NO: 53)

BG1197

BG1197

(SEQ ID NO: 224)

GAACATCCTGCTGTTTCAGTTCTATTTTTCTTGTGTAGTCAAAAAGACATTATTACCCCATTGGAAATTA

CAACAACACATTAGTTGTGAGCGCCAAGATAAGGCTAAAGACGTAAAAACGCTCTGAGTATTCATTCTTT

CAGGTCAGGTTCAGAAACTAGTTTCGTTT[CATTTCATTTCATTTCATTTCATTT]CATCCACCTCCTCT

TCACTTGAGAAGTTCTGTCTTTTGCGATCCTTGTCATTTTTGTAAAGGTGAGTCGATCTATATATGGTCA

CTAGTATTCTGGAAATGATGCTATTTTAATACTCAGTTCGAACATTCTGTTATCAAATCCGGTTCTAGTT

AGTTGTTCGCGGGATAGGGTTTGCTTGAGATCATTTCGCTTCTTTATTTTTTTTAATGTCACTGATGGAT

CTGGTAATCTTCCTTATCGAATTAGGAAAATGAATCTGTATTAAGTGGACTAATCTCAAATCTAGGTAAA

AAAAATGGGAGGAGGAGGAGGAGGAGAAGGAGTTGGCGATTTCAGAGCCAAAGTATGGAGCATGTCTGGT

GGGCCTTACTGTAGGCCCAAGCACTGGCGTCGCAACACCGCCTTTGCAATGCTCGGCGTTTTCCTTGTCT

GCATCCCCATTGCCATGAAGTCTGCCGAGCTCGAGG

BG1230

(SEQ ID NO: 54)

AGAGAGACCTCCAGTCACCTCGTCTCTTCAGGCCTCTTGTGTTTCCTCCAACTTCCTTTACCAAAAAAAA

ACAAATCAAAATCAGATTCAAAGGAGAGAAAGAGAGAGGGAGAGAGCACTACAAGAGTGGAAAAGAAGAG

AATCAGGTCGTG[GAGAGAGAGAGAGAGATGG]CG[GATGGTGGTGG]TGATGAATCTGAGATGCGATGG

TGGTCGTGATGAATCTGAGATGC[GATGGTGGTGG]TGAGGAATGATGGCGGATGGGAAAGATGGC[GAT

GGTGGTGGTGG]TGACGAGTGAATGAGCGG[TGGTGGTGG]TGACGAGTGGGAGGAGAGATGGCGGTAGT

GGTGGTGGTGGTGATGAGGAGGTCAAACCTGATGGATTGGAGGAGAAAAGGAGGCGTCACAAAGAGAGAG

AGAGAGATTTGTGTGTTAGGTTAAAGATTGCACATTCAGAAATGTGCTTAGACAATGATCTGAAGTGGTC

TTGGTCGAGGTAGTCCGTACATGTCCGTACACAGTGC

BG1241

(SEQ ID NO: 55)

GGATGGGATGAGTCAGCTGCTGGTGATAGGCCCAGTCGAGTTTCAGTTTGGGACATTGAACCAGTTTTAA

CTCCTTTCTACATATGTCCTCCTCCATTTTTTCGACCTCGGTTTGCTGGACAACCAGGAATGCCAGGTAA

AGTCTTTGTACAGTTTCATTTTGCACATCATCTTTGAATCTCCTTAGAGATGGCAATTCTGGTGGTCTTG

CAGATGATGGGACTGACATGGAGTCTGCGTTGAAGAGAGCAATGCCGTGGCTTGACAATGGCCTAGAGAT

GAAGGACCCTTCCAGTACGATATTTCCTGGTCTGAGTTTAGTTCAGTGGATGAGTATGCAACAGCAGAAC

GGCCAGGTCCCTTCTGCCGCTGCACAGCCTGGTTTCTTCCCGTCAATGCTCCCTCCAACCGCGGCTCTGC

ACAACAATCTTGGCGGGGCTGATGATTCCTCAAAGTTACTGAGCTTTCAGGCGCCTCCAGGGGGGGTTTC

CTCATCAAACCTCCAATTTAACAAACCGAATCCGCAAGCGGCAATGTCCCAGTTACCTCAGCCACCAACT

ACGTTGTCCCAACAACAGCAGCTGCAGCAGTTGTTGCACTCCTCTTTGAACCATCAGCAGCAGCAGCAAT

CACAGCCTCAGCAACCACAGTCGTTGCAGCAACAACAACAACCGCAATCCCTGC[AACAACAAC]AATCA

CTG[CAGCAGCAACAAC]AATCACTACTG[CAGCAGCAGCAGCAACAAC]AATCTCTGCAGCAGCAGCAG

CAACAACAATCTCTGCAGCAA

BG1244

(SEQ ID NO: 56)

ATGATGATGAAATAGCTCTGAAGAAGAAGTTAATTAAGGAATTGTTGCTGTCTAATTAGGTGTTCTTGTT

GTTTGGTTAATTATGTTTGGTTCTCGGATTTGAAAGCTCTGTTAAAGAGCTTCAGTTTTAACTTTAATTA

TCGGATTTGAAAGCTCTGTGAAGAGCTTTATTTTTCACTTTATCTGTAATTGTTCTCCTGTTCTTGATGA

TATAAAATATTTAAGTTGTTCTTGTGTTGTTCAGTTATATTTACAGTTGTTGTTTATGATATATCATGTT

TCTTTGTCTTGTAGAGAAGTCACGGAGTCCACAGAGATGCTTGGACTGAAGGGAGTCACGGAGTCCACAG

AGAGATGTCATGTACCATGTCTTGTAGTGTGCAGGGTCTGTAACGAGTCACGGACCATGTGTTTGTATGT

GTCAGTATGTGTTTGTACGTGTCTTGTATGTGTCACAGAGTCCATGTTTTTGTTTGTGTCTGTATGTGTT

TGTATGTGTCGATGTCTTGTAGTCACGGACAGTATTTTTGTAGTCACGGACTTTTACCAAACTCATCTTC

TATTTATAACATCAATCTCATCTTCTATTTATATCAACCTTCTCTCTCGAAACATATACAACGAACTCTT

CTTCTCTGCTTTACAACAACAAACT[CTTCTT]CT[CTTCTT]AACAACAACAAACT[CTTCTT]ACCAT

ATTTATATTTTTTCCCCTTATTATAAACACCAAAACCATCTTTATAAAAACTTTATATGG[CTTCTTCTT]

CTC[ATGATGATGATG]CGTTTG[ATGATG]CATTTG[ATGATG]TTTTTG[ATGATG]TCTATGATCA

ATATTTTGATCAAGCATTTGAGAATTTGACCATTTGTCGTGATCAAGAAGAACGAAGAAAGAAAAGAAAA

AAACGAGCGTATATCGAAAGACATCGTGAGGAA

BG1286

(SEQ ID NO: 57)

AGTCCGAGACAGAGTATGCTA[AGCAGCAGCAGCAGCAGCAGC]TGAATACTGCATATGATGCGTCACAG

ACAAATGCTCAGAATCAGATGCAGAATCTTGCTTCTTTATCAAATGTGATGGTAAGCTACATGTGCATTA

TTCATATTTGAAGTGATCCACCAATGACATTCTCCAATGGCATTGCTAACATTGGTACTTTGTTTGTGTG

TTTTTGACTCAGCAGGGATATCCACACTCAGATCCCAACAGTTTATTGGCACAAAACGCTAGGGAGCTTG

AGTTCCAGTATTCCAATTTTGCACAGTCTATGCAGTCAAGAAATAGCAATAATGCTTCTTCACTTGGTGG

TCAAAGCATTTCCATGCCAGAGGTAAATAACCACTTTTGTCTTCTTTTTTTTTAAGAAACACAAGATGTC

TTGTTAATTAGGTTTTGCTCGACTATGGAGTGATCTATATGTATCCAAATCTATACAACAAGAGGAATTT

ATATGATTTTGATTATATATTTTCTTACATTGTAGGCGCCCCGAGGCAGTGGAATCCAAGCGACGCAGCA

AAACTTACAAGGTGCTAATATCGCCACTGGACCAGCACTTCCTCAACAGCTT

BG1288

(SEQ ID NO: 58)

ATAAGCATACCAATGAAGATATCAAAGAATGCAATATGTATGTTTTGTGTTGTGAAAGCTAAAGGATTCT

ACTTTATTTGTGTTGAGTGATGGTTCTTTAGTTTGGTGTTAATGTCTTGTGAATTGTGTTTGGCAGGTAC

AAGCTAGGTTTTTGTCCCAACGGTCCTGATTGTCGGTACAGGCACGCGAAGCTGCCTGGACCGCCGCCTC

CAGTTGAGGAAGTTCTTCAGAAGATACAGCAGCTGACTTCGTATAATTACGGGCCTAATAGATTCTATCA

GCCACGGAACGCTGCTCCGCAGTTGGGAGATAGTAATAAGCCTCAGGTGCAAGTTCAGACGCAAGAGGCG

GGTAACTTGC[AGCAGCAGCAGCAGCAGCAGCAGC]AACAACCTCAGCAGTCACAACATCAGGTCAGCCA

GACTCAGACACAAAACACTGCTGACCAAACGTCTCATCCTTTGCCTCGTGGGGTAAATAGGTGTGTTCAG

AGTTTCTAAAGTTTTTAATTGGGTTGTGTAAACTATGCTTCTGTATATCTGTCAAGACATTGTTTATTG

BG1321

(SEQ ID NO: 59)

ATGAAGATTGATGTATATTCGAATTTATAAAGTCTACGTTTAGTAAAGGTATACAAATCAGAGGTCTGAA

TTTGTTCAACTTCCTCCTCATTCCCCCATCCCCAAAAGAATCCGAGTTTTTTTGGATCAAGCCTATATAG

ATCCAAAAACCAACATAATGGCCCATTAAAGATGCATAGACTCGAACCAAACCGGATTAATACACTGCGG

GTGAAACCGGTTTGGGAATTTTCACAATTGACTGAAGAATCAGGGTTTAAGGAGAAGTCACAGACCCAGG

[AAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGCAGAAG]AATGGAGTCAGAGCACCAAACGA

TGGAACAGTTCCTACGATGGGCAGCAGAGCTTGGCGTATCAGATTCCATCGATCCTTCTCGATCTCAAGA

TTCATGTCTCGGCCATTCCCTTTCCGTCGCCGACTTCCCTCTCGCCGGCGGGTGCGTAGAAACAAAAATC

ACATCTTTTTATCATTCAAATTCCTAAACTTTTTCGACCATTGATGGGAAACTAGGAGAGGGTTGGGGGC

TGTT

BG1368

(SEQ ID NO: 60)

CTTCAAGTTTCTATTATTATCAGTTCAGGAAGTGACACTCACTAGACCAACAGAAGAAGAAAAAAATCAA

CATACAGAAAACAGATAAGCACTGCTCATATTAATCATGAATCGTTCAACAAATTTGATCCGAACATTAC

AGAAACTATACGTGTTTGATCCAACAACGAAAGGAGCACAAACAAAATGAGATCAATACGATCGTTCTTC

ATTGTCGTTCTATTACAAAACTGTGCTTGCTTTGTTGGTTCGAACTCGAACATACAACAACATAGATAGT

TATGTCGGGATATACTTATTTATATTTAGATTTAATTATGGATAACGACGGCGAGAGATTCTCGGCGACG

GAATATCAACTGTTTCGCGATGAATGCTTCGATCGTTTTCTGAAACTCTTCGTTTGTCAGATTATCCTCC

GGAAACGGCACTTCTTCGCACGACGCGACGGTTTCACGGCACTTCTCCGTCTCCGATCGCCGGAGCGTAG

GTTTCGTCACCGTCTCCGGACTCTGTTTCGCAGAGATTTCCGTTTTGCTTCTTTTATAGACCTT[CGTCG

TCGTCGTCGT]CGGAGGATGATCATTCGTCGATTC

BG1392

(SEQ ID NO: 61)

CTCAATCTTTGTGTGGGTGGAGCAATACATAAATTTTGTGTGGTTGGAAGGTTATGAAATGAAACTTTTA

GTGGAAACGTGAATAGATCATCCAACTTATTTTGTGTGTTTTAAGAAAGATTAGATGAATTTGACTCAGC

TCATTGGAGAGAGAGAGAGAGAGAGAGAGAGAGGCTTCACAGAGCCCATCGAATCCTATGCGCGTGTGAA

AAGCACGATCCAATCACGAAGCTAAAATCTTCAGCTTCGTTGTATAAAAAAAACTTATTGAAACAAACCT

CAAATTCCAATTACACCCTTGACAGCGATACACACTCTCTCTCTCTCCAACTAAAACATATCTGGAAATT

ATAAATAAAATTTATACTTTATCTGGTAACCCATCAAATAAAGCTATTAGTCACATAATAGATGACAAAA

AAAAAACAAATAAAGAAAATTTAGGAAACAAATCTACTGAGATTAGGCTGTAAATCATACGTATATCTTT

CCCGTATACAGAGTGCCGTTTTAAGTATAATGTCGACACGTGTCGGTCAGAGGCTCGGCTTCCAAGGGTA

AGATTGTAAAATCACGATCGTCATCTCTCTTTAAGAATTTCCAGAGTGCT[GAGAGAGAGAGAGAGAGAG

AGAGAGAGAGA]GGTGCTTTCCCATAGCCATTCACGTC[GAGAGAGAGAGAGAGAGA]GGAAGGAGATGG

AGGATATACAGGAGGAAGAGAACGGTACGGACGAGGAGGTGCTGGGATCGAGCTTGACCATGGAGAAAGT

GGCGGCAGCTAAGCAGTACATCGAGAATCACTACAAAGCTCAGAATAAGAACATTCAGGAGAGGAAAGAG

AGGTATTATAAATCGTCTCTTTCGTTGAGTGAGAGATTTGAGATTTGGAATATCGTTTTTTTTTAGAGAC

TAGTTAGGGCGAAATTAGTTGCGTGAGCTTTGATTAGTCTCTCGTATTTGATGATAATCATGGT

BG1442

(SEQ ID NO: 62)

GAACAATAACCTGATCAAGGCTCTGCTCAGCTGCTTTACGCTCGTCGGGATTGGGACTGCAAGCAGCCGC

AGCGATGATTACTGCCAGGTTAGACAGATCCATCGGGAAAATTCAGCAACAGATTCTCCGAGGAATAGTC

GGCGTGTCAAGAATTTTCCGGTGAATCGAAGAGGCGAGGAGGAAGAAGATGACCGACGAAGACGAGGACG

AG[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA]GGTATAAGAAGGAAGGTCTAAGCTAGGGTTTT

AATGTATGGTTCGTTGGTCTGTTATGGTAAGTCTATCGCACGCACGCGCGTGGTAAAAAAGTGAAAAAAA

AGAAAAATCGACGTGAGACACGATACACAACCCAGACTTGCCTGGACCTCTAGTCACCTATTTATTTTCA

CCGCCTGCTCGTTTAGTTAGTTACGGTCAATCGATTGACTTTTGGTTATTTTCTATGTGTTTTCAATATA

ATCAAATTCAAATGATTTTTTAATCAAATCAAATGTAAATAATAAATAATAAAAATCCAAATGGAATTAA

TTAGTTTAAACTTATTAAACTATTTTGTCATCTTTTTAGTTATTTAAGAATTATATTAAAACTTGTAAAA

TTCTAGTTACAAATATAATTTAATGCATAAATCTAATGAATTTGGGAGAAAATAG

BG1449

(SEQ ID NO: 63)

GGTTTCCGCCGCAATGATTTGGTAGTTGTGGTGAACAGGCTGCATATGTTTATTTATTAAGGTCCATAAG

ATCATAGAAACAATGGTGAAA[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA]TTCATAC

AGTGGTGTTGGCGGAGACTGGGATGGAACGAGTTGGGGTGCCAATTTCTGGGAAGAGGTAGGTGGAGTGG

TTATCATCATCACTGGATGAGTAGTATGCTCCTCCATGAAGTGGACTCCGAGGAGGGCTGTCCTGCCGCA

TAACGTAGCCATCTGGCTCATACATTGCTTCCATTTGCTCATCATTATGATTGTTGCTGCGACGGTCAGG

CTGCGATTTGTTTAGGAGAGCTGATTATTTATTGTCAAGAATATGTTTTATCACTAGAGAGAAGCTCAGG

CTTGAGAATGTTGTTTGAGTACCGTTCTGTATTGTTGTGGTGGTGGCGAACGGGTCGCAGCTGGATGAAA

TGGGCTTGGTGGAGAGCTGTTTCGTGGTGTTACTTCGCCGTCTCGCTCATACACTTCCTCGGATTGCTGA

TTATCAGGGTGGTTTCTACGACGATCAGGCTGTTAGAATTAGAATAACGAACTTTATTGTCAGGTTAAGT

GAATCACTTTATCTGTTTCTGGAAAATAAGGCTGAGTTTTATGGTACCGTTCTGTATGGTTGTGGTGGTG

GTGGCGAACGTCTTGCGGCTGGATGAAAGGGGCTCGGTGGAGAGCTGGTTCGTGGTGTTACCTCGCCG

BG1453

(SEQ ID NO: 64)

AGTGCTTTAGGAGAGGAGGATCTTGATACAAAATTGTTCTCAAGAATTGAGAAGAGGAAGAAGAAAAAAG

GGTATTAAAAGGAAGAAGAGGAGGGCTTTATTAATCATGTATTGCTAAAGAGGAAGATGAGGAACTCATC

ACCTTCCTTCCTTCAATGGCAATGAAATGAAAA[GAGAGAGATATGAATGAGAGA]GTGAGTGAAAAA[G

AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA]GTTGGGTCTACCATGA

AAAGCAGAGGAGGTGGGTCAAAGCAAAAGTTTTCGACTCTTTTTTTAGGGCTTTTCAATTTCCTTTTTTT

CTTTACTTTCCATGTTTGTATATTTCCCAAAACTCGAATTCATACACAAGTATCTCGGGAAAACGGCTTA

TTCATGCCCGAACTAGGGGTTGCTGAGAGAATACATACCTCAACTTTTACTGCAAGTCGAAACATAC

BG1513

(SEQ ID NO: 65)

ATAGGTTCTGTGTCGTAACCATTAGAGTCTTAAAACCGTCAATTTCGATTCTCACATCCAAAAGGTTTTA

ACATTTATATATATAGTAAGATGGGTAAAGTTTGTAACCGCTCGTGTATAAAAATCAGGTTTACGAAAAT

CGCATTACTTTTGATTTTCTGATCGAAACAGAGCTTCGAAAAGGAACTACTACGCAGTAAATAAACTTAC

TTGAAGAAACGAAACTTACTTCGAAAAGGAATTACTTTCTGAAGGTTGCGATAGCGAAGAGAAACTT[GA

GAGAGAGAGAGAGAGA]GCGAGCGAGAGAGATATGAGAGTGATACCGCGGCGGAAATGAAGAAAAAAAAA

AAAGGTTTAGGGTTTAACGACGACTGTTGCAAGTTGTAACCTTTGACTTGTTTTTTTTTAATAAATCTTT

TTTTCTTTAAATTAAAAGAATAAACTCTAGAGTGGAAACCCCCTGATAAGTAATAATGTTTCAGTTCCGA

CCCCAAAGTAAAGTTTCAAATAATTTAACCCAACTTTATTGTAAATAAAAAAAATATTTCGATGAAACGA

ATATGTGCAAAATTTCATATATCCATAATTCATTACGATGTGTTTTATCAAAAAAAAAATCTTTT

CA0105

(SEQ ID NO: 66)

GGTTAGTCATCCAAGAACAGAAAAATCTCTAAAGAAAACAAACAAACCTTGATGTAATAAGCCTTGCGAA

TCTCTTCCTCAGAAGCGGAGGGAGTGACACCAAGAACATCATAATATACTGTTTCCTTCACCATGATCGG

ATCAAAGCAAGAGATGTAACTTTTTTGTGTAGATAAGAAAATAAAGGTTGGTTTTGTGGATTGTGTGTGA

AGCTTTTAGGAGATATGGG[GAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAA]GTTGGGACACAAG

AGAGGTAGGGTGTGTGATCTGCTTGAAGAAGCAAATAAAGAGATGTCTTTACAGTTATGCACTTTTGATT

TAATAAATAAAAAACTTTATAGCGGGGAGGCTACACTACACTTTCACCATCTCTTTTTAACTGTCCACTC

AATTGCTTTATTGATCTCATGCCTCCTTTTTTATTATTCCATTGCTTTTCTGTATTGTTGAACATACTGA

AGAAAAAGAAGATGATGATTTTTGCAATACGATTGTGATCTGGTGTATCTTATCATTTAGCCAAATGGAT

ATTAAGTTGGAAAAAAATTCAAAAATATAGGTCCTACCGGGAGTCGAACCCAGGTCGCTGGATTCAAAGT

CCAGAGTGCTAACCACTACACCATAGAACCTTGTTGTCTTAACTTTACTTTATTTATTTATAATGAAACA

TTATAT

CA0120

(SEQ ID NO: 67)

TTTAAGCCAAAATGTCATAACACAACAAAATGAGACAATAATAACATTACTGTAACAAATACATAGTTTC

TAATTAGAACAAAGACTAAACCAGACCAAGAGAAAAGTCGACAACAACTTTTAACTCTGTCCTT[CCACC

ATCATCATCATCATCATCATCATCATCATCATCATCATCA]TCCTCATAACTTATTGTTGTACCAGAACA

CACCTTTCTTCTCACCTTGCCTATCCGGTTCAACATAGATACACTCCTTCGCCTCCCTCCACATCGCCTT

AACCACCGGCGTTCCATCAAACTGGTAATACTCTCCAAGTATCGGCTTTATCGCCTTGGTCGCTTCCATC

GCGTTATAATGCGGCATCGTCGAGAACAGATGATGCGCCACGTGCGTGTCCGTGATGTTATGAAACACCT

TGTTCAAGATTCCATAGTCTCTATCCACAGTAGCCAAAGCTCCTCTCAACCAATCCCACTCCGAAGAATA

TAGTGAGGCAGCGAAGGGTGCGTGTGCTGCAAGTAAGTGATCAAGACGAGGAAACAGTTGACAATCATAA

GCGGAACTCCGTAGACACAGACCATCGAGGCCACTCCTCGCGAACCAGCGTAGCGGTAGAGACCGTAACA

TACGGAGAGGACGCCAGCGTCAGAGATGTATATCTGGAGACGCTCGCGGTCGTTGTAGATGGGAGCGTTC

GGGTGGAAATGGCAAGCGAAACCGTCGCTGTAAGGTCTTCCAGAGACGTTGAAGGCTAAGTACAACGGCC

AGCCGAGCGTGAACTGGACGGTTAGCATCACCGTGCGTCCTAGCGGG

CA0163

(SEQ ID NO: 68)

TGAGCTATTAAAACTACATTATCTTAACGTCATATCAATTTGACATTTGCTTAATATTCATTTTCTTAGA

ACCGCTTGTCAAAAGCTTTCCCAGTTTTCCATTAACTAAAGAATGCTATCAGGAGAAGTTTCTGAAATTA

GATCATCATCATCATCATTTCTTAGCAACTTTTCTGAGATTGAGATCATATATTATCATCACCATCATCA

CCACCACCATCATCATAATCATATTTGCTTAGCAAATTTTTCTAAGAATCGTATATTATAACCACAAAAT

CTATATTTACTAACTTACAAGATAGATCCCATAAATTTATAACATTCTGCGATTACTCATTCCCTATATA

AATAACGTTCCATCTATTATATCCTACATTAT[CATCATCATCATCAT]CACCATCACAAT[CATCATCA

T]CACCATCACAATCATCACCATCACAGTCATCATTTTTTCATAGCAAACTTACAATTCGAAGAAACGAG

CGCCAAAACATCCGACCTTCTCCAGCAAGACTGAATCCAAAAATCCGAAATCGACAACATCTCCAGCTCA

TCACGAACCCTAGGCAGCCACACCCGCACGAATTCGACATCGGTAAGGTACAATTCGTCGGAATCGTCTT

CGACCAAGTCGTCGAACGACATCCCGCTGAGCGCCTTATCAACCCCAACCGATATGACCCTAACGTTGCG

AGAATCTTCGATCAGATTCCTGAAGACCGTCTTGAACGGCGTGACGGAGGTCGAGCGCGATTTGGAGTAC

CGTGACAACGTGCATAGG

CA0221

(SEQ ID NO: 69)

TGTTCCTCTGTTTCTTCAACCTCAAAAGCTCTTTCCACAGAACAGTGCAGTTCGGTGGACGAGGAGAAGG

GTCGTCGTCAAGAAACCAGCTTCCTCTGTTATTATTCTCCTGCTCCTTGGTGCTGTTGTGAACAGTCTCC

TCTTTCTTAACTCTACGAT[CTTCTTCTTCTTCTTCTTCTTCTT]CTACTACTTCTACTTCGTCTACGTC

GACGACTTTTGTTTTCAAGGTAACGTTTTGAAGCTTCTCCGAGTGTTTGGCTTGCCAGAAAGGAAGCAGC

TTTCCTTCGGAGAACAGCTCGTCTGCGGCGGTGAGCATCGTTTGTGTGTTCGACAGAAACTCAAAGTCTC

CAGCTTTCACTTGTTCTTCTTTTCCCCTTAGGAGATTCTCAGGGTTGATGCAGATGTAGTCTCCCTCGCT

GTCTGATGATGACAGATCGGCGGAGAAGGAAATGCGAGGTCCTTCCGTCGTGAAAACCATCGTAGCCTCC

GCCGTTTCCGCTACTACCATGATCGTACAAATGTGTAGTTATGAAGTGAAAGACAAATCAAGTGGA

CA0226

(SEQ ID NO: 70)

TCTTAAATTTAAATTCTATTCCTCAAACACTAAATCTTAAATCTACACTCTATATCTAATACTCTATACC

ACAAATTTAAACTCTATATACAAAATCATAAACTCAATCTTTACAAACTTATAATAGAAACTTTAAATTT

AAACCTAAGTATATTATAAAACTCAAATTATATACTTAATCCTAAATCTTAACCCTAACTCATAAACCAC

ATATTTCATAAAATATTAAATCTTAATTTTTAAATAAATTATAGTTCCATAAATAAATTAAAATTTCAAA

TAAAAAATTTAGATTTTAAATTTGAAATTATGATATCAAAGTATTTAAAACTTAAATAATTTTTATAATA

GCTATAAATAAATAAGAAGATAATTTGTATTGTTTTTTTATACCATTGATTGATTTGAATCAAAGACTCA

AGATAGCTCTTGTATCTATTTTCGCCTTTTTTTCTTATCGGTAGTTGTTGTTTATGGCATGGATCACCTG

CACCCTTAGATAATATTGAACCAGAGATTAATTGTTCTTTTATTCTTTTTTTTTTAATTTACTTTTCTCA

GATCTACGAAA[GAGAGAGAGAGAAGAGA]TGGAGTTCAAGGTAGAGAAGGAGAACGCGACGGCTGTTCG

TC[ACCACCACCACCACCACCACCACCACC]ATCGTTCGTCACTACCACCTTCGCTTCTCAGATACGTCT

TCACCGGAGTCGCCAGAACCACCGTCACGCTCGTCATAACCAACATCGCTCGTTCCCACAACCACCGCCA

TCTCTCCCATCAGCCATCGCTCGTCACCACCACATCACTTCTCAGATCCGTCTTCACCGGAGTCGCCACC

ACAACCGTCACGCTCCTCATAA

CA0233

(SEQ ID NO: 71)

TTTTTTTGGTTCTTTTAACTTTTAATAATTAATTCAATAATCTGTAACCTCTCTATGTATCTCTTCCTCA

TTGTATCTGATTGAGTTTGAATCAGTTTTTGAGCAGGACGTGGTGATGCCAGAAGATCTAGCGAATGTCC

TTAGGACAGCGAAAGAGATTGTCGTTGCCACAGTCCTTCCCGTCACACTTTGCTTTTTGTACATCTCTAT

GCCTTGACGCCGCCGTAGGAACACGCTATCAAAGCTTGCGGGGTTGGAGGGAGCTCGACGACGGAGGAGG

CAAGCATGGTCTCGTCAGATACAACAGAGTCAGCATTATCTCAACATTCCGGTCATGTAAGCGGTGTAGT

CGTTTATACAAATTTGATTTTCAGATCTGAGATTCGCTTTTTGACTTTACAGTTTTTGTGTATATTTTTG

TAGGATAGGATGAAGGG[GAAGAAGAAGAA]GGAGGAGACGAAGACGAGAGGCTGTGTGGTTGGCGTCAG

TTATAT[CACCACCACCACCACCACCACCAC]TACCGTCACGCTTGTCACCACCATGCTCCTTTCCATTC

GAGGCGGCTGGAATCTTTTTTTTTCTAGGTTTAGA

CA0328

(SEQ ID NO: 72)

ATGGTACAAACAATCATACAGACACGCATTCTCTTCTAGTTCAGCTGCTGCTTTCTCTAAGCTTGTCTTC

ATAAAATTGTTACAAACTTTGGAGCATCCTTCCTTGGGCCATCAAGGGACCGATACATGTACAACGTCTC

CACATAATCTGCATTGT[CGTCTTCGTCGTCGTCGTCGTCTTCGTCGTCGTCGTCATCATCAT]CTGACC

CAAACACCT[CCACCACCA]CACTAGGCAGAGTATGAGCAACATCCCTGCAGGCTCCCCGGGACAACCTA

CATGACGACATCCAAACAAACCTCATCTCGTTATACCTATGCATACCAGAGCGCAGTCCAACATCACCAA

AGGGACTGTCCCTGATCTCAAGCTTCTCTAGTTTAGGACACCCCTCGAGGATATATCTCAGACCCATGTC

ACTGTCCCCTGCAAAAGCTACAGATAGAGTACGTATCAGTTTCCCATACTCTCCTATAAGGCTAAAGGCT

TGGTCCGTTAGTAATCCAGATACTGCAAGCCTGGTTAGCTTCTTGCAGTTTTTAACAATGGCGCCAAATC

CATCGTCCATTGGCTTCCTTGTCACGTGGTCAGGCCTATGGCGACCCATTATGCAAAGCCTAAACACGGT

AAGCTGGGGACAGTTCTCAGACATGGCTGTCACAGCTACATTTGTCATCCGCTGGCAGAAGTAGAGAATA

GACTCAAGTTTCTTACAACCTTTCTGAAATTGCTTGGAGGCCTAATCCCGAGACAGGACCTTCACTGTCT

TCACTAGGATCCAAAGGGAAAATCCCTAGCTCACGGAGCTCCTTGCATG

CA0410

(SEQ ID NO: 73)

CGCCGCGTCTCCTCACTCTCCGGATTACTCTCCGTCTGAATCCTCTCCTTCTCGCTCGCGATCTCCCTCT

CCTCCTTCCCGCGACGCTCCCTACCGTCTCCGATCGAAAGCCGCCGCCGCCTCCGCGAATCAAGGAGCTG

GTGGTAATCCATCGGGAAGCCGTACTACTAGGAGCCGTCAACAAGCTGGGAACATCCGTACGTTCGCCGA

TCTGAACCGTTCCCCCGCTGACGGCGCGGATAGTGATTCCGACGAAGGCCAAGAGTACTATACTGGTGGA

CAGAGGAGGTAAAATTGTGTTTATATTGAATGATCATAAACTGAGTAATGTGGAATCATGGAGAATTGTG

CTATTGATTGTTTGTGTTGGCTTCTCTTTAGCTAATGGATTGGGCCTTGTGTGTTTAGTGGGATGATGGT

TCAAGATCCTACTAAGAAAGCAAAAGATGTTGATGCACTCTTTGAGCAAGCTAGGCTTTCAGCTGTGGAC

AGGCCTGTTGAGCCATCGAGATCAGCTTCTACAAGCTTCACTGGAGCTTCTAAGATGTTATCTGGTGAGC

CTGTTCCCTCTGCTACTCCT[CAGCAGCAGCAGCAGCAG]CAAGACCAGCCTCAGTTGGTTATGCACACC

ATCACTTTCTGG

CA0423

(SEQ ID NO: 74)

CCCTATTTCGCTGAATCTGCTTTCTAACCCTAATTTTCTCGATTTTTCTGCTCAAGCGTGTTGGCAATGT

CGGAGGACATGGTGATGCATTTCTCCTCCAATTCCTCCAATCAGTCCGATCACTCCCTGCCCGACAAAAT

CGCGAAGCTCGAGGCTCGCTTGACCGGCAAAACCGCCTCCTCCGCCAAGCCGCAGCCTC[AGCAGCAGCA

GCAGC]TCTCCGTCTGGTCATCTGCTTCCGCCCCTGCCAAAGTCGCGGCGGGTTCGTCGGATGTCTCTAT

CAGTGATTCCGACGACGAGGTAACTTCCGATGATTTTTTTTTATTATTTTTTTTTTTAAGATTTGATGTC

TAATAGTATTCTCGTTGTTACTACTGTCTCAGAACACAGGAGATTTCCTGATCCGAGCAAATACCAAGAA

GCGCCAGAAAGTTCAAGACTTTAACAACAACAACTCCACTCTTGTTGATCATGCTGAGGTAGTGAATTTT

CAGTTTAAATATCGATCTTTTCGTCCCTTGCCTGGTTCGTAGTTATATTGATATGGTAACTAAGGTTGTG

CGATACTGAAACAATCTGATATGATGCAAGTTTTGTATTCCCTTTTGATGAATTATTATAATGTCGAAAT

TGAAGCCGCAAGAGGCAGCATATGATGGAAGGAAAAACGACGCTGAGAACCAGACAGGCGTCGATGTGAG

TAAGAAGAAGCAAGGTCGAGGTCGAGGTTCATC

CA0456

(SEQ ID NO: 75)

CTTCTGTTAGAATTCTACCG[TTGTTGTTGTTGTTGTTGTTG]TCTTGGTTGTCTTAGAAGC[TCAATCA

A]CGCCTCCGCCTTTAGCTTAGCTCGACTCTTACTACTCTGCGACAAAG[CCACCA]CAGGAGCAATCGC

ACCTTCTCGCGCCACCATGGTTCGATACACCACACT[CTCCTC]ACAAAGCTGCAGCAATATCGACACGC

CCATCTCCTTCTGCCTCTGCGTTCCCACCTCCACTATCTCCACAAGCACCGGAACTCCTCCTTCCTCCAC

CACCGCCGGCTTCGACTCCGGCGCCGACATCAGCAGATTCATCACGTACGCCGATTTATCCACCATGTTC

GAATCGAAATCCGCCATCAGCTCCACGAGCGGCTTCATAACTCCCGATTCCACGGCCCTGGTCTTGTTCT

CCTTGGCCGAGCAGAGCGAGTAAAGAGCCGTCGCCGCGTCCTTCTTCCCCCTGAACCCGCCGGTTTCCAG

AAGGTTCACCAAGTGAGGAATCGCTCCGGATCTCCCGATCGCGATCTTGTTGTCTTCGATCTGCGATAGG

CGGAGGAGAGCGCAGGCGGCGTTCTCTTTCGCCGTCGGCGTTCCCGATTTCAAAACCCTAACGAGCGGTT

TAATCGCGCCGGAGGAAGCGATCAGCTCCTTGGTCTCGTCGCAGAGGGAGAGGTTCAGCACAGCGGTG

CA0488

(SEQ ID NO: 76)

GCCAAGCTCTCCGACTTGTCACCGGTGCCAAGAAGCAGCCACTGAGAAAATAAAACTCATTCAAGATTCA

AAATCCTTGTGTGCTTCTTCAATGCCATTTTAGTTTGACTTCTTCATTTGCTACAGTTCATTAGTTATTT

CCTTATTTGCAAAAGAGCCCTCGAGTTTGTTAGAAACGTGAAATAAAGCCATTAAATACCAATTCCCTCC

ACTTTGAAGGGGTTTTGAATATCTTTCCCTCGACTCCAAAATCCTCGCCGGCGATAAGCAAACCCTAGAT

TCGATTCGCCGTCTGTTCATCCAGCAATGTCGTCGTTCAATCCATTCTCTACCCCACAGCGACATCAGCA

GACGCCTCAGCCGCAGAGCATCTCCTTCTTCTCGCCACCGCAGAGCACTCC[CTTCTT]CTCTCAACTG

[CAACAA]CAGCAAACGCCGTCGTTTCAGCCGCACCAGTTCCAGCAG[CAACAACAACAACAACAACAA]

AGTCAGCAGCAGCTGTATTTGTTCACGAACGATCAAGCTCCGGCGAGTTACAGCACCGAATGGGAGTGAT

CTTTCATCCGATTCTCAGAAACTTCTCCTTGAGATTGAGTATTGCTCTTCTTTC

CA0546

(SEQ ID NO: 77)

ATCAAACCATAGAAATACATGACTTACTACATTCACTCATCTGTGTTGGAAAACTTTTCAAATTTATTAT

ATCTTAATTTATATTATTTACAAATGTTTATAATTGCATGATTTCAATTATCCCCCATCACAACATATTT

TAAAAAATTTAAAAATTATTTTTAAGATATACAATATGAGAAGATTTTCAGAAGGCTTCTATGAGTATGT

TCTTAAAAATACATTCTATTTTTTTTTTTTGGTCTAATGGACTATTTATAATTTCAGTAGCATTTTAGAT

TAATTTTGCATTTGATCCATGAGGTATATCTTTGTGTTTAAAACCAAGTTTTAGGTTATATTTGGAAATT

TCCTCTTGATAGTTTGAAGGTTTGAAGTTTTGATGCGGATAGCAATGGATAATAAAACGGATTTTGGATC

TAGGACAATAATTCGTCCATCTCCTACGTGGGGTCTTTAGTGATAATGAAAAAACTCTTCTGGTAAAAAC

AAAATGTTTTAATAAATATGGGGCTCATCCATAAGTGAAAAATACCTCTCTTCTTCACTGCAAATGAATT

ATAAACCCCTTCCTTATCCACACACACACAGACTTGTTCGCTCTCTTAAACCCCT[GAAGAGGAAGAAGA

AGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAA]GGCGAATCATGCAGATTTGCCAAGCAGCGGTAACC

TTCACCTTCACGAACCCAACAAACCCTAATTTCTGCAAACCCAAACCTCTCTTCCCAAGCTTCCAACCCC

CTCGCCGCGTCGCCTTGCCGCCATGCCGTGGCTTCAGCTCCGACGAGTTCCCCGTCGACGAAACCTTCCT

CGAGAAATTCGGACCCCAGGACAAAGACACAGAGGACGAAGC

CA0552

(SEQ ID NO: 78)

GATTTAAAATGACAATTTTTTATTGGTTGGTTCCGCTAGGGGGTGAACCAAGAATAACT[CTTCTT]TTA

TTTCTACGTTCTCATTCTTT[CTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTT]CTACGATTTTTAATT

TCATTCAATGAAACAACAAAGTAGATCTGATTTTTATTTGAGTTTGGGTCCAAGAAGTGAAGAAAAATAT

TGGAGAGGAGGATCGACGCCTCTGTTCAATAGCCATGGAAACTCTGTACGCTTCCTCTCAAGCTCCGTGG

AGAAGAATACCAGGAAGACGGCGAGCAAGCTCCGTGGAGAAGAAGACCACGAAGACGGCGAGCGACTATG

AAGGTGGCTGGATGCAACGACAAGTACGCCTCATCTCTGAAGCTTGACTCTCAAATCCCACAGCGAAGAA

GAAGAATCAAGGCGGATGTGAAGAGCATAGAAGAGACAACGACGAAGGTGGTGGTGGGGCTGGTGAGGAC

TGGAAGATGAAGAAGACCACGTAGGTGGTGCCATGGTGGTGTGCGGCGGCGTACAAGATGAAGAAGACGA

CATATGGTAGTTAATTAGAAATTAGGTTTAAGCTTGGTTTAGGGTTTTGGTTTATTTGGTTTGGTTTTAG

TACTTTTTTATCTAATTAGATTTTTTTTAATATTTTTGTAAAACAATTAA

CA0603

(SEQ ID NO: 79)

CATCATCATCCATTCATCATCATCATCATCATCATCATCATCATCAGCCACCTTTCTACTTTGTCTGTTT

CACAACTGCCCAATCTACCTCCCAAAGTCGTCTCTTCCATGTGATACACTTCGCTTGGCTTCTTCTGCCT

TTAGCTGTTCCCGTCTCAACGTTCGTACGTTAAAGGTAAAGT[CTTCTTCTTCTTCTTCTTCTTCCCTT]

ACGTATTTTCGTTTTCCATCTAAAGATTCATTCTCTCCTCCGAGTTTCGTCCCCTGTCTACTCTGTTTCT

GTGATGTTGACCTCTCTCTTAAGCTGATCTGATATGTGTTCTTCTTCCTCTTTGATGCTTCTGTCTCTGT

AATTCTTTGACTACTTTAGATATTTTATCTTATGGGTTTCATTAAACTCGCACAAAGCTCGTGACTTTGA

GTTATATAACCAGTTCAGCTCTATTAAAGTTTTCTTGTAGACCAAACACTCATGAGTTACAGTGTCTTGT

TCTTAATCTTCCTTTTGACTATTTTATGAAAAGTTCTTGATCTTCGTTACTTTTCAATAGTCTGATTC

CA0614

(SEQ ID NO: 80)

CGAGGGACACGAGGATAGGACCTGGGATGCCTTGAGTGCAGGCTGTGCAGCACCATAGGCGGGCACCTTT

TTAATATTGTATGTTGTAATATTTCATCTAAAATTATAAGATAATAGGGTATACATAACTTATTTGCGTG

TAAATAGATCTCATTTCTACATTTGAGAATCATGAAAAATATATATGTTCCAAGTGGTTCTGCAATGTGT

TAAA[TATATATATATATATATATATATATA]GATATTATTTTCAATTAATATACTCACAAAGTTGGTTA

TGACTTATAGTACAAAACAAAATGTGGAGTTCATTAACTACACGAAACCCATTTGTCCACAATATTGAAG

TAGTCTTTTGTGATGATTGACATAAATTCTCATTTTAATTGCCCTTTATTGGGATAGCTGACAACAACAA

AATAAGTAATTCTTTTCAGATTTGAGAAAATTTCAACTACATATGTAAACAATTCAAAGAACATAAATAT

AATAAGAAATGTGCACAAAAAAAAATATAGAGATATATAAGAAATAGGTAAATGAGGCCAAAGATTGTTG

TTATATAGAAAGCAAGTCACTGCATCATAATATCATGTGGTGGTTCAACTTTATGACGATAGTGAATAGG

TCCTCTCTTACGCCTTGAGATTTTGTTTCCGTTGAAGCTGCAGATAACACACCTACACCTATATCTTGGA

TGATAGTGATGATGATAATTATGATGATGAGGATGATGACGCAAGTGATGATGGTTTTTCCTAAGAGCGC

AACAAACCGAGCTGTCTGATACC

CA0636

(SEQ ID NO: 81)

CTCGGAAGAACGAATCGGGTCAGCTCAATGCTCCATCGGGTCAACCTTATCTCTACTCGGATCTCTCTCT

CGGGTCGAACTTCACTTGCGCGACGCGAAGCTCGAACAGCTCTGTGACTTGCTGGGGAGGAGGAGCGGAG

AGGTTCAACAATGTAACCGAAAAGATCTCATTCGAGTCAGTTACATCCGGGTCGGGTCTAATCTGCGGGT

TGATATCCGGTAACCTCTCGGTCATGTGTTGGAGCCCTAATAACTTCTCAAGAATCTTCCTTCCTTTCCC

AGATATCTTACCAGGTCCTTGCGTTGAATCATCTATTTGCAAATGTGGTGTGTATCCACGATCTGATCAG

CTATGCTCCGGCTCGGGTTCGATCTGCAGCAAATGCAAAATCTCA[CCTCCTCCACAACCACCATCACCA

CCACCACCACCA]CCGTCAGATTCATCTCCATCTCCATCTCCGCCGCCGTCGAAGGCGTTAACGAGAGGA

TTACTAGCGTTTGCGATCGTTGGATCAGTAGGAGCGTTTGCAGGGATATGCAGTGTGGTGTACTGTTTGT

GGACCGGAGCTTTCTTGGGGAAAGAGAAAGTTCATAACTCGGTTCAACCGACGATAACCCGCGGCGGTTC

GAGTACCCGGTCAAGCAGCTCGCCGCCTTCTCGGTCCTTGACGAATAGACGTCAGGGATCGAGAATATTT

TCGATGAGAA

CA0681

(SEQ ID NO: 82)

TATACTGCTAACTATTTACATGTAAAATCTCTGTCAATTATCTTTTCCTATTCTATACAATTTTCCACAG

TTATTTTTGTAGTTCTGTTGCTGAACTTGAAGCTGAGTTTGTGGTGAAGAAACAATAACACCAAACACAG

CATATCACCTCCTTCCTTCTTCTAATGCATCTCTTCTCCTCTTCAACCCCACACTGGATGCGAACCTGTT

GTTCTTGCTTCAATCGTTTGAGCTTGGATTTTTTCAATCTCCATCTCCCCTCCTCTTCCACCAACATTAC

TCTCTTCATCCCCATCCAATACCAACTTCTCAAACCTACACTGTTTTGTTGCAAAACACACAGTCTCAGC

CACATCATTAAAAAAAAGGAAGAGG[AAGGAAGGAAGGAAGG]AAAGTTTTACATTTTCACATTCCCATA

AGGGTAGGTGAGACGGCTGATACATTCGCAACTCTGTTTGAGACACGTACAGCTGATGGTTGTTATCCAC

CACACGAGGGGCCTTTCCTTTCTTAGTTGCTTTCCACGGCTCCTCAGCACTCTGCTTTATAACAACCTCC

GGAAACACGTTACTTCCAGAACCCGAAGTTGATCCACCTCCTCCATATACGTCACTCTGCTGATCTTGCA

TCTCTCTTCTAGATCGGTTCTGGACCATGTACCACCTTCGTATCCTCACAACCGGTCCTTCAGTCTCTGC

ACCAGACTCGGAATCAATCCCCGTCATCACAAAGCCTCACCACCATCCCCAGCCTTCAACGCGTACTGTG

TCATACATCCAGAAGGAGCAAAAACAGTAGATTCCTCGTTACGCTATTATCAGAAGCAGTA

CA0719

(SEQ ID NO: 83)

GCTACATATGTCCCTAAAGAGTGAAACTAAAGCGAATTGCGAGGATTATTGAACAAGTTTCCTTCCAACT

TTCTAACGAATCAGCCATCATAGTAGCTCGCAATCAACATTTAGTTTCTGGGAAGATGAACAAACACAAA

TTACCCAAGAACACGAGACACCCAGAACATAAACAAAATCAAATACATCATGAATCCGATTAAAAAGAAC

GAAGATGGAGCAAAGTACCTTTTCTCGATTCGACTTGGAGAGAAACTCGAACGAAAGGGAATAAAACCCG

AGGAGTGACTTAATTGGGTCACATAATTTTGTTAACCGGAAAGTTACCGAACCGGAATCATACAGCTCGT

TGTGTAGT[GGTTGGTTGGTTGGTT]TTACAACTTCCACAGACTAAAAATGACATGAAAAATTAATCAAT

TATTTACCTGAAATGTACGATTAGCCAACAATTAGTTCTGTTATTCATAACAAAAGAAAACATTTTAATT

ACAGAGGTGAACGTATCCTAAAGAGAAATCTTTTTTTTCAAAACAATTAAACTTCCATTCATTAACATTA

ACCATCGCAAATACAAATCAAGGTCCAATCACACATATACGACTCAGACTCAGGATCTGACTGGTTCAAA

CGCAGC

CA0736

(SEQ ID NO: 84)

GCTTTTATCCCGGTTTAATAACTTTGAAATTCAATCCATCGACGTGGTCGGTTCATGGTCGAACCAATTA

CTGGACCCAACCCGACTATACAACCGGTTCATGGTCGAACCCGGTCCAACCATCGGGTCGGTCCGGTTTT

AAAAACACTGCTCTAAATGGAAAAATATAGTCCTTTTTAACTATTGTTTAATTCAAAATCTGTTGACTAT

AGTGATGGATAATACAATTTATATGGGATTTGAATTTATGTATTAGATGTAAAAATTGAAAAAGAAAACA

TATTATATACGCTCTACGAGCTTTTTAAATAGATTTATTGGACCTAAGTATTTCATAAGTTTTGAAAACA

TGGGCTTACAAAACCTTTTTAACGATGTTCAACCCGGGCTTAGACAAACTTTATGAGATCACGGCTAGGC

CTTTGAGTGCTATTATTTTATTTTATTTATTTCTTGAATTTTAGGGATTAATAAATGTGAGAAGGAGTAG

ATAGTACATAATTAGAGATTGATGGAACAAATTGCAATAATTTAAAAGTAAAAGGATTTAAAATGCAAAA

AAAAATATGAGGACACATGTCAACAAACCCTCCTTCTATATGTCATAAGAAGGGAAAAAATCAACTTTAT

ATAT[ATAGATAGATAGATA]GATATGTATAATTTGGGATAAAAGTCGGTATAGCCGTACAGGCGTTTGT

GCGATCAATCGGTCATCAACTAAACAAAATTTTAAAATGATTTTTTAAACAAAAAAAAATATTATTTAAT

ATTTATTAAATAATTTGCAATTTTTAATAAAAAATAGTTTTCATATGGGATAAAATTTATCAATCTCATC

TACTATATAAA

CA0739

(SEQ ID NO: 85)

GATCTTATCACCAAATTTTATATTGTCACGTTTAAGTATATCTTTCGTAGAAACATATTTTCCTCGAAAT

GAAACTATATGATATAATATTTTTTTTGTCTAAACATTTTTATACTAAAAACTGATAAGATTATTGTTGG

TAACTACAATTATATTTACCTTGATAAATATATAAAG[ATATATATATATATATATATATATATGTATGT

ATGTATGTATGTATGTAT]ATATATCATCTTTTATTGAATTTGGATAATAGCAGATTAATTAATATTTTT

TAGATAATGATAATATATAATTAAATTTTGATTTACTCAATTATTATTTATGCAAGTTTAACTTTTATTT

TTGGGTGATTTATTATTGTATATGAATATATAAATATATTATGAATAAATATAATTATCTAATTATTAAG

CATTATAAATATAATTATTCATTAAATGTAAAATGACTCTAATTACTCTAGTTTTTAATGCGATAGTTCA

GATCAAAAATATCAATCAGAAACTAATAATATCACAATTTTATATTAGAGTATATTTGTTTAATTAACTA

ACTAATACAATCTGTGAATTTGTATCATTACCAGAAACAACCAATTGAGCAAGTCGGTTAAAAAGTTCAT

GCCATGTTTTAATTTTTGAGCTCATACATTTTTCATTTACTCAGGATTCAC

CA0753

(SEQ ID NO: 86)

GAGTATCAGCTCTACAACATCGTCCGGAAGCAATTGCAACTCTTCGCGATGCCTTTTCAGTGTTCTTGTT

TTGAACGGAAACATCCTTCTCGTCCTCGTCGTCTCTAAATTTTATTGAATTTGATCAACAGATCTGATAT

ATATATATAGATACAGGCAGCTAAGGAATCTGGAAACACAAAAAAAAAAAAAAGAGAAAATTTCCTCTCC

GTTTAAGTAAGATTTCCTTTTTTGAATTTAAACAGAATCGAAACATCAAATC[TAATAATAATAATAA]T

ACAAAT[ATACATACATACATAC]ATATTACCTCAGACTCAGGCAATGAACAAGCCTTTTCTAATCCTCA

GGAATCATCCA[TCTCTCTCTCTCTCTCTCTC]GTTTTGTTTGTGAGCATCGATG[CGGCGGCGG]CGCT

TAGACAAAGACATCTCATCGGGACGCCTCTTTTACAACTCCTCCTCCTGTCTTCTTTTGGGCTTTCTGTA

AGGCCCGACCCGGTTTCCCTTAACGCCGGTACGTCCTTAGTTCGCTTACCTCGACCAAACTGGCCCTATC

CGAATTTATTCTCTTAACTTAGGATTATTATGCAATTTTCCTCTAAGAGGTTCAGTTCAGTACACAAGGT

TCGCTAAGTCTAATCCAGCAACTTAGCAGTCTACTAGTAATCGCAGCATAATGAACATGTACCTACTGCC

TCTGTACTTTGGTATCTGATAATCCATCCATACACTCCTTCA

CA0834

(SEQ ID NO: 87)

GCCATTTTCCTGATTCGAATCCAGATACTGCATAATAAATTTGAGAATAATAATCACTTTTTATTCACTG

CGACATTGTAAGAGTGACTGTTATTCATAAGGCTGTTACTGTTAGGGTCGAAGGCAACTATTATTCTTTT

TTTTGTTAAAGCCTTTATTCTTTCTTTTTCTTTTTGAATCTTTAGTTCGTAAATATTCTCTTTCATATTC

ATAAAAAATACACAACACAACATATGTATTACTATTAGAGGCATAACCATTAACATTGGATTTATTGAGG

TTAGTAATTATTATGGTTGTTTGACAACAAAAAAAAAAGTAATTTTTTTTTGAGCAAACAAAAAGTAATT

ATCTGACAATAGTAGAAACTAAAAAATGCAACCATGCAATACGTGGTTTTATAATCATTCTATTGTTAAA

TATGATGAT[AATAATAATAAT]GAT[AATAATAATAATAATAATAATAATAATAATAATAATAATAAT]

ATTACAGAATGTTGATGTAATAAACAAAAATAGTTTGTTAGCTAACGCCTCAGATCGATCAATGAGTAAT

TCATTCAGTTACCACATAAAGAAACAAATAAAAACTATGATAAAAAAGTTTTGACATCATTTTTTATTGA

CATGTCAATATGTGATAATACACTCTCTGCAGCAGTGACAACAAATACTACAAAC

CA0837

(SEQ ID NO: 88)

TTGGAGTCCTCCGACTTGGTCTTGATACAAGATTGTGAGGAAATCACTGTCCGTGTGTGGCATCAAACCA

TACACCTCCGATGGTTTCGGACATGGTGGATAACGGTTCATCCTTAGATAACATGTGTTTCGCACACAGG

TTTTTTTGAAGAAACTTGATTTCCGTCCTGATTTCTCTGCAAGGACCTCTGCCAATGAATATGCCAGAGC

CTCGGATTCTGAAGCAAAATTTTCCATTGTTGAGCTGCGATATAAAATGTACATATTATAACTAAGGTTA

ATTTATTATAGAGACAAATAAATCATGTTAAATAAATTAGGTGAAATAATTGCGAAAGCCATGAACCTTT

CTTGTTCCTTTTGTTTAATCCAAGCCATGAATTGTTCATTTGTTTTAACGTAGAATTGCTAAGATTTTTT

TTTTTGTAAACCATGAATTGAAGTTATGATAAGAAAAGAAATGGAAAAT[ATTATTATTATTATTATTAT

TATTATT]TAATGGTAGAATGATATAGTATAAATAATTATTTCACGGTAATTATTTGATTTGGTAGAAAA

TTGCGGAAATTATTTGATTTGCTGATTTTTTTTGTGAAGAACAAAATTCACCTAACAAAAGAAACACGTA

AGATTATTTGTTATTTGTGGTACATAT

CA0896

(SEQ ID NO: 89)

TAAATTTACATGTAAATTACCACGAAACATTTTCTTTGTAACTTTACTACGACCTTACTACGAAATTCAG

TTTTGTCGTAAAATCGTAGTAATTTTCTCGTAAATTTACGAGGAATATATTTCCTCGTAATTTTTCCTTG

TTATAGGCATGTTTTCTTGTAGTGTTTATGTTGCCGTTGTTCTGACCACGATAGTTATGATACCTTTGTG

ATTTTCTGGTCACATATTCACTTAATTATTTTGTATGCTGACATACCTCATGGGAGGTTCGCTTGATATA

AATCATCACTTACAAACAAAAAATATTCATAAAAAAAAATATTCACACGTTTACAAAATCAAAAAGAGTT

ATATATAAATAGCTAT[AATAATAATAAT]GATACTAATATTAATAACAGT[AATAATAATAAT]GTTTA

GAAAGCTAAACAACAAGGATTAGAACATGTATTTTTACAATTGCAAAAACAACAACAAAGTCGTAGCTTA

GGACATTTAAAACAAGATGACCATTTGATCTTGCAGTTGCAGCTGCAACATGAGCTCTTCTTATTAAGAC

ATGATGGTCGACTGCAACTGCGGAAACATGTGGGTATGCAACAACATAAGTCCGGACAAGAACAACAGCA

ACCTAGGAAGCATGAACAGCTCGGGCAGCTCAGGCATTTGGGGCAGCTCGGTTTCGGGCAACAACAGCTG

TTTGAGCAGCAGGACCCGTTGCAGCATTTGGAACAACGCAGCTACAACATGTGGAAGTGCAGCATTTTGG

CTTCCTCAGATGGCACGAACACTCGGGTCGG

CA0991

(SEQ ID NO: 90)

GGCCGACGGCGATCTGTATCATCGCCCTGTGGTGGAATTCCTCCTCCGAGGGTATAGAAGGAAGCTAACT

TGGTCTGCTTGTGAAGCTAAAATGAAAGGCTCGAATTTGTTGTACCTTCGTCCACCGTTGACATCAATAA

CACCGAATTTCTTAGACCGAACACCTCTGTTGACGACGGGGCAATGGAATGTATGAAAAACTTAAGGCAG

TTTCAGGTTACGTTTGCAATGGATTATTCTCAATTGGTGAAGATGGTTTTCTGAACCAGATGAATGACCA

GCATTTGAAAGCTACCTGGAAGATATTAAGCTTTTGCGACGAAGTTTCGTCAACTCAGATATTATTCATG

TTCATAGGGCGGAGAACATAAGGGCGGATAGCTTGGCACACGTAGTACTCAGAAACAACCGTCTTTCGTC

GTGCATATGGACGCAGAGTTGCCACATTGGTTTACAGAGTCTACATGAGTCTGTAAATATTTGCTGTTAA

[AATAATAATAATAATAATAAT]ATATATCTGTCTATCAATTTTTAAAACACAATAAGTTTACGGTATAT

TTTTCATTGAATAGATTGTTTTCAACTTTCACATGTATTTGTATCTTCTTCTATATATATATTTTCAGAT

TATTATTTCATTATTANAATCGTAACAATATGTATAAAAATTAGTAAAATATTGTTTTGTTGTCATATTC

AAAGATA

CA1027

(SEQ ID NO: 91)

AGCGGCGATCTGATTCTCGCCCTGTGGTGGATTCTTCTTTCTTTAGTCTTTCCATTATTCTATGACGGTG

TAATTCCCTATATATAAAAGGCTCCTTATATTTATGAATAATATAGAAACATAGATTTCATTACGACTAT

ATTATTAGTATATCAGTCTAGGCGTTTACCAATACCAATATACTTAATATATTTAGTATAATATCTTATG

ATTTACAATTATTTTCATATGATTTTGTACTATAATATGTCAATTATTATAATTTATAAAAAACTT[ATT

CATTATTTATTATTATTATTATT]ATAAACCTACAACCTTTCAACTTAATTAGAATTCACAACCTTTAGA

ATTAATTGAGATTCTTATTATTAATAGATATTATAATCTTTTAAATGGTATATAAGATAATCACCACGGT

ACTAGAAAGCCTAGAGCCAAAGCACCGCCTAAGCCGCCGCCTAGAACAATTACCTAATTTAAAGAAAAAC

TAATACTTATATTTGATTTTGAAATTTTATTAAACTTTGCAAAAAAAGAAGAAGATGGAAACATGTTAGA

AACATATATCCAAATATAAAAATATAAGAATAATTTTATAAAAATTAATGATTAAAAACATATGCAAGAT

TTCGTATGAAAAAACTATTCTGCACAAAAATAATTTATAATATTAGTTTAATATTTACATATTTC

CA1032

(SEQ ID NO: 92)

NGGCGACGGCGATTGATTCTCGCCCTGTGGTGGAATTCCAGTTTGACCAGGACTGTCGTAAGCTTAGTTT

AATTTCACCAGCAGACACCGACTCAATAGCACCCTGGAAAAAACACACTCAAACCAGAAGCAAATGAACT

ATAATAGTCCAAGTAGAAGAAACACAATCAATCATCCAAGAAAAGATACTACTACATCACCAACAATACT

GCTAGATAATGTAAAAAATGGACAGAAGAAATAAAACTACACTGGTCTTCCACCGAAAGAGTCCAAATAG

AAACACAAGGAATAAAGCAAAAGAAAACTAAAATTACCATAGCACTAGCAAGATAATGTAAAAACCTACA

TTGATCTTCTACAGAAGCAGTTTGTTTTATTTTTTCTCCGTTTAGAGAATTTTGGGGTGCTTCTCACCTT

ATTGAACTTGACGACGACATCCCTGAGGCATTTCCAACCGCCAAAACGGAACACAACAGATGCTCCCAGC

ACTCGGCTAAGAATCCATGCAAAGAATCTTGAACAGAGTCTGGAGAGTCAAAATG[AAATAAATAAATAA

ATATAATAATAATAATAATAAT]AAGACCACTATAGCAGCATAGTCCAGCAGCTAAATCATGCAATCTCA

GCTACTGAAGGAAATTAGAGAATGTGCAAACCGAACTANAATCATCACTAGAACTAACTCACACGAAGAT

CATCCACAAGACCATGGAAAGAATCAGGAAC

CA1034

(SEQ ID NO: 93)

AACGGAGACTGATATCTCGCCCTGTGGTGGAATTCTCTGACAATAGTAGAAACTAAAAAATGCAACCATG

CAATACGTGGTTTTATAATCATTCTATTGTTAAATATGATGAT[AATAATAATAATGATAATAATAATAA

TAATAATAATAATAATAATAATAATAATAAT]ATTACAGAATGTTGATGTAATAAACAAAAATAGTTTGT

TAGCTAACGCCTCAGATCGATCAATGAGTAATTCATTCAGTTACAACATAAAGAAACAAATAAAAACTAT

GATAAAAAAAGTTTTGACATCATTTTTTATTGACATGTCAATATGTGATAATACACTCTCTGCAGCAGTG

ACAAACAAATACTACAAACTCTTATTTTTAATCGTTCAAAGATAAGAGTCTATACTAGTAGACTAGAAAG

TGGGGGGAAACAAATAAATTTAGGAGGATTCATTGACAATTTAAGAAGACATTTTTGATACGCCTCGTCT

TATTAGAATTGGGAATGGCCTATGGAGAGGATATGAATGTGATGGGCATAGTGATAAGGTAGAGGAGATA

ATGCAGAAAAGCGAGAAGAAGAATCTTAAACTATCATTTATGAATTATGAGTTAACCTCAGAAAGCCAGT

TTACAAAAAAAAAAAATTATGATATCTCCACTCGTTTCTATTAACTTATTCCTCCATGATTGGTCGTTTT

TGTAAACTTCTGATGATTC

CA1035

(SEQ ID NO: 94)

AGCGGAGATTGATTCTCGCCACTGTGGTGGAATTCAGTAGCTATGTGCTATTAGTGCATTGATTGTTCTT

TTGTGTGGTGTAATAGACACCTGTTTGTTCCATGCTAGAGCTAGGCCTAAATTTTTGTAGTGCTATTAAC

TAAGTCAGTGGTTTGTGGTTTAGCATCCCATACCTCACTGAGTGACTCCCTTATTGCTCACCCCTCCTTC

GTTCTCCCAGGTGAGACCGACAATCATGAGTGATTTTATCGGATTGGTACTTTTGAGCTTTTATCGTTAC

TGAGCTTTTAGACCTTTGGACTTTTATCTTTTATGCTATTTCATATTTCAGACTTTCGGTTTTATATTGC

TATCTATATTTCAGATGTTATCGGACCTTCTGATATTGACTTTTGTATTATGAAGTGGAG[ATTATTATT

ATTATTATTATTATTATTATTATTATT]ACTAGATTCCTTTTCCGCGCTACGCGCGGATAGTATCTTATA

AATTTTAAATTTATTTTTAAAAAAAAAAATATTTAAAGTTTAATTTACATTATTTTATACCAAAAATCAC

AAATATGGCTAAGAATTGGTTGATTTTATTTGTGATTTTTTGACAATATTAAATTGATTTATTTATTGCT

CATAGTTAACAGATTTGTTTGATTGGTTTTCAGTTCATAGTAATGTATAGTATTATATTTGGTGAGTGTA

TATA

CA1066

(SEQ ID NO: 95)

TCTCAGCGCTGAGCTCCATGTGGTGGAATTCATCCTAAATCTCTTTGATGCGATTGATCTCTTTTATAAC

TCATTTTGACTCTTTTGGAATTGGAAGAGATGGACTGGACGGCTATTGCAAAGCCTTTGGAAGAGATGCA

AAGGAGCTGGCTATTGCAATCTTCTACGCTCAAGAATAAGAAGACATATGTCAAGAAGAAGAAGATATAT

GCCTGGACTCTTGCCTTCATCGGCGTACTTGTGGTTATTGCATTTAGTTTGAACATAAAGCTCTTAGGGG

CTCATGCATAACGCTTTCTTAATTAGCTCTGTTTTTTCCAACTGATGTTTACTCTTTCTGAT[ATTATTA

TTATTATTATTATT]AATTGTTAGTTGCTGTTGACCGGTTGAGGCTGTTTTTGAGCCCTGGATATCTTTC

TGAGTTGAGAAAAATTTCATAACCAAATCGAGATTGTTATGTGCTCTTTCTTGCCTCCTTTCAACAAGTT

TAATAGAACCAAAGGCAAATAGTTTGTCTTTATTCTATACTAGGATTGCGAATCCCCGCGGTCCGCGGGG

AAAAAAAGATGTTTTACCGCAAAAAAAATGATGTTAAAACTTAAATGTAATAGTAAAATTTTAGTTTGTA

ACAATCAACCGGTTGAATGGTTAATAATCAAATATTGCAATGTTAACTATTAAAAATTACAGTGGAACAT

TTAAAAGTTGTGAATATTTATATAAAA

CA1080

(SEQ ID NO: 96)

AGCGGCAATTGTATCTCGCCCTGTGGTGGAATTCTGTGATGATCAAAAACAAGTTTCAATCCAAATATCC

TGATTTTGCAAATTATTTGACAAAATCCACATCTCTAATGGTGTTAGAACACTAATAAG[ATTATTATTA

TTATTATTATTATTATTATTATTATTATTATTATT]AACAGTCTTTCCAAAATAAATGATTGATAAAATA

TTGAAAAAGAAGGCAAAGAAGAGGTTGAGAAACAATTCTTATTTCAAAATTTTACAAAAATACAAATTGT

TCGCGTAACATTTTCATTTTCTATTTAGTTTAATTTTTGTCATTTAAAATTATCTTGTTGGTGTTGTTGG

CGTAAGCCCAAAACCGATGTAGCCTACACTGGGCCAATCTCCTGCGCAAGCCCAAGACATAAAGCATTAG

GGTTTTGTTGCTAGCTCATATGTAAACAAAACTTAAGCTATCTTGTTGCCTAAGGTTTTAAGTTTTCTAA

GATACAAAGCTTGTACATACACAAGCTAGATCATAGTTGTGATCACCTCTGTACTCTCTTATTCATAGTG

AAGTTTGGGAGGACAGTCTCCCACGAGACGTACCGGTTAGAGGCCGGGAACTCGTTAAATTGTGTGTGTT

CTTATTGCTTTAGTTTAATCTCTTCTTAAACAACCATAAGCATGATAAGAACTAGTTA

CA1090

(SEQ ID NO: 97)

GGAAACGGCAGTCTGATATCATCGCCACTGTGGTGGAATTCTAACATAACATATGATTGATAGTACATTG

TATATTCTAATTCAAATTAAAAAATATAAATATACAAATATAAATCACCAATATAGTATTTTTACATATT

AATTTTATAATGCTTTATTCTAATATTTTCTTATACCTACTTATTAATTTTTAACTTATTAATTCTAATC

AATGGTCTATTCTGTATTTATTCTCATAAAGAGAAAATAAAGATCTACCAGAAATTGATATTTATGTACA

TTCATTACATGTACAGTAATAAGACCCACATAATCATTTTGTTTTAGCTATGGCTCGTGTTAGGAAAAAT

CTATAAGATGTATCTAATAGTGTGTTGCAATACTAGACTACCAGTTGATCCAGTTGTTTCAAATAATTAG

TTATGTTTCGGAAACTTTGTAGATTGGCTTATTTTCCATGCAGCTTTTTGTTACGACAGAACAAATTACG

CATAAACCTTTAGGCTGAGCAAAGTTGATGACTTTAACCAAGATTGAGTCTTGAATATGTGTCATTACAT

CGAAATATCGAATGTTAAAAATA[TAATAATAATAATAATAATAATAATAATAA]CAATAACGGGTTCAT

ATATCATTACAATTATAACGTAATAGCTGAAAATTCAAAATTGACTAAAATAATATTATGACCCGTCCCT

TTTTATGGTTCCCCCGTTCGTGTATTTGCATTGTTGGCCGTTGGTGATCCCATGNCGCACTTACCCTCCA

AGTCTTCA

CA1097

(SEQ ID NO: 98)

TCTGGAGCTGAAGTCCATGTGGTGGAATTCTGATTTTTGAAAAAATTAAGCGTTATTTTTGTGATTTTTG

ACTTTGAGTGCTAATTTGGAAACAAAAACTTGATTTAGAGATATTTTTGTCTTTTTTTCTTCTCCAAGCG

TGTATCTTTATTTTTATTTTTTATAATTAAACAGGCGGCTTTTTATCCTAATTCAATTCAGGTGGGGTTT

TGTTCTTTTACACATGTCAATTTTGTTCTTTTAATGGTAAAAATTTAAGATAAATTATAAACTGAACCGG

AATCGCAATTGGTAATAAACTGAACAAAATTCCTAATAAGATATATTCCTGAAAAAATCCCCGGAAGATT

TTGATACGTATTAAAATCATATTAAGTTTGAAATATCAAGTTTTATATAATAAGATATACATTATATGCA

ACATCTTTGAATAACTCTCAACCTTTTGGTCATATCACAATAAAGTGGTGGAGCTTTTTCCAGTTACTGA

TGAATGAGTTAAAAAGTACTTAAGTTGCAATAATCTATTCATATTCCATGATCAAAAGCTCTTACAAGAA

ACAAAAGATTACATGAAAATGTCCAAAAGGGTACTTT[ATTATTATTATTATTATTATTATTATT]ATCA

GGATTGAGACCCACGTATACGTAATAAGAAAATATATAACTAATAGCGAATGCTACCTTATGTCATATAC

ACGTAAACACATCCCACTGGTCTTGGCAACACAAGGTGTCATCCTTCTCTTAAACATTCAAC

CA1107

(SEQ ID NO: 99)

AGACGGAAATCTGATATCATCGCCACTGTGGTGGAATACTAAACAAGGCATATAGCATAATATTATTTCA

TGATATAAAAGCAAAAAAAAAATAGAATAATTAAATATACAATAAAAAAAATAAACAATAACTAAATATA

CAATAGCAAAAATGAAAAAAACTAAATGAAACATCTATCTGAAAAATGTATAATAAATAAATAATAAGTA

AATATATAATATGAGAAATAAAAATATTACACTAAATATCTATCGTAATATTAAAATAAAAATGGAGGTG

GAGGCGTTAATATGGGCAATGGAGTGTATGAGGAATTTGCGTCAGTTTCATGTCACGTTTGCAACAGATT

TTCCTCAATTGGTGAAGATGGTTTGAGAACCAGAAAAATGACCAGCATTTGAAAGTTATCTAGAAGACAT

CAAGATTTTGAAAGAAAGTTTCATCAACTCAGAGATCATTCATGTACCTCGGACGGAGAATTTAAGAGCG

GAGAGTCTAGCACGTAGTGTCAAAAAACATTTGTCTTTCATCGTTCACATGGATTTAGAGTTACCAGTTA

GGTTTACAAAGTCGGTATGAGTCTGTAAAAGTCGATTACAA[AATAATAATAATAATAATAATAATAATA

ATAAT]AAGTAAATATATGATACAAAAAATAGAAAAACTACATTGAAAATGTGTAGTAAAATAAATAATA

AATAAATATAAAATATGATGACAAAAAAATGACAAAAAAGTAAATATAAAATATAACAT

PE0012

(SEQ ID NO: 100)

ATACGCCAAGCTTCAATCAAAGGAGGGAAGTGGTGAGAATACAAACCTAGCCTTATTCTCTCTATGTATC

TTCCTCAACTCTGCATCTTCCAACTGTGCCTGAAAAAAGAAAAGCAAAACCCATTAGACGCTAAGCTAAT

GCAATTTCGAGTTTAATGTTTCAGCTTAATCCACATAAAGACGGAAACATACCTGCTCCTGGGTGAATTC

CTCCGGTGCACCGTCGGAGTCTGAATCGGAGTTGTGATCTTTGTTATCCGACATCTGATTATTAT[CTTC

TTCTTCTTCTTCTTCTTCTTCTTCTT]CGTTGCTTTCTTCTCCCTCAGACGACACACACTANGGTTTAAC

GGCTCTTCAGTTTCTCAAAAACAGAAGATTTCTATTCTGAGAGTTAATTGCTTCTCTCCTTTATGGTGGA

TTCTATTGGGAAGCTTGCATGCCTGCAGGTCGACTCTAGANGATCCCCGGGTACCGAGCTCGAATTCACT

GGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCTGGGCGTTACCCAACTTAATCGCCTTGCAGCACAT

CCCCTTTCGCCAGCTGGGGTTANTANCGAAAAGGCCGCACCGATCGCCTTCCAACANTTGCGCANCTGAA

TGGCGAATGGCGCCTGATGCGGTATTTTCTCTTACCTCTGTGCGGTATTTCCNCCGCNTATGGTGCCTCT

CANTACAATCTGCCTGATGNCGCNTANTTAACCANCCCGAAANCCGCCANNNCCGCTGAANCCCCTGAAG

GGGNTGTTCGGCNCCGGNATCCGCTTTAAAAAAANCTGTTAACGTCTCCGGAACCGCTTTTTTCAAGGTT

TTCCCGTCTCNCNAAN

PE0017

(SEQ ID NO: 101)

CGCCNNGCTTCCTTTAAGAGTTAATTTCATACAAATAAGCCCACTAAACAGCGTTGTTTAAACTTTAAAC

CCTGTCTTCTTACCAAAGCTCCTCTTCTTACATAGTAGCTCTGCTAAACCTTCGCCGCAATAAACCCAAA

GTCGTAAGAAACCGTCGCCATGCCTGCTCTTACGCGTAACAAGCAGAAGGGAGCTAAGTCGCAGACTCCT

CCACTGATTAAGCGGACTAAATCGAATCCCACGCCTCCACCGAAGAAGGCGATGAAGTCCCGTAAGCCTC

CGTTGAAGAAACAGAGGAAAGGTGTTTCGGATGAGAAGCCTGAAGTTTCTAATGATGAGGAGGAAGAGGA

AGAAGAAGAAGAAGAAGAAGTGAGTGAAGAGTCTGATGACGGGAGATGAATTGGGTTCTGACCTTTTCTC

AGATGGTGACG[AAGAAGAAGAAGAAGAAGAAGAAGAAG]ATGATATAGAGCCTTCGGATGACGACTTTC

TTGGTGGTAGCGATGAGGAAAAGGGAACTTTGGGTTCTGATTCTGACTCTGATGAGTCAGATAAGCTTGC

ATGCCTGCAGGTCGACTCTAGANGATCCCGGGTACCGAGCTCGAATTCCTGGNCGTCGTTTTACNACGTC

GTGACTGGGAAAACCTGGCGTTACCACTTAATCGCCTTGCAGCACATCCCCTTTCGCCNGCTGGCGTTNT

ACCNAAAAGGCCGCNCCGATCGCCTTCCACAGTTGCGCNCCTGAATGGCGAATGGGGCCTGGATGCGGTT

TTTTCCCCTTCCCCTCTGTTGCGGTTTTCCNCCGCCTNTGGTGCCTCTCNTTCATCTGCNCTGATGCCCC

TTNTTTANCCNNCCCGAACCCGCCANNCCCGTGAACCC

PE0063

(SEQ ID NO: 102)

ATCTCTATCGCTATACTTGTCACTTTGTTCACTTTCTCAGCAGTTCCTTCCACATGGTGATGCAACCATC

TCGCCATTACACTCAGCAAGCCTCCCTTAGCCTTCTTGCATCGTTCTGACCTGTCGTTATATCCATAATT

CTTTGCAATTTTTGTCATTCT[CTTCTTCTTCTTCTTCTTCCTCTTCCTCTTCCTCTTCCTCTTCCTCTT

CTTCTT]TATGAACATGAGCAGCCATTTCCTGTCAACTCTTTGACGGATGTGCACCAATTGACAAGAGCT

TACTCTGTTACACCACTCCAACAAACTCTTCCTTCGTCTCTTAGATATTTTCTCCATTTGTATTCCCAGT

GTCTCAAAGTTATCATTATCCTTTTCGCTCAAAGTATCATCCAAATGCTTTTCAATATCTAGAAAAGTTT

TTTTCCATCTCTTTCTCTTCCTTGTTACTCTTACACCTCCCTCAACCATTTGATCGAACACATGGTGGGC

ATACTTCTCCTTCTTGGTCTCAAGCAACTCCTCTCTTGCATCNTTACCCCTCTCAAAGT

PE0091

(SEQ ID NO: 103)

CGGCAATAATGGACCACTGGTTTGGTTGAACCACTAAAAAGAGTGC[GTGTGTGTGTGTGTGTGTGTGTG

TGT]GAGGGACTCTATTTAAAAGCACTGCTTAACTCAATAATTATTCCATCGCTCCAAAATAAAANAGAA

TAGCTAAAAGATGGCTCTCGAAGTCTGCGTGAAAGCCGCCGTTGGTGCCCCTGATGCTCTCGGCGACTGT

AACTTCCCTTCTCTCTCTCTAGCTCTTTTTTTTTATATCAGATTATGATCTCTGATGATCTTCAAATGTA

AAATTTATAATACATGATTTGTCTGTCGTTTCAGG

PE0131

(SEQ ID NO: 104)

CCGATAAGAGCCATCATCTCGAGGAGGGGTATTAAGGGATATGTATCCCACATTGGAAAATCAATGGGAC

ATTAAGTAATATATAAAGGGTTAGGGCCAATCCACTAATAGCCAATTGGTTTTGAGTTGGAAGCCCATAA

TAAACCCGAATCTAACAAGATTTTAGATTGATTAAGGAAATTAATATATTATATGCAATATTTCATGGTT

AACGTCAAAATAAGTCCAATTTATAAACAAGCGGATAAACATTTCCCTATATATATGGGAAGGTTTGTGG

TTGCCAAACTCAAAGCACATTGGGTCTTATCTCTCTCTAA[CACACACACACACACACACA]AACTCACG

TATATATTTAGAGCTA[GAGAGAGAGA]TGGGTGAAGAGATGAAAGAAGTGAGAGTAATCGAGGAGTGGT

CTCCGGTTATAGTAATGGTGAT

PE0133

(SEQ ID NO: 105)

CCTCCTGTGCCAAGTTTTACAAGGCACCCAACCTGTGAACCTAAATTGCTTTGAAAGAGAAGTTTCTCTA

TCTATAT[CACACACACACACACACACACA]AATCTAATCTTTCTCTTTCAACCGTAAATTTTGCTCACC

ACCAAGGCAAGTTTCCTTCTTTTGCTCCCCTAGCAATATTAATTGCTACTAAAATATCTTGCTAAGGGTA

ACCAAATCTTGCTTCATTCCTCTGTAATATCACAGAAAGAAACTAAAATTTAGGGTTTTTTTTTGGGTTC

TTTCCATGTGATGTGAGCATTTTTGGGTGAGAAAGATGAAGACTATAATTAAGTTAGGGATTGGGTTGAG

TTTGGTGTTTGGGTTTCTTCTCTTAGCACTTATTGCAGAAGTCTATTACCTTCTGAGATGGAAGAAGCAC

AAGAAGAGAGTCATAAGCCAAGAGAGTGAGGAAGAGAAAGAAGAAGAGCAACAACAACAAACTGGGT

PE0177

(SEQ ID NO: 106)

TACCCAAGCTTCCACGATCGGAGGGATCTATCGGGACTTCTCGGAGATTTGTTTCACGGTGATGTCTATT

GATCTTTGGCTCTCTGTTTCGATTGTTGTTGTC[TTCTTCTTCTTCTTTTTCTTCTTCTTC]TCGAGAGG

TTGCGGGGTTTTGAAATCGTCTCCTTCGTCCATGTAGTCGTTTTGGTGTTTGTCCGCCAT[GAGAGAGAG

AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA]GCGAAGACGTTACGAAAAACTTCGATAGAGAGTATA

AGAGAGAGATGCTGAAACTGCTTAAACCCTAATTTTGATCGAGTGTGTTTTGGGAAATTTGCAGAGTAAG

TCCTTATATTTGAGCCGAATTAATTAAAGTAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGG

GTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAAC

TTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTTATAGCGAANAGGCCCGCACCGATCGCCC

TTCCCAACAGTTGCGCNCCTGAATGGCGAATGGCCCTGATGCNGTATTTCTCCTTACNCNTCTGTGCGGT

ATTCCCNCCGCATATGGTGCCTCTCNTTACATCTGCTCTGAAGCCGCNTNTTTANCCAGCCCGACACCCG

CCAACACCCGCTGACCCCCCTGAAGGGCTTGTCTGCCCCCGGGNTCCCTTNCAAACAACTGTTNACCGNC

CCCGGGAACCGCNTNTTTCAAANGTTTCCCCCGCCTCCCCGAAACCCCCAAAAAAAGGGGCNCTNANACC

CCNNTTTTNNGGGTTANGTCNGAAAANAANGGTTCCTAAANTTCGGGGGGCCTTN

PE0187

(SEQ ID NO: 107)

ATCTAGGACCCCATGATCCGAATAAGGATAATAAAAAAATGGATCTGCCGAATAAGTATCTGGATAACTT

AAAATTCCCTAGATACCCCCCCCCCGC[CACACACACACACACACACACACA]TCAATTTTCCTTGTAAT

TTTTCTCTTGTTCTCTATTTTTCTAAACTCCAATAAAGCAAGTCTTTAACATATACTCCACCTTTATTTG

AGTAAATAATCATGGATTTAATCTCTAAAGTGAAGGACACTTTGTTTATGTTTTCTGTTTTTCTAAATTG

TAAAATCTATTTTCTACCTTTTTAATGTGCTAATTTTAGGAAAAATTATATCAATATTTTGTGTCGTAAT

TAAATCTGTCAACATGAAGTAAATCTGTGTCAAAAAGAAAAAAAAATCTATAGAAACATAATTAAAGTAA

ATGTATGAACATATAAAATAAATCTATGAATGATGTATAAATCTATCAAAATTAAATAAATATGTGG

PE0203

(SEQ ID NO: 108)

CTTTAATGCTTAAAGCTCGTTCCAGCTCAGGGGTTGGGAGTTTAAGCCTAAAACTCGAGATCTGCTCATC

CGCAGTTGCTGATTTGGCAGCTTATTCACCTTCGCATACCTACAAATCAACGAATACAACGCAAACGTTC

CTCCTG[CACACACACACACACACACACA]TATACTTAGAACCACATAACACCACTTTTTACAAAAAAAA

ACAAGGATTAGGGATGTAATATTATTACCTTCGCCATTGTCGTTGGCTTTAAGGACAACAAAGACATACT

TTGCTAAAGGAATGACAGCAATGGTGTAGATAACGAGAGACAAGGCACCAAGAACATCAACTTCTGATCT

GATAGGAACTTTACTGAAGACATCACTAAACACATACAAAGGGCTTGTTCCCATGTCTCCATACACAACA

CCTAACGTCTGAAACGCTATCCCAATCGT

PE0250

(SEQ ID NO: 109)

ACTCAATAACATCAGGCTTTTCTGGATGCAATCTTGCATTGGTTTGGTCCAGTTTGCATCGGGACAAATA

TTGTTGCTGGTGTATATGGGATCGGAATCTTTGCTCCGACATTCTCTTCGGTGTCTT[CATCATCATCAT

CATCATCATCATCATCATCATCATCAT]CAGTATCAGTATCATCTCTTATTCCAAACTTCTTATATTGTA

GATGTTCTTGACCGGAGTGGCAAATGAAGACATCTCATATATGGCATTGCTTTTAGGGTTTTTAAGAAGA

TGTGGTGTTGCTATGTTGGTCTTTGATGCAACCACGGTGGTCGGAATGGGCGAGAATGGTCTGCAAACTA

ATGGAGATTGTAGGTGTTCACATGGGTTCATCGACTATTACTTTGGGAGAAGTCCAACAACATCAAGCCC

TCTTCTTCCTGCAGGAAAATAAAATATTTCGTTCATCAATTAAAGTAAGAGAATTAACTCATCACTATGG

TGATATGTTAGTTTCTGTTTATTGTAAGACTTAAAATTACCAG

PE0281

(SEQ ID NO: 110)

ACAAAACCGCTTCCACCTGCGTTTCGTCGCAGNCAAGCATTTGCGTTTCATTGGGTTTTCTCGGTGAACC

AAGCTCGCGTTAGTATCCAAACATGTTTTCACAGAATCTCGTAGTAACGACATTTCTTGTTGAAGTTGTT

GGATCTGTGTTCTCATTTCGCTTATCAGCTCCATTTCCTGAAACGTTTTTAATTAAGCGGTTCAATGATT

CTCTTCTATGGGGATAACAATAAAATCTAAAAACCTTACAGGTGACGGAGGGTTGTGAACAGACAAGACA

GGAGTTGAAGTTACTTCAGTGTCTTGACAACTCCATGATCCTGCAGGAGACGATGCAAAGATCGGCGAAG

AAGACGAGTCATCTCCATCGTCTTGCTCTTCACCTTCTTCAGTAAATGGCT[CTTCTTCTTCTTCTTCTT

CTTCTTCTTCTTCTTCTTCTTCTT]CAGTTCCTCCACATTTTCATTCTATGGTCTTCCTCTTCTTCTTCA

TGTTGCAATTCCCATTTTTCAGAATGTTTGTTCGAATGTGTCTGCACACGAGACATCATGAGCCTATCGA

TCTGATCTCTTAAACCGGTCTGGGAGAANGTCTGTGACCGGTCCTCTGTAATATCCAAAAACCACACATT

TTTCTTAGTNAATGGGTACCCAATTAGGAAAATGGGATTTAAAAGATTGGATCAG

PE0283

(SEQ ID NO: 111)

AATGAACGAGACAGGAGGTGCCACCCTATTGTCTTTGATGGACCAGAGTCTAGCTATCCGGTAAACAGTA

TTTTATAACAAAGACATGATCATGAAATGATTTTTTTCTTCTGAAGTTTAACTGATGACTCATATATCTA

TATCTGACTAGTTCATCGTGGACATGGAGCATTCACAGGACCAGAGCACACATCGAAGCTGACATACAAA

TGAATCTCTTATTGCCTCTAGTTTCACACGTAAATATTCAGTTTCTAG[GATGATGATGATGAATTGATG

ATGATGATGACGATGATGAT]CAGTCAAAAGTACTGTAAATTGATGGTTATGGTTGTCTTGGCTTTGCTT

AATCATGT

PE0286

(SEQ ID NO: 112)

ACAAGTTACCATTTGAGGATATATCAGAGAAGAAGGAGTTGCTTGAAGATGACGAGAAAACCAAAAAGAA

GATGAGTTCTAATGGTCGTTGGTACGAGGAGCTTGATGTCTTCATAGAGAAACCTGAAACTGGTGTTCTT

ACTGGTGATGGTGCTGTGGTGGACGCATGACTGGGAACGAACCTGTTGATGGTGACGAGTTGGATGTTGA

GCAACAAGATGATAATTCTGATGGTGATCATGGTGATCATGAAGCAG[GAGAGAGTGAAGA]TGAGTATC

AAGCGAGTGATGAATCTGATAAAGAAGAGGATATTGACAGAAATTTTGAAGAGGATGTTGAGATGTTCCA

GGGATGAGAACTACGATGGAGGAGATTCCAGACGAGGAGGAGGTATATTCTGACACGGAGGAGTCATCTG

ATGATGAAGAGGAACAAGCTGAGAAGGATGCTAATAGGGGTGAATTAGATGGCATTTTTAAGTCTTAGGC

AGGAANTTGCAATGCCTGCAAGTCGACCTCTAGAGGATNCCCGGGTACCGAGCTCGAATTTCCACTGGGC

CGTCCGTTTTACAACGTCCGNGACTGGGAAAAACCCTGGGGTTAACCCAACTTAATCGCCTTTCAGCACA

TNCCCCNTTCGCCANGNTNGGGGTAATAGCCNANAAGGCCCGCAACCGATNGGNCCTTTCCCAANAGTNG

CCGCACCTNAAATGGNGNATTGGCGCCTTANGNGGGAANTTTNNCCTTANGNATT

PE0324

(SEQ ID NO: 113)

ACATAAGCCCTTTTTATTATCTCTGCATATCATTACATTCATTTTATGTCACATATGTTTATTGCTCTTC

TCTTCAGATTACTATTACATCGCAAGTAAAACAAAAGAGTTAGAAAATAAAGTAAACACTCCATACATAG

TCAAAGTATCTCCATTACTCCTCTTCTTCGTGTTAACAAGTCTTTAGGCGTTTCTAAACCGCAGAAACCA

TCATAGCCGGTGATGCACCAACCATCAAGT[CTTCTTCTTCTTCATCATCATCATCATCATCAT]CCTCT

GCTTCCCACATGAAATGAGCGTATGATCCCAAAACCATACTACAAAAGTCACAAACCTTTAACATTCTGA

AAAAAAAACTCATCAAAGAATCCAAACTCTACATATAACATAACATACCAATCATCAGGAGAAGCGTTAA

CAGCTTGATCAAAGTAACACTGAGCTCTCTTCTCATCTCTCTTCGTCTCCCAAATCAGCTTCCCATACAT

CGACAACGCTTCACCATCACCTGGATCCGCAAGTATAGCTCTCCCGTAATACTCCTCCG

PE0340

(SEQ ID NO: 114)

CTTAGTGACCCAAAAGCCATTGGTGTATGATAGAAAAGTTAGTTAAATACCGTTACTCGCAAGGAAGACC

ACACATTTTTTAATTCTATCTCACTTAGTCAGACCAGCTCGGATCCTTCTCTAGAAC[CACACACACACA

CACACA]CTCAGAGTGAGAGATTCATCAATGGCGGTTTCTTGCAGCCACTCATCGATTCTCTTGCCCCCA

ACCACCTCCTCCGTTGGCTTCAACCGCTTCCCTTGTCTCCAAACGCTGCGTTTCAAATCCAGAAACGTTT

ATCAGAAAGCGAGGATCTCTACAGTGTCGGCGTCATCTTCACGGTCTCTCGAAGCTCTGATCTTCGACTG

CNACGGTGTGATACTCGAATCGGAGAATCTACACCGTC

PE0355

(SEQ ID NO: 115)

TCAAAGAGCACTTACAAGGATCCAGACGATGGAAGGCAACGATTCTTACTCGAACTTGAGTTCATTCAGT

GTCTCGCGAATCCTACTTACATACACTGTAAGCTCTTATGATTCCTTATCACATAGTATCTACTTATAGC

ATTTAGGAAGTGATAAGAGATCTT[GTGTGTGTGTGTGTGTGTGTGT]TTTATGCTCTATGATGAACTTA

CCACTTAGCTTTTNGATTCTGTTTTGGCAGACCTAGCACAGAATCGTTATTTTGAAGATGAAGCATTTAT

TGAATACTTGAAGTATCTTCAGTATTGGCAGCGACCAGAGT

UB0015

(SEQ ID NO: 116)

ACTTTAGTGGTCGAAAATCAAAATATTCTTCCATCATTTTAGTTTTTTTTTTTCTCTATGTTCGCATCAA

GAAAACGAAATGAAAGGGATTATAAAAGGAAGAAGAACTTGTGAATCACGGTAAGTTTCGGGGTTTGTTG

TGAGGAGATTTCGAGAGAATCAAGAATAAAATTATATCACGAGATTTTTTTGTTTGAAGTGAGAAAGAAA

TCAAAGATTTTATTTTTTCTCTTTTGGTGAGTGATA[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG

AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA]CGTGTTTTGGAACTACGG

TGATTTTTACTATTTTGATGATGTTTTCAACTTTGAAGAAGACCTTCTCTCATGCTCACTCTTAGCATCC

TCCTCATTTATAGGATTAGATGGGAGAGAGAGAGCGTTTTAGCCATTAATACTTTAATAACAAAATGAAA

AATCTGATATTAACATTTCTTTTTTCACTTCTCCATCAGTGGCATTTTCGATATTTT

UB0126

(SEQ ID NO: 117)

ACCTCCTGATGACTGCTTAAACAGCGCCTGCGAAGATCTGGATTCTGTAGTTAACCAGGCTAGGGAGTTC

TTAGAGGACTGGTCCCCAAAGTTGAGCAAGCTCTTTGGTGTAAGTTGATGAACAAGCTCTCATTTTCAGT

TTTCTTT[CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT

CTCTCT]TTGCATCATTCATTGAGTTGTGTGTGTGCAGGTGTTTCACTCCGAGCTTTTGTTGGAGAAGGT

CCAGACTTGTTCACTGGAGATTAATCGCATACTTCTTCAGTTATCACAGTCAAGTCCTGTAACTTCAAGT

GT

UB0163

(SEQ ID NO: 118)

ACAGAAACAGTAACATCAACACACACAACAAACAGCTCGCGAAATGAATTACAGATTCCTCTCCGAAATC

AAAACAGGAAACGGACACA[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGATGAGA]AGGTGA

TACCGTCGAGAGGTTTGATGTTGCCGTCGCGGCGATCGACGGAGAAGCCTTCATGAGGCGAATCGACGGG

TTTGACGACGT

UB0181

(SEQ ID NO: 119)

ACAGACGCGATGATGACACTTCTGGTCCTAAGCGTTGTGTTACGTTTGATACTCCTGCGCTTGTTTACTT

GGAGTATTCTGATGTGGTTGCTGATAAGTATGAGAATCTGAGTTTGGACAGCTTGGTTGAAGTTAGGCTT

GATCTTCAGTTGACTGCAGATCAAATCATGCGCAAGAATGCTACAGACAGTGTTGGTTTTGTTCCCGGTG

ATGTTTCAACTTTGTTCATGGGGGTCAAGAACGTCAAGATCCTCTGCTTATCTCCTGATTCTTTAGATGT

GAGTCCAGTCCTTTTTAAGTTAGCTTCATCACTGTGTAGCATTTGTTTTTTTTTTAAATTTGATTGGTTA

GTGATGATACAAAATATTTGATTCTG[GTGTGTGTGTGTGTGT]TTGTGAAAGTTCAGTCCCTTTAACTT

AGCTTCATGAGTGTGTAGCCTTTGTTTTTTAATTGGTTACTGATGATATGGTGTGTGTGTGTGTGTGTGT

GTTTCAGACGCTCTACTACCGTGGTGGTGACATGCCGGTGTTCAACAATCTGATTT

UB0196

(SEQ ID NO: 120)

ACATGAGAACAAGATGGGTTCGAATTACCTCTAGCCTAGATCTGGATCTGGAAACAAGAGACAAGGGAGA

GACGAGATCTTGTCACCACCACCAATCGGCTGCCACCACCACCACCACAACACGGCGCCAGCGAAAGGGA

GATA[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG

AGAGAGA]AGAAGAAGAAGAGAGAAAAGGAAAAGAGAAGCTTGATGGCTAGGGTTTCTTAGTCTCTCTAA

ATCTCTGCAGGGCTTTGCTCAAGTTTCAGAATGAGAGAAAAAAGAGAGGAGGCAACTTTATTTATAGGAA

ATGGAGGGAACCCTAGGTCATTTACCTTAATGGGCTGCAGTCCTAACGAGCTCTCGTTAAAAAAATTTGG

GCCGGGTATCGGGATGTTACACTAACGGTGTGTGGCGATGAAGGCTCTTCGACTCTCAAAATTAATGATG

TCCATAACTAAATAAAAACTACTCGACTTTATTAAGATATAGCTTCAATGATTTAAAATTAAATATAGAA

CTCT

UB0307

(SEQ ID NO: 121)

ACCTNTGGGTAAGTAACTGTGGTGGC[CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT]CAATACAC

TCTTCACTTAATAAATGTGAANACGTTAACTNGTTTCTTTTNTCACTTCTCAGTTATGTAGCTCCAGAGT

ATGCGAACTCTGATCTTCTGAATGAGAAAAGTGATGTCTATAGCTTTGGTGTTGT

UB0315

(SEQ ID NO:122)

CACGAAAGCAGGCCCCACCCAATAAGCGATGAGCTGTATATTTATTTTGTCTTGTTTTCACAAAAAATAA

CCCTTCATGTTTACAGTTAATTACACAACAGCCCCTTTCTTTCCTCCATGACCAACGACAAGGTCGAATT

T[CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT]CCGTCGTCTTCATTCAATCTATCTCAG

TGATTTACTCGCAATAGAAGTCGCCTCTTAATCTCTCGAGAGAGAAGCTCAAGT

UB0331

(SEQ ID NO: 123)

CGTGGACTAACGCTCGTGTGCAGGAAACGATGTTCGTGAAAAGGTATCCGATCAGAGGAGCCTCCGCCGG

TAAAAACCCTTCG[CCGCCGCCGCCTCCG]TTGAATGGTAATAACTCTTGTTCTCGCTTTCTCTTCAAAC

TCCCTTTTTTTTCTCTGATTATTTTTGTTGGTTA

KK66

(SEQ ID NO: 124)

GAATGGGAAGCATCACAATGATAATGCTAATGGCGGTTTTGGTCTGGTCCATTACTCTAGAGACCTGCAT

TGCTAGAAGAGGAAGACATTGGAGACATAACCACCGAAGCTCCTC[A/T]GACTTGTCTGATTCCTTGTC

AAGCAAGAAACCAAAAAGCCACAGTCACCACCACAGCTCTCA[C/T]AACAACAACCATAATCATCACCA

CAAGTCTAAACCTAAACCAAA[A/G]CCAAAGCTGAAAACGCCGCCAAAAAGTGACCACA[A/C]TAAAT

CTCCGGTGGTTTCACCGCCACCAAAAGTCCAACCACCGTCTCTTCCGCCGCCAAAGGGATCCAAAGTTTT

CAATGTGATGGATTTTGGCGCAAAGGGTGATGGCAAATGTGATGACACTAAGTCGTTTGAAGCGGCTTGG

GCAGCAGCTTGCAAAGTGGAGGCATCCATGATGATCATACCGCCTGAATACACTTTCCTTGTGGGTCCAA

TCTCATTCTCTGGTCCTTATTGTCAAGCTAACATTGTGTTTCAGCTTGATGGTACTATTATAGCTCCAAC

GGATTCAAAATCATGGGGAAAAGGGTTAATGTGGTGGCTTGAATTCACAAAGCTGAAAGGAATTAAAGTA

CAAGGTAAAGGTGTGATTGATGGAAGAGGCTCTGGT

KK98G

(SEQ ID NO: 125)

GACAGAGATAGCCCTAACTTAGTCACTCTCTCTCACACACACTCCAGTTCAAAGTTCAAA[A/C]AATGG

CTCCTCCACAGAAGCTCTTTCTCGCCGCCATTGTCGCTGCCGTCATTGTAGCCGCCACCACCGGATATGC

ACCTAATAGTGCTGCGGAAGATATTGTGCATTCCTCATGCGTGCACGCGAGCTATCCATCGCTATGCGTC

CGTACACTCTCTACCTACTC[C/T]GGTCCAACCATCACAAACCGTCGCGAGCTAGCTCAAGCCGCCGTC

AAGATAAGCCTCTCCCACGCTCGAGCAGC[C/T]GCTAAGAAACTCGCGGCTGTGAGAGAAACCGTGGG

[A/G]AAGAAACGGGTGAAAGCGGCGGTTGTGGACTGCGTGGAGATGATTGGAGACTCGGTGGACGAGCTG

[A/C]GCCGCACGCTAGGCGTTTTAAAGCATCT[A/C]CACGTTTCGGGCGTTTCGGCGAACGAGTTCA

[A/G]GTGGCAGATGAGCAACGCGCAGACGTGGGCTAGTGCGGCGTTGACGGATGACGACACGTGTCTCG

ATGGGTTTAAAGGGGTCGAGGGTAAGGTTAAAACGGAGGTGAAGCA[G/T]TGGATGACGAAAGTGGCGA

GGGTTAC[A/G]AGCAACGCGCTTTACATGATCAACCAGCTAGATGAATCACGTGGCTAGCCCCACGTAG

TACGTTCTTGATGTTATGATGTGCTTGTCCTAATGGACAGTTATGATTTGGTGTTAGTTTTTTTCGTGTT

TGCTTAATTGCGAGTTATCTACTATTTAAAAATGAGAGGCATTGTCCTTTTAAGTAGTTCTGATAATGGT

ATACTAAATAAATGGTTTATCTCTTTTTTCGGACGGTATGTCATTGTATCGTATTGTGTTGTTCCCTTCG

GATTCGATAGCATGTGATTTTGTCTTGACGTGTAGTAGCGCCTTGGCTGAGCTAATGCTCTAAATAAAAG

TTTTAAGTGGC

Table 14 below sets forth additional information about the markers of QTLs associated with whole field plant resistance to Sclerotinia, as well as exemplary sets of forward and reverse primer sequences for each polymorphic region.

TABLE 14

List of SSR and SNP markers and primer sequences

used for amplification of loci associated with

Sclerotinia whole plant field resistance

Seq

Seq

ID

ID

Marker
Repeat
Forward Primer Sequence
NO
Reverse Primer Sequence
NO

AG0023
(CTT)
ACAAGGAGATGATCGCGGTTTC
126
CCAATCTGTGTAAACCAAACGGG
127

AG0045
(GAA)-(GAA)
ATAAGGCTTGAGGGACATGCCA
128
TGGCTCCACAGAAACAGCTTTG
129

AG0047
(CAT)
GAAGCTGCAATACTGAGGCACC
130
GCAATTCTTACCTGTTGTCCCAAA
131

AG0070
(CCA)-(GA)-(GA)
AACTGGTCGAGCGGGATTTTTT
132
TAGGAAACCCTAGCCGTCAAGC
133

AG0086
(GA)
CAATGTCGGTAAGCACCGGAAG
134
TGCCGGAAAATGCTGACTTGTA
135

AG0093
(CAG)-(CTTTT)
GCTTCAGCCAAGGGATTTGAGA
136
AGCTCTTTTGGTGCGATTCGAT
137

AG0125
(CAT)
CCACATGCCTTAGGTGATTGGA
138
TTCTTCCGGCTTCTCAAAGGTG
139

AG0148
(CCA/T)
CATCCTTGTCCAACGTCCCTTC
140
TTCCTCTCTTCGAGATCGGTCG
141

AG0171
(CT)
GGACTCGAACATCTCCAATTTAACT
142
TGAAAATAGAATACAATTAGGGCTT
143

AG0203
(CTT)-(CTT)
GCGTTGCCCCTCTCCTCTACTT
144
CGCAATCTACAAAAGATACATCAAAAAG
145

AG0239
(CTT)-(CTT)
AAGAAAAGAGAAACGATCCCACG
146
TGAGAGTGAAGAGGAGTTGGGTC
147

AG0243
(GAA)-(GAA)-(GAA)
GATTGGGGGATGAGATTGTTGG
148
GCCGTCCAAAAGTCAAAGGTCA
149

AG0272
(CTT)
AGATGATCGCGGTTTCCTCAAG
150
GAGGCAAAGCTATAAGAACAACTCCA
151

AG0304
(CAT)
CATGTTTGGTTGCTACGGTGGA
152
GAGGTTGAGACGGAGAAGCACC
153

AG0323
(CAT)
GACCAATACAAAAACCGGGCAA
154
TTGATGGAGAGTGGGTTGTGCT
155

AG0324
(CAT)
GTCTCACCAACTCCAAACTTGTTAA
156
CAGGTTCCTTACCAAAGATAAAGAG
157

AG0328
(CTT)
AGCTCGTCTCCTTGCTGTCTCA
158
GGAAGTGAAGAAGAAGCCGGTG
159

AG0359
(GGT)
TGCTCAAAACCCTAGTCGTCACC
160
TGAGAGCGAAAACCAAGAGAGGA
161

AG0369
(GGT/C)
GAGTGGGCTGTACCTTGTAGTTGA
162
TTTGTAAACATCAGAATCACCACC
163

AG0370
(GTTTT)
TCCATTATTACAACCACCCGCC
164
GTTCCGTTGCCCTCTCCTTTTT
165

AG0378
(CCG)
GTTGCTGGTGGAGTTGCTGCT
166
TTTGTAGATGCGACTGCTTCATCTT
167

AG0391
(GGA)
CGACGCTCAAGAGGAAATGCTT
168
CAGTGTCCACCGGAGTAGCAGA
169

AG0410
(GGA)-(GGA)
TGATCCATATCGGGGAAAATCG
170
TTTTTGCTTGGTTTCCGACAGA
171

AG0441
(GA)-(GGA)
CCGGAAACCTCCGATTGAGTAA
172
GGATTAGTTTAACATAGATGGGCCG
173

AG0477
(AAT)-(CT)
GGGAGATAATGTTGGGAATCTTAATCG
174
ACTGAGCCATCCTTCCTCCTCC
175

AG0482
(GGT)-(CGG)
TCCGTAGAAAGAACAGGCTCGG
176
GAACCGCCGCCAAAACTAAAAT
177

AG0504
(CAT)-(GGA)
ATCAAATCCACCCACTGCACCT
178
GCCTTCTCCTCACATCTACGCA
179

AG0510
(GGA)-(GAA)
TCATCTGATTCATCGTCATCATCA
180
TGACTCTTGTCAACACCACCACG
181

BG0031
(AGG)-(AG)
GAGGAAGCATAGGAGGAGGAG
182
ACATAACCCAAATCCCCAAAT
183

BG0106
(AGGG)-(GAT)
CGAAATTATGTGTGTGCGCTCC
184
CCCGGTTAGGAAATTACGGATCA
185

BG0111
(GAT)-(ACC)
TCATTTTGACTTTTGGCGTTTGG
186
TGCATAAGTACGTTGAAAAGGGCTC
187

BG0119
(CGG)-(GAT)-(GTT)-(ATTA)
ATGAGGAGAAAGGATTCGCGGT
188
CGGTAAACGTCCAAACCTCACC
189

BG0181
(ACC)-(ACC)-(ACC)
GCAGAGCAACGAAGTACGCCTT
190
TCGTGATGGTGTCTCCAATGGT
191

BG0228
(AGC)-(CAA/CAG)
GAGACGAAGCCATTGGTAGGGA
192
CGGCTTGTTGTTGTTGCTGTTT
193

BG0255
(GAA)-(GAA)-(GAT)
CCAAACTCAGCACAGCCTTTCA
194
ACGTTTGCCACATTCACAGCAT
195

BG0278
(CTT)-(ATT)
TGGCTGTTCAGTTGTTTAGCTGGA
196
CCCATTTGAAGGCAGAGAGTGC
197

BG0295
(AGGG)-(TCC)
CACATCTCTCCGATTTCATCGC
198
AAGAGGATTTTGTCGGTGGGTG
199

BG0452
(TGG)-(GAT)
TCCCTTTGGTGATGTTGGACTG
200
CACATAATCTGCATTGTCGTCTTCG
201

BG0516
(GAT)-(TAA)-(TTGGT)
TGGTCAACAGAAAATGGCCTGA
202
TGGCATGTCCTTTCATGCTCTC
203

BG0647
(CTT)
ATGCAAAGATGGGCGAAGAAGA
204
CGAGAGCGGGTTACGAGATCAG
205

BG0651
(CTT)
TGCATAACAAAAGATTTGAACCCG
206
GGAGCAAAAGAGCGGAACAGAA
207

BG0713
(CT)
ACCGCCAAAGAAGACGAAAATG
208
CTCGGCGGACAGATACACTCAG
209

BG0864
(GA)
GAAGCTAAATGCGTTGCGTTGC
210
TTTGGCTGTAAAATGAAGTGAGCA
211

BG0869
(CT)-(CT)
GAGTCGGCCACAAATCAAGGAA
212
TCGGAGGAGGAGGAGATTGAGA
213

BG0988
(CT)
CCCAAGACTCCAACCGGAAAAT
214
TCGCATTAAACGAGGACGTGAG
215

BG1062
(CT)
CCAACTCGTTCCATCCCAGTCT
216
CGGTGGCTCGTACTGCTTCTCT
217

BG1090
(GTA)-(GA)
CCGTTGAGGTAGGTTTCTGCCTT
218
TTGCCTCGCTCACATCTTTTTG
219

BG1101
(CAT)
ACATGCTTGTGGATAAATCATCAT
220
GAAAATGAGAAAACCCAAACTAAA
221

BG1123
(GA)
AGGCCACCTTTTGTCACCAGTC
222
GGGGGGTTAATTTGTCCCATTT
223

BG1127
(GA)
CCGCCATAACAAAAATCTTCCC
224
TGTTTCCTACAAAGGTATAACCGGC
225

BG1149
(GAT)
TTGCTACACTTCCCTGTGGGTG
226
CCTCTCTCACTGAGTGCATTTC
227

BG1182
(AG)
CGGCGCACTGATGATGTTTCTA
228
GGGAGAGAGTTTGTTCGGTGCT
229

BG1197
(CATTT)
TTGTGAGCGCCAAGATAAGGCT
230
AAACCCTATCCCGCGAACAACT
231

BG1230
(GA/TGG)
GGCCTCTTGTGTTTCCTCCAAC
232
CACCACTACCGCCATCTCTCCT
233

BG1241
(CAG/AAC/CAG)
TTAACAAACCGAATCCGCAAGC
234
GATTGTTGTTGCTGCTGCTGCT
235

BG1244
(CTT)-(CTT)-(CTT)-(AGT)
CAACGAACTCTTCTTCTCTGCTTTACA
236
TCACGACAAATGGTCAAATTCTCA
237

BG1286
(AGC)
GTCCGAGACAGAGTATGCTAAGC
238
TCATTGGTGGATCACTTCAAATA
239

BG1288
(AGC)
GATTCTATCAGCCACGGAACGC
240
CACCTATTTACCCCACGAGGCA
241

BG1321
(AAG)
TGCATAGACTCGAACCAAACCG
242
TCTGATACGCCAAGCTCTGCTG
243

BG1368
(CGT)
CGACGGTTTCACGGCACT
244
TCATCCTCCGACGACGAC
245

BG1392
(GA)
GCTCGGCTTCCAAGGGTAAGAT
246
GATGTACTGCTTAGCTGCCGCC
247

BG1442
(GA)
CGAGGAGGAAGAAGATGACCGA
248
ACGAGCAGGCGGTGAAAATAAA
249

BG1449
(GA)
AGTTGTGGTGAACAGGCTGCAT
250
CCAACTCGTTCCATCCCAGTCT
251

BG1453
(GA)-(GA)-(GA)
TCACCTTCCTTCCTTCAATGGC
252
TGACCCACCTCCTCTGCTTTTC
253

BG1513
(GA)
TGAAGGTTGCGATAGCGAAGAG
254
TTGGGGTCGGAACTGAAACATT
255

CA0105
(GAA)
TGTTTCCTTCACCATGATCGGA
256
CACACCCTACCTCTCTTGTGTCCC
257

CA0120
(CCA)-(TCA)
GACTAAACCAGACCAAGAGAAAAGTCG
258
TGTTGAACCGGATAGGCAAGGT
259

CA0163
(CAT)-(CAT)
CCATCATCACCACCACCATCAT
260
CTTGCTGGAGAAGGTCGGATGT
261

CA0221
(CTT)
GGAGAAGGGTCGTCGTCAAGAA
262
TCGAACACACAAACGATGCTCA
263

CA0226
(GA)-(ACC)
CATGGATCACCTGCACCCTTAG
264
GCGACTCCGGTGAAGACGTATC
265

CA0233
(GAA)-(CAC)
GGCAAGCATGGTCTCGTCAGAT
266
AAAAAAAGATTCCAGCCGCCTC
267

CA0328
(CGT)-(CAT)-(CCA)
ACTTTGGAGCATCCTTCCTTGG
268
GATGTTGGACTGCGCTCTGGTA
269

CA0410
(CAG)
CTGTTGAGCCATCGAGATCAGC
270
CCAGAAAGTGATGGTGTGCATAA
271

CA0423
(AGC)
AATCAGTCCGATCACTCCCTGC
272
GGAAGTTACCTCGTCGTCGGAA
273

CA0456
(TTG)-(TCAA)-(CCA)-(CTC)
TTCTGTTAGAATTCTACCGTTGTTG
274
AGCTTTGTGAGGAGAGTGTGGT
275

CA0488
(CTT)-(CTT)-(CAA)-(CAA)
CAGCAATGTCGTCGTTCAATCC
276
CTGTAACTCGCCGGAGCTTGAT
277

CA0546
(GAA)
CCCCTTCCTTATCCACACACACA
278
TGGGTTCGTGAAGGTGAAGGTT
279

CA0552
(CTT)-(CTT)
CGCTAGGGGGTGAACCAAGAAT
280
GCGTCGATCCTCCTCTCCAATA
281

CA0603
(CTT)
CCTCCCAAAGTCGTCTCTTCCA
282
GTAGACAGGGGACGAAACTCGG
283

CA0624
(TA)
TTCCAAGTGGTTCTGCAATGTG
284
TCAGCTATCCCAATAAAGGGCAA
285

CA0636
(CCT)-(CCA)
CCAGGTCCTTGCGTTGAATCAT
286
ACCACACTGCATATCCCTGCAA
287

CA0682
(AAGG)
CCTCTTCAACCCCACACTGGAT
288
TTGCGAATGTATCAGCCGTCTC
289

CA0729
(GGTT)
CCGAACCGGAATCATACAGCTC
290
TGCGTTTGAACCAGTCAGATCC
291

CA0736
(ATAG)
CCGGGCTTAGACAAACTTTATGAG
292
ATTGATCGCACAAACGCCTGTA
293

CA0739
(AT)-(GTAT)
AAAAACTGATAAGATTATTGTTGGTAAC
294
CCAAAAATAAAAGTTAAACTTGCATA
295

CA0753
(TAA)-(ATAC)-(TC)-(CGG)
CAGGCAGCTAAGGAATCTGGAAA
296
TAAAAGAGGCGTCCCGATGAGA
297

CA0834
(AAT)-(AAT)
AAAATGCAACCATGCAATACGTG
298
GTTTGTAGTATTTGTTGTCACTGCTGC
299

CA0837
(ATT)
TGCGAAAGCCATGAACCTTTCT
300
TGTTCTTCACAAAAAAAATCAGCAA
301

CA0896
(AAT) -(AAT)
CTCATGGGAGGTTCGCTTGATA
302
GCAACTGCAAGATCAAATGGTCA
303

CA0991
(AAT)
CACATTGGTTTACAGAGTCTACATGA
304
ACAAATACATGTGAAAGTTGAAAACA
305

CA1027
(ATT)
TCAGTCTAGGCGTTTACCAATACCA
306
CTTAGGCGGTGCTTTGGCTCTA
307

CA1032
(AAT)-(AAT)
TTGAACTTGACGACGACATCCC
308
GATTGCATGATTTAGCTGCTGGA
309

CA1034
(AAT)-(AAT)
AAAATGCAACCATGCAATACGTG
310
TTTGTAGTATTTGTTTGTCACTGCTGC
311

CA1035
(ATT)
CTCCCAGGTGAGACCGACAATC
312
CGTAGCGCGGAAAAGGAATCTA
313

CA1066
(ATT)
CTCTTGCCTTCATCGGCGTACT
314
ATCCAGGGCTCAAAAACAGCCT
315

CA1080
(ATT)
TGACAAAATCCACATCTCTAATGGTG
316
AGGCTACATCGGTTTTGGGCTT
317

CA1090
(TAA)
CCATGCAGCTTTTTGTTACGACA
318
GGCCAACAATGCAAATACACGA
319

CA1097
(ATT)
AAAGTGGTGGAGCTTTTTCCAGTT
320
GCCAAGACCAGTGGGATGTGTT
321

CA1107
(AAT)
TAGTGTCAAAAAACATTTGTCTTTCA
322
TTGTCATTTTTTTGTCATCATATTTT
323

PE0012
(CTT)
CACCGTCGGAGTCTGAAT
324
GAGCCGTTAAACCNTAGTGTG
325

PE0017
(AAG)
ATTGGGTTCTGACCTTTTCTC
326
CTTTTCCTCATCGCTACCAC
327

PE0063
(CTT)
TCGTTCTGACCTGTCGTTAT
328
GGAAATGGCTGCTCATGTT
329

PE0091
(GT)
CGGCAATAATGGACCACTGG
330
CGGCTTTCACGCAGACTTCG
331

PE0131
(CA)-(GA)
TATGGGAAGGTTTGTGGTTGC
332
CACTCCTCGATTACTCTCACT
333

PE0133
(CA)
TCCTGTGCCAAGTTTTACAAG
334
GGTTACCCTTAGCAAGATATT
335

PE0177
(TTC)-(GA)
TCTATTGATCTTTGGCTCTCT
336
CGTAACGTCTTCGCTCTC
337

PE0187
(CA)
GACCCCATGATCCGAATA
338
AAGACTTGCTTTATTGGAGTT
339

PE0203
(CA)
TACAACGCAAACGTTCCT
340
TTGATGTTCTTGGTGCCT
341

PE0250
(CAT)
TGGTGTATATGGGATCGG
342
GTTTGCAGACCATTCTCG
343

PE0281
(CTT)
GAGACGATGCAAAGATCG
344
TGCAGACACATTCGAACA
345

PE0283
(GAT)
CATTCACAGGACCAGAGC
346
CAAAGCCAAGACAACCAT
347

PE0286
(GA)-(GA)
ATGGTGACGAGTTGGATG
348
CCTCGTCTGGAATCTCCT
349

PE0324
(CTT)-(CAT)
CAGAAACCATCATAGCCG
350
TGATTTGGGAGACGAAGA
351

PE0340
(CA)
CGTTACTCGCAAGGAAGA
352
TTCGAGAGACCGTGAAGA
353

PE0355
(GT)
ATGGAAGGCAACGATTCT
354
TTCTGTGCTAGGTCTGCC
355

UB0015
(GA)
TCGGGGTTTGTTGTGAGG
356
GAGGAGGATGCTAAGAGTGAGC
357

UB0126
(CT)
ATGACTGCTTAAACAGCGCC
358
CTTCTCCAACAAAAGCTCGG
359

UB0163
(GA)
ACACACAACAAACAGCTCGC
360
AACATCAAACCTCTCGACGG
361

UB0181
(GT)
AAGAACGTCAAGATCCTCTGC
362
ACCACCACGGTAGTAGAGCG
363

UB0196
(GA)
CATGAGAACAAGATGGGTTCG
364
CTGAAACTTGAGCAAAGCCC
365

UB0307
(CT)
TGGGTAAGTAACTGTGGTGGC
366
AGAGTTCGCATACTCTGGAGC
367

UB0315
(CT)
TCCATGACCAACGACAAGGTC
368
AAGAGGCGACTTCTATTGCG
369

UB0331
(CCG)
GTGTGCAGGAAACGATGTTC
370
GGGAGTTTGAAGAGAAAGCG
371

KK66
—
CCACAGTCACCACCACAGCTCTCAT
372
GGCGGTGAAACCACCGGAGATTTAG
373

KK98G
—
GCCTCTCCCACGCTCGAGCAGCC
374
CCCTCGCCACTTTCGTCATCCAA
375

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.

Number	Date	Country
61426170	Dec 2010	US
61449776	Mar 2011	US
61566064	Dec 2011	US

	Number	Date	Country
Parent	13992780	Jun 2013	US
Child	15416050		US

QTLs ASSOCIATED WITH AND METHODS FOR IDENTIFYING WHOLE PLANT FIELD RESISTANCE TO SCLEROTINIA

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

Provisional Applications (3)

Divisions (1)