Patent Grant
10612102
The present invention relates to a marker associated with resistance to smut whereby a sugarcane line resistant to smut can be selected, and a method for use thereof.
Sugarcane has been cultivated as a raw material for sugar, liquor, and the like for edible use. In addition, sugarcane has been used as, for example, a raw material for biofuel in a variety of industrial fields. Under such circumstances, there is a need to develop novel sugarcane varieties having desirable characteristics (e.g., sugar content, enhanced vegetative capacity, sprouting capacity, disease resistance, insect resistance, cold resistance, an increase in leaf blade length, an increase in leaf area, and an increase in stalk length).
In general, the following three ways may be used for identification of a plant variety/line: “characteristics comparison” for comparison of characteristics data, “comparison during cultivation” for comparison of plants cultivated under the same conditions, and “DNA assay” for DNA analysis. There are many problems in line identification with characteristics comparison or comparison during cultivation, including reduction of precision due to differences in cultivation conditions, lengthy duration of field research that requires a number of steps, and the like. In particular, since sugarcane plants are much larger than other graminaceous crops such as rice and maize, it has been difficult to conduct line identification based on field research.
In addition, in order to identify a variety resistant to a certain disease, an inoculation test is carried out using a causative microorganism of a disease after long-term cultivation of sugarcane, and then disease resistance data are collected by observing lesions and the like. However, transmission of the causative microorganism to an external environment must be securely prevented when the test is carried out, and thus it is necessary to provide, for example, facilities such as a large-scale special-purpose greenhouse, a special-purpose field or isolation facility from an external environment. Further, for creation of a novel sugarcane variety, first, tens of thousands of hybrids are created via crossing, followed by seedling selection and stepwise selection of desirable excellent lines. Eventually, 2 or 3 types of novel varieties having desired characteristics can be obtained. As described above, for creation of a novel sugarcane variety, it is necessary to cultivate and evaluate an enormous number of lines, and it is also necessary to prepare the above large-scale greenhouse or field and undertake highly time-consuming efforts.
Therefore, it has been required to develop a method for identifying a sugarcane line having disease resistance with the use of markers present in the sugarcane genome. In particular, upon creation of a novel sugarcane variety, if excellent markers could be used to examine a variety of characteristics, the above problems particular to sugarcane would be resolved, and the markers would be able to serve as very effective tools. However, since sugarcane plants have a large number of chromosomes (approximately 100 to 130) due to higher polyploidy, the development of marker technology has been slow. In the case of sugarcane, although the USDA reported genotyping with the use of SSR markers (Non-Patent Literature 1), the precision of genotyping is low because of the small numbers of markers and polymorphisms in each marker. In addition, the above genotyping is available only for American/Australian varieties, and therefore it cannot be used for identification of the major varieties cultivated in Japan, Taiwan, India, and other countries or lines that serve as useful genetic resources.
In addition, Non-Patent Literature 2 suggests the possibility that a sugarcane genetic map can be created by increasing the number of markers, comparing individual markers in terms of a characteristic relationship, and verifying the results. However, in Non-Patent Literature 2, an insufficient number of markers are disclosed and markers linked to desired characteristics have not been found.
Meanwhile, as a marker associated with disease resistance, a marker associated with black root rot resistance in sugar beet disclosed in Patent Literature 1 is known. In addition, a technique of selecting a Zea mays variety using a maker linked to a desired trait is disclosed in Patent Literature 2.
The level of infectiousness of the causative microorganism of sugarcane smut is high, and therefore the onset of smut quickly results in the infection of the entire field. Crops of sugarcane affected with smut cannot be used as raw material for sugar production, and even they die. Therefore, the development of smut will cause a significant decline in yield within the following year or later. Damage due to smut has been reported in more than 28 countries, including Brazil, the U.S., Australia, China, and Indonesia. Smut can be prevented by sterilization treatment prior to planting; however, preventive effects are limited to the period of early growth. Thus, cultivation of a sugarcane variety imparted with smut resistance has been awaited.
In view of the above, an object of the present invention is to provide a marker associated with resistance to smut, which is a quantitative trait of sugarcane.
In order to achieve the object, the present inventors conducted intensive studies. The present inventors prepared many sugarcane plant markers and carried out linkage analysis of quantitative traits along with such markers for hybrid progeny lines. Accordingly, the present inventors found markers linked to quantitative traits such as smut resistance. This has led to the completion of the present invention.
The present invention encompasses the following.
(1) A marker associated with resistance to sugarcane smut, which consists of a continuous nucleic acid region existing in a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 14, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 15 and the nucleotide sequence shown in SEQ ID NO: 22, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 23 and the nucleotide sequence shown in SEQ ID NO: 32, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 33 and the nucleotide sequence shown in SEQ ID NO: 51, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 52 and the nucleotide sequence shown in SEQ ID NO: 62, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 63 and the nucleotide sequence shown in SEQ ID NO: 72, or a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 73 and the nucleotide sequence shown in SEQ ID NO: 85 of a sugarcane chromosome.
(2) The marker associated with resistance to sugarcane smut according to (1), wherein the continuous nucleic acid region comprises any nucleotide sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NOS: 1 to 85 or a part of the nucleotide sequence.
(3) The marker associated with resistance to sugarcane smut according to (1), wherein the continuous nucleic acid region is located at a position in a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 5 and the nucleotide sequence shown in SEQ ID NO: 9, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 18 and the nucleotide sequence shown in SEQ ID NO: 22, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 25 and the nucleotide sequence shown in SEQ ID NO: 32, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 33 and the nucleotide sequence shown in SEQ ID NO: 42, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 57 and the nucleotide sequence shown in SEQ ID NO: 59, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 64 and the nucleotide sequence shown in SEQ ID NO: 66, or a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 72 and the nucleotide sequence shown in SEQ ID NO: 80 of a sugarcane chromosome.
(4) A method for producing a sugarcane line having improved smut resistance comprising: a step of extracting a chromosome of a progeny plant obtained from parent plants, at least one of which is a sugarcane plant, and/or a chromosome of a parent sugarcane plant; and a step of determining the presence or absence of the marker associated with resistance to sugarcane smut according to any one of (1) to (3) in the obtained chromosome.
(5) The method for producing a sugarcane line according to (4), wherein a DNA chip comprising a probe corresponding to the marker associated with resistance to sugarcane smut is used in the determination step.
(6) The method for producing a sugarcane line according to (4), wherein the progeny plant is in the form of seeds or a young seedling and the chromosome is extracted from the seeds or the young seedling.
This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application Nos. 2011-101050 and 2012-94995, which are priority documents of the present application.
According to the present invention, a novel marker associated with resistance to sugarcane smut linked to a sugarcane quantitative trait such as smut resistance can be provided. With the use of the marker associated with resistance to sugarcane smut of the present invention, smut resistance of a line obtained by crossing sugarcane lines can be tested. Thus, a sugarcane line characterized by improved smut resistance can be identified at a very low cost.
The marker associated with resistance to sugarcane smut and the method for using the same according to the present invention are described below. In particular, a method for producing a sugarcane line using a marker associated with resistance to sugarcane smut is described.
<Markers Associated with Resistance to Sugarcane Smut>
The marker associated with resistance to sugarcane smut of the present invention corresponds to a specific region present on a sugarcane chromosome and is linked to a causative gene (or a group of causative genes) for a trait characterized by smut resistance. Thus, it can be used to identify a trait characterized by smut resistance. Specifically, it is possible to determine that a progeny line obtained using a known sugarcane line is a line having a trait characterized by the improvement of smut resistance by confirming the presence or absence of the marker associated with resistance to sugarcane smut in such progeny line. In the present invention, the term “smut” refers to a disease characterized by lesion formation due to infection with a microorganism of the genus Ustilago. One example of a microorganism of the genus Ustilago is Ustilago scitaminea.
In addition, the term “marker associated with resistance to sugarcane smut” refers to both a marker linked to a trait characterized by the improvement of smut resistance and a marker linked to a trait characterized by the reduction of smut resistance. For example, if the presence of the former marker in a certain sugarcane variety is confirmed, it is possible to determine that the variety has improved smut resistance. Further, if the presence of the former marker and the absence of the latter marker in a certain sugarcane variety are confirmed, it is possible to determine that the variety has improved smut resistance with high accuracy. It is also possible to determine that a certain sugarcane variety has improved smut resistance by confirming only the absence of the latter marker.
The term “sugarcane” used herein refers to a plant belonging to the genus Saccharum of the family Poaceae. In addition, the term “sugarcane” includes so-called noble cane (scientific name: Saccharum officinarum) and wild cane (scientific name: Saccharum spontaneum), Saccharum barberi, Saccharum sinense, and the earlier species of Saccharum officinarum (Saccharum robustum). The term “known sugarcane variety/line” is not particularly limited. It includes any variety/line available in Japan and any variety/line available outside Japan. Examples of sugarcane varieties cultivated in Japan include, but are not limited to, Ni1, NiN2, NiF3, NiF4, NiF5, Nib, NiN7, NiF8, Ni9, NiTn10, Ni11, Ni12, Ni14, Ni15, Ni16, Ni17, NiTn19, NiTn20, Ni22, and Ni23. Examples of main sugarcane varieties used in Japan described herein include, but are not limited to, NiF8, Ni9, NiTn10, and Ni15. In addition, examples of main sugarcane varieties that have been introduced into Japan include, but are not limited to, F177, Nco310, and F172.
In addition, a progeny line may be a line obtained by crossing a mother plant and a father plant of the same species, each of which is a sugarcane variety/line, or it may be a hybrid line obtained from parent plants when one thereof is a sugarcane variety/line and the other is a closely related variety/line (Erianthus arundinaceus). In addition, a progeny line may be obtained by so-called backcrossing.
The marker associated with resistance to sugarcane smut of the present invention has been newly identified by QTL (Quantitative Trait Loci) analysis using a genetic linkage map containing 3004 markers and 4569 markers originally obtained from sugarcane chromosomes, and sugarcane smut resistance data. In addition, many genes are presumably associated with sugarcane smut resistance, which is a quantitative trait characterized by a continuous distribution of sugarcane smut resistance. That is, sugarcane smut resistance is evaluated based on the incidence of smut characterized by such continuous distribution. For QTL analysis, the QTL Cartographer gene analysis software (Wang S., C. J. Basten, and Z.-B. Zeng (2010); Windows QTL Cartographer 2.5. Department of Statistics, North Carolina State University, Raleigh, N.C.) is used, and the analysis is carried out by the composite interval mapping (CIM) method.
Specifically, seven relevant regions included in the above genetic linkage map with LOD scores equivalent to or exceeding a given threshold (e.g., 2.5) have been found by QTL analysis described above. That is, the following 7 regions have been specified: an approximately 18.7-cM (centimorgan) region including the relevant region, an approximately 39.2-cM region including the relevant region, an approximately 19.2-cM region including the relevant region, an approximately 32.0-cM region including the relevant region, an approximately 39.5-cM region including the relevant region, an approximately 53.4-cM region including the relevant region, and an approximately 38.0-cM region including the relevant region. The term “morgan (M)” used herein refers to a unit representing the relative distance between genes on a chromosome, and it is expressed by the percentage of the crossover rate. In a case of a sugarcane chromosome, 1 cM corresponds to approximately 2000 kb. In addition, it is suggested that a causative gene (or a group of causative genes) for a trait that causes the improvement of smut resistance could be present at the peak positions or in the vicinity thereof.
The 18.7-cM region is a region that comprises 14 types of markers listed in table 1 below in the order shown in table 1 and is linked to a trait characterized by the reduction of smut resistance.
The 39.2-cM region is a region that comprises 8 types of markers listed in table 2 below in the order shown in table 2 and is linked to a trait characterized by the improvement of smut resistance.
The 19.2-cM region is a region that comprises 10 types of markers listed in table 3 below in the order shown in table 3 and is linked to a trait characterized by the reduction of smut resistance.
The 32.0-cM region is a region that comprises 19 types of markers listed in table 4 below in the order shown in table 4 and is linked to a trait characterized by the improvement of smut resistance.
The 39.5-cM region is a region that comprises 11 types of markers listed in table 5 below in the order shown in table 5 and is linked to a trait characterized by the reduction of smut resistance.
The 53.4-cM region is a region that comprises 10 types of markers listed in table 6 below in the order shown in table 6 and is linked to a trait characterized by the reduction of smut resistance.
The 38.0-cM region is a region that comprises 13 types of markers listed in table 7 below in the order shown in table 7 and is linked to a trait characterized by the improvement of smut resistance.
In addition, in tables 1 to 7, “Linkage group” represents the number given to each group among a plurality of linkage groups specified by QTL analysis. In tables 1 to 7, “Marker name” represents the name given to each marker originally obtained in the present invention. In tables 1 to 7, “Signal threshold” represents a threshold used for determination of the presence or absence of a marker.
In addition, the peak contained in the 18.7-cM region is present in a region sandwiched between a marker (N804607) consisting of the nucleotide sequence shown in SEQ ID NO: 5 and a marker (N815502) consisting of the nucleotide sequence shown in SEQ ID NO: 9. The peak contained in the 39.2-cM region is present in a region sandwiched between a marker (N803928) consisting of the nucleotide sequence shown in SEQ ID NO: 18 and a marker (N826906) consisting of the nucleotide sequence shown in SEQ ID NO: 22. The peak contained in the 19.2-cM region is present in a region sandwiched between a marker (N829378) consisting of the nucleotide sequence shown in SEQ ID NO: 25 and a marker (N821999) consisting of the nucleotide sequence shown in SEQ ID NO: 32. The peak contained in the 32.0-cM region is present in a region sandwiched between a marker (N915070) consisting of the nucleotide sequence shown in SEQ ID NO: 33 and a marker (N918161) consisting of the nucleotide sequence shown in SEQ ID NO: 42. The peak contained in the 39.5-cM region is present in a region sandwiched between a marker (N911103) consisting of the nucleotide sequence shown in SEQ ID NO: 57 and a marker (N918344) consisting of the nucleotide sequence shown in SEQ ID NO: 59. The peak contained in the 53.4-cM region is present in a region sandwiched between a marker (N918761) consisting of the nucleotide sequence shown in SEQ ID NO: 64 and a marker (N900663) consisting of the nucleotide sequence shown in SEQ ID NO: 66. The peak contained in the 38.0-cM region is present in a region sandwiched between a marker (N901524) consisting of the nucleotide sequence shown in SEQ ID NO: 73 and a marker (N915180) consisting of the nucleotide sequence shown in SEQ ID NO: 80.
A continuous nucleic acid region existing in any of 7 regions containing markers shown in tables 1 to 7 can be used as a marker associated with resistance to sugarcane smut. The term “nucleic acid region” used herein refers to a region having a nucleotide sequence having 95% or less, preferably 90% or less, more preferably 80% or less, and most preferably 70% or less identity to a different region present on a sugarcane chromosome. If the identity of a nucleic acid region serving as a marker associated with resistance to sugarcane smut to a different region falls within the above range, the nucleic acid region can be specifically detected according to a standard method. The identity value described herein can be calculated using default parameters and BLAST® or a similar algorithm.
In addition, the base length of a nucleic acid region serving as a marker associated with resistance to sugarcane smut can be at least 8 bases, preferably 15 bases or more, more preferably 20 bases or more, and most preferably 30 bases. If the base length of a nucleic acid region serving as a marker associated with resistance to sugarcane smut falls within the above range, the nucleic acid region can be specifically detected according to a standard method.
In particular, among the 14 types of markers contained in the 18.7-cM region, a marker associated with resistance to sugarcane smut is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 5 and the nucleotide sequence shown in SEQ ID NO: 9. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 5 and the nucleotide sequence shown in SEQ ID NO: 9. In addition, among the 8 types of markers contained in the 39.2-cM region, a marker associated with resistance to sugarcane smut is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 18 and the nucleotide sequence shown in SEQ ID NO: 22. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 18 and the nucleotide sequence shown in SEQ ID NO: 22. Further, among the 10 types of markers contained in the 19.2-cM region, a marker associated with resistance to sugarcane smut is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 25 and the nucleotide sequence shown in SEQ ID NO: 32. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 25 and the nucleotide sequence shown in SEQ ID NO: 30. Furthermore, among the 19 types of markers contained in the 32.0-cM region, a marker associated with resistance to sugarcane smut is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 33 and the nucleotide sequence shown in SEQ ID NO: 42. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 33 and the nucleotide sequence shown in SEQ ID NO: 42. Moreover, among the 11 types of markers contained in the 39.5-cM region, a marker associated with resistance to sugarcane smut is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 57 and the nucleotide sequence shown in SEQ ID NO: 59. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 57 and the nucleotide sequence shown in SEQ ID NO: 59. Among the 10 types of markers contained in the 53.4-cM region, a marker associated with resistance to sugarcane smut is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 64 and the nucleotide sequence shown in SEQ ID NO: 66. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 64 and the nucleotide sequence shown in SEQ ID NO: 66. Among the 13 types of markers contained in the 38.0-cM region, a marker associated with resistance to sugarcane smut is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 73 and the nucleotide sequence shown in SEQ ID NO: 80. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 73 and the nucleotide sequence shown in SEQ ID NO: 80.
In addition, a nucleic acid region containing a single marker selected from among the 85 types of markers shown in tables 1 to 7 can be used as a marker associated with resistance to sugarcane smut. For example, it is preferable to use, as a marker associated with resistance to sugarcane smut, a nucleic acid region containing a marker (N802870) consisting of the nucleotide sequence shown in SEQ ID NO: 6 located closest to the peak position in the 18.7-cM region, a nucleic acid region containing a marker (N826906) consisting of the nucleotide sequence shown in SEQ ID NO: 22 located closest to the peak position in the 39.2-cM region, a nucleic acid region containing a marker (N821999) consisting of the nucleotide sequence shown in SEQ ID NO: 32 located closest to the peak position in the 19.2-cM region, a nucleic acid region containing a marker (N916186) consisting of the nucleotide sequence shown in SEQ ID NO: 35 located closest to the peak position in the 32.0-cM region, a nucleic acid region containing a marker (N918508) consisting of the nucleotide sequence shown in SEQ ID NO: 58 located closest to the peak position in the 39.5-cM region, a nucleic acid region containing a marker (N913735) consisting of the nucleotide sequence shown in SEQ ID NO: 65 located closest to the peak position in the 53.4-cM region, or a nucleic acid region containing a marker (N901163) consisting of the nucleotide sequence shown in SEQ ID NO: 74 located closest to the peak position in the 38.0-cM region. In such case, the nucleotide sequence of a nucleic acid region containing the marker can be specified by flanking sequence analysis such as inverse PCR analysis using primers designed based on the nucleotide sequence of such marker.
Further, as a marker associated with resistance to sugarcane smut, any of the above 85 types of markers can be directly used. Specifically, one or more type(s) of markers selected from among the 85 types of such markers can be directly used as a marker associated with resistance to sugarcane smut. For example, it is preferable to use, as a marker associated with resistance to sugarcane smut, a marker (N802870) consisting of the nucleotide sequence shown in SEQ ID NO: 6 located closest to the peak position in the 18.7-cM region, a marker (N826906) consisting of the nucleotide sequence shown in SEQ ID NO: 22 located closest to the peak position in the 39.2-cM region, a marker (N821999) consisting of the nucleotide sequence shown in SEQ ID NO: 32 located closest to the peak position in the 19.2-cM region, a marker (N916186) consisting of the nucleotide sequence shown in SEQ ID NO: 35 located closest to the peak position in the 32.0-cM region, a marker (N918508) consisting of the nucleotide sequence shown in SEQ ID NO: 58 located closest to the peak position in the 39.5-cM region, a marker (N913735) consisting of the nucleotide sequence shown in SEQ ID NO: 65 located closest to the peak position in the 53.4-cM region, or a marker (N901163) consisting of the nucleotide sequence shown in SEQ ID NO: 74 located closest to the peak position in the 38.0-cM region.
<Sugarcane Marker Identification>
As described above, markers associated with resistance to sugarcane smut were identified from among 3004 markers and 4569 markers originally obtained from sugarcane chromosomes in the present invention. The 3004 markers and the 4569 markers are described below. Upon identification of these markers, a DNA microarray can be used according to the method disclosed in JP Patent Application No. 2009-283430.
Specifically, the 3004 markers and the 4569 markers originally obtained from sugarcane chromosomes are used with a DNA microarray having probes designed by the method disclosed in JP Patent Application No. 2009-283430. The method for designing probes as shown in
Next, adapters are bound to a genomic DNA fragment subjected to restriction enzyme treatment (step 1c). The adapter used herein is not particularly limited as long as it can be bound to both ends of a genomic DNA fragment obtained by the above restriction enzyme treatment. For example, it is possible to use, as an adapter, an adapter having a single strand complementary to a protruding end (sticky end) formed at each end of genomic DNA by restriction enzyme treatment and a primer binding sequence to which a primer used upon amplification treatment as described in detail below can hybridize. In addition, it is also possible to use, as an adapter, an adapter having a single strand complementary to the above protruding end (sticky end) and a restriction enzyme recognition site that is incorporated into a vector upon cloning.
In addition, when genomic DNA is digested using a plurality of restriction enzymes, a plurality of adapters corresponding to the relevant restriction enzymes can be prepared and used. Specifically, it is possible to use a plurality of adapters having single strands complementary to different protruding ends formed upon digestion of genomic DNA with a plurality of restriction enzymes. Here, a plurality of adapters corresponding to a plurality of restriction enzymes each may have a common primer binding sequence such that a common primer can hybridize to each such adapter. Alternatively, they may have different primer binding sequences such that different primers can separately hybridize thereto.
Further, when genomic DNA is digested using a plurality of restriction enzymes, it is possible to prepare and use, as an adaptor, adapter(s) corresponding to one or a part of restriction enzyme(s) selected from among a plurality of the used restriction enzymes.
Next, a genomic DNA fragment to both ends of which adapters have been added is amplified (step 1d). When an adapter having a primer binding sequence is used, the genomic DNA fragment can be amplified using a primer that can hybridize to the primer binding sequence. Alternatively, a genomic DNA fragment to which an adapter has been added is cloned into a vector using the adapter sequence. The genomic DNA fragment can be amplified using primers that can hybridize to specific regions of the vector. In addition, as an example, PCR can be used for a genomic DNA fragment amplification reaction using primers.
When genomic DNA is digested using a plurality of restriction enzymes and a plurality of adapters corresponding to the relevant restriction enzymes are ligated to genomic DNA fragments, the adapters are ligated to all genomic DNA fragments obtained by treatment with a plurality of restriction enzymes. In this case, all the obtained genomic DNA fragments can be amplified by carrying out a nucleic acid amplification reaction using primer binding sequences contained in adapters.
Alternatively, when genomic DNA is digested using a plurality of restriction enzymes, followed by ligation of adapter(s) corresponding to one or a part of restriction enzyme(s) selected from among a plurality of the used restriction enzymes to genomic DNA fragments, among the obtained genomic DNA fragments, a genomic DNA fragment to both ends of which the selected restriction enzyme recognition sequences have been ligated can be exclusively amplified.
Next, the nucleotide sequence of the amplified genomic DNA fragment is determined (step 1e). Then, one or more region, which has a base length shorter than the base length of the genomic DNA fragment and corresponds to at least a partial region of the genomic DNA fragment, is specified. Sugarcane probes are designed using at least one of the thus specified regions (step 1f). A method for determining the nucleotide sequence of a genomic DNA fragment is not particularly limited. A conventionally known method using a DNA sequencer applied to the Sanger method or the like can be used. For example, a region to be designed herein has a 20- to 100-base length, preferably a 30- to 90-base length, and more preferably a 50- to 75-base length as described above.
A DNA microarray can be produced by designing many probes using genomic DNA extracted from sugarcane as described above and synthesizing an oligonucleotide having a desired nucleotide sequence on a support based on the nucleotide sequence of the designed probe. With the use of a DNA microarray prepared as described above, the 3004 markers and the 4569 markers, including the above 85 types of markers associated with resistance to sugarcane smut shown in SEQ ID NOS: 1 to 85, can be identified.
More specifically, the present inventors obtained signal data of known sugarcane varieties (NiF8 and Ni9) and a progeny line (line 191) obtained by crossing the varieties with the use of the DNA microarray described above. Then, genotype data were obtained based on the obtained signal data. Based on the obtained genotype data, chromosomal marker position information was obtained by calculation using the gene distance function (Kosambi) and the AntMap genetic map creation software (Iwata H, Ninomiya S (2006) AntMap: constructing genetic linkage maps using an ant colony optimization algorithm, Breed Sci 56: 371-378). Further, a genetic map datasheet was created based on the obtained marker position information using Mapmaker/EXP ver. 3.0 (A Whitehead Institute for Biomedical Research Technical Report, Third Edition, January, 1993). As a result, the 3004 markers and the 4569 markers, including the aforementioned 85 types of markers associated with resistance to sugarcane smut shown in SEQ ID NOS: 1 to 85, were identified.
<Use of Markers Associated with Resistance to Sugarcane Smut>
The use of markers associated with resistance to sugarcane smut makes it possible to determine whether a sugarcane progeny line or the like, which has a phenotype exhibiting unknown smut resistance, is a line having a phenotype showing the improvement of smut resistance. The expression “the use of markers associated with resistance to sugarcane smut” used herein indicates the use of a DNA microarray having probes corresponding to markers associated with resistance to sugarcane smut in one embodiment. The expression “probes corresponding to markers associated with resistance to sugarcane smut” indicates oligonucleotides that can specifically hybridize under stringent conditions to markers associated with resistance to sugarcane smut defined as above. For instance, such oligonucleotides can be designed as partial or whole regions with base lengths of at least 10 continuous bases, continuous bases, 20 continuous bases, 25 continuous bases, 30 continuous bases, 35 continuous bases, 40 continuous bases, 45 continuous bases, or 50 or more continuous bases of the nucleotide sequences or complementary strands thereof of markers associated with resistance to sugarcane smut defined as above. In addition, a DNA microarray having such probes may be any type of microarray, such as a microarray having a planar substrate comprising glass, silicone, or the like as a carrier, a bead array comprising microbeads as carriers, or a three-dimensional microarray having an inner wall comprising hollow fibers to which probes are fixed.
The use of a DNA microarray prepared as described above makes it possible to determine whether a sugarcane line such as a progeny line or the like, which has a phenotype exhibiting unknown smut resistance, is a line having a phenotype showing the improvement of smut resistance. In addition, in the case of a method other than the above method involving the use of a DNA microarray, it is also possible to determine whether a sugarcane line, which has a phenotype exhibiting unknown smut resistance, is a line having a trait characterized by the improvement of smut resistance by detecting the above markers associated with resistance to sugarcane smut by a conventionally known method.
The method involving the use of a DNA microarray is described in more detail. As shown in
Next, a plurality of genomic DNA fragments are prepared by digesting the extracted genomic DNA with restriction enzymes used for preparing the DNA microarray. Then, the obtained genomic DNA fragments are ligated to adapters used for preparation of the DNA microarray. Subsequently, the genomic DNA fragments, to both ends of which adapters have been added, are amplified using primers employed for preparation of the DNA microarray. Accordingly, sugarcane-sample-derived genomic DNA fragments corresponding to the genomic DNA fragments amplified in step 1d upon preparation of the DNA microarray can be amplified.
In this step, among the genomic DNA fragments to which adapters have been added, specific genomic DNA fragments may be selectively amplified. For instance, in a case in which a plurality of adapters corresponding to a plurality of restriction enzymes are used, genomic DNA fragments to which specific adapters have been added can be selectively amplified. In addition, when genomic DNA is digested with a plurality of restriction enzymes, genomic DNA fragments to which adapters have been added can be selectively amplified by adding adapters only to genomic DNA fragments that have protruding ends corresponding to specific restriction enzymes among the obtained genomic DNA fragments. Thus, specific DNA fragment concentration can be increased by selectively amplifying the specific genomic DNA fragments.
Thereafter, amplified genomic DNA fragments are labeled. Any conventionally known substance may be used as a labeling substance. Examples of a labeling substance that can be used include fluorescent molecules, dye molecules, and radioactive molecules. In addition, this step can be omitted using a labeled nucleotide in the step of amplifying genomic DNA fragments. This is because when genomic DNA fragments are amplified using a labeled nucleotide in the amplification step, amplified DNA fragments can be labeled.
Next, labeled genomic DNA fragments are allowed to come into contact with the DNA microarray under certain conditions such that probes fixed to the DNA microarray hybridize to the labeled genomic DNA fragments. At such time, preferably, highly stringent conditions are provided for hybridization. Under highly stringent conditions, it becomes possible to determine with high accuracy whether or not markers associated with resistance to sugarcane smut are present in a sugarcane sample. In addition, stringent conditions can be adjusted based on reaction temperature and salt concentration. That is, an increase in temperature or a decrease in salt concentration results in more stringent conditions. For example, when a probe having a length of 50 to 75 bases is used, the following more stringent conditions can be provided as hybridization conditions: 40 degrees C. to 44 degrees C.; 0.21 SDS; and 6×SSC.
In addition, hybridization between labeled genomic DNA fragments and probes can be confirmed by detecting a labeling substance. Specifically, after the above hybridization reaction of labeled genomic DNA fragments and probes, unreacted genomic DNA fragments and the like are washed, and the labeling substance bound to each genomic DNA fragment specifically hybridizing to a probe is observed. For instance, in a case in which the labeling substance is a fluorescent material, the fluorescence wavelength is detected. In a case in which the labeling substance is a dye molecule, the dye wavelength is detected. More specifically, apparatuses such as fluorescent detectors and image analyzers used for conventional DNA microarray analysis can be used.
As described above, it is possible to determine whether or not a sugarcane sample has the above markers associated with resistance to sugarcane smut with the use of the DNA microarray. Here, as described above, as the marker associated with resistance to sugarcane smut, a marker linked to a trait characterized by the improvement of smut resistance and a marker linked to a trait characterized by the reduction of smut resistance are provided. Markers associated with resistance to sugarcane smut designed based on the three aforementioned regions identified in tables 2, 4, and 7 are linked to a trait characterized by the improvement of smut resistance. Meanwhile, markers associated with resistance to sugarcane smut designed based on the four aforementioned regions identified in tables 1, 3, 5, and 6 are linked to a trait characterized by the reduction of smut resistance.
Therefore, if any one of the markers associated with resistance to sugarcane smut designed based on the three aforementioned regions identified in tables 2, 4, and 7 is present in a sugarcane sample, it is possible to determine that the sample is of a variety with improved smut resistance. Further, if any one of the markers associated with resistance to sugarcane smut designed based on the four aforementioned regions identified in tables 1, 3, 5, and 6 is absent in a sugarcane sample, it is possible to determine that the sample is of a variety with improved smut resistance. Preferably, if any one of the markers associated with resistance to sugarcane smut designed based on the three aforementioned regions identified in tables 2, 4, and 7 is present in a sugarcane sample, and if any one of the markers associated with resistance to sugarcane smut designed based on the four aforementioned regions identified in tables 1, 3, 5, and 6 is absent in the sugarcane sample, it is possible to determine with high accuracy that the sample is of a variety with improved smut resistance.
In particular, according to the method described above, it is not necessary to cultivate sugarcane samples to such an extent that determination using an actual smut resistance test becomes possible. For instance, seeds of a progeny line or a young seedling obtained as a result of germination of such seeds can be used. Therefore, the area of a field used for cultivation of sugarcane samples and other factors such as cost of cultivation can be significantly reduced with the use of the markers associated with resistance to sugarcane smut. In addition, the use of markers associated with resistance to sugarcane smut makes it possible to reduce the cost of facilities such as a large-scale special-purpose greenhouse, a special-purpose field, or isolation facility from an external environment, without the need to actually cause infection with a causative microorganism of smut (Ustilago scitaminea).
In particular, when a novel sugarcane variety is created, it is preferable to produce several tens of thousands of seedlings via crossing and then to identify a novel sugarcane variety using markers associated with resistance to sugarcane smut prior to or instead of seedling selection. The use of such markers associated with resistance to sugarcane smut makes it possible to significantly reduce the number of excellent lines that need to be cultivated in an actual field. This allows drastic reduction of time-consuming efforts and the cost required to create a novel sugarcane variety.
Alternatively, upon creation of a new sugarcane variety, firstly, it may be determined whether or not a marker associated with resistance to sugarcane smut is present in a parent variety used for crossing, thereby allowing selection of a parent variety with excellent smut resistance. It can be expected that a progeny line with excellent smut resistance will be obtained with high frequency by creating a parent variety with excellent smut resistance on a priority basis. The use of such marker(s) associated with resistance to sugarcane smut makes it possible to significantly reduce the number of excellent lines that need to be cultivated. This allows drastic reduction of time-consuming efforts and the cost required to create a novel sugarcane variety.
The present invention is hereafter described in greater detail with reference to the following examples, although the technical scope of the present invention is not limited thereto.
1. Production of DNA Microarray Probes
(1) Materials
The following varieties were used: sugarcane varieties: NiF8, Ni9, US56-15-8, POJ2878, Q165, R570, Co290 and B3439; closely-related sugarcane wild-type varieties: Glagah Kloet, Chunee, Natal Uba, and Robustum 9; and Erianthus varieties: IJ76-349 and JW630.
(2) Restriction Enzyme Treatment
Genomic DNA was extracted from each of the above sugarcane varieties, closely-related sugarcane wild-type varieties, and Erianthus varieties using DNeasy Plant Mini Kits (Qiagen). Genomic DNAs (750 ng each) were treated with a PstI restriction enzyme (NEB; 25 units) at 37 degrees C. for 2 hours. A BstNI restriction enzyme (NEB; 25 units) was added thereto, followed by treatment at 60 degrees C. for 2 hours.
(3) Adapter Ligation
PstI sequence adapters (5′-CACGATGGATCCAGTGCA-3′ (SEQ ID NO: 86) and 5′-CTGGATCCATCGTGCA-3′ (SEQ ID NO: 87)) and T4 DNA Ligase (NEB; 800 units) were added to the genomic DNA fragments treated in (2) (120 ng each), and the obtained mixtures were subjected to treatment at 16 degrees C. overnight. Thus, the adapters were selectively added to genomic DNA fragments having PstI recognition sequences at both ends thereof among the genomic DNA fragments treated in (2).
(4) PCR Amplification
A PstI sequence adapter recognition primer (5′-GATGGATCCAGTGCAG-3′ (SEQ ID NO: 88)) and Taq polymerase (TAKARA; PrimeSTAR; 1.25 units) were added to the genomic DNA fragment (15 ng) having the adaptors obtained in (3). Then, the genomic DNA fragment was amplified by PCR (treatment at 98 degrees C. for 10 seconds, 55 degrees C. for 15 seconds, 72 degrees C. for 1 minute for 30 cycles, and then at 72 degrees C. for 3 minutes, followed by storage at 4 degrees C.).
(5) Genome Sequence Acquisition
The nucleotide sequence of the genomic DNA fragment subjected to PCR amplification in (4) was determined by the Sanger method. In addition, information on a nucleotide sequence sandwiched between PstI recognition sequences was obtained based on the total sorghum genome sequence information contained in the genome database (Gramene).
(6) Probe Design and DNA Microarray Production
50- to 75-bp probes were designed based on the genome sequence information in (5). Based on the nucleotide sequence information of the designed probes, a DNA microarray having the probes was produced.
2. Acquisition of Signal Data Using a DNA Microarray
(1) Materials
Sugarcane varieties/lines (NiF8 and Ni9) and the progeny line (line 191) were used.
(2) Restriction Enzyme Treatment
Genomic DNAs were extracted from NiF8, Ni9, and the progeny line (line 191) using DNeasy Plant Mini Kits (Qiagen). Genomic DNAs (750 ng each) were treated with a PstI restriction enzyme (NEB; 25 units) at 37 degrees C. for 2 hours. Then, a BstNI restriction enzyme (NEB; 25 units) was added thereto, followed by treatment at 60 degrees C. for 2 hours.
(3) Adapter Ligation
PstI sequence adapters (5′-CACGATGGATCCAGTGCA-3′ (SEQ ID NO: 86) and 5′-CTGGATCCATCGTGCA-3′ (SEQ ID NO: 87)) and T4 DNA Ligase (NEB; 800 units) were added to the genomic DNA fragments treated in (2) (120 ng each), and the obtained mixtures were treated at 16 degrees C. overnight. Thus, the adaptors were selectively added to a genomic DNA fragment having PstI recognition sequences at both ends thereof among the genomic DNA fragments treated in (2).
(4) PCR Amplification
A PstI sequence adapter recognition primer (5′-GATGGATCCAGTGCAG-3′ (SEQ ID NO: 88)) and Taq polymerase (TAKARA; PrimeSTAR; 1.25 units) were added to the genomic DNA fragment (15 ng) having the adapters obtained in (3). Then, the genomic DNA fragment was amplified by PCR (treatment at 98 degrees C. for 10 seconds, 55 degrees C. for 15 seconds, 72 degrees C. for 1 minute for 30 cycles, and then 72 degrees C. for 3 minutes, followed by storage at 4 degrees C.).
(5) Labeling
The PCR amplification fragment obtained in (4) above was purified with a column (Qiagen). Cy3-labeled 9mers (TriLink; 1 O.D.) was added thereto. The resultant was treated at 98 degrees C. for 10 minutes and allowed to stand still on ice for 10 minutes. Then, Klenow (NEB; 100 units) was added thereto, followed by treatment at 37 degrees C. for 2 hours. Thereafter, a labeled sample was prepared by ethanol precipitation.
(6) Hybridization/Signal Detection
The labeled sample obtained in (5) was subjected to hybridization using the DNA microarray prepared in 1 above in accordance with the NimbleGen Array User's Guide. Signals from the label were detected.
3. Identification of QTL for Sugarcane Smut Resistance and Development of Markers
(1) Creation of Genetic Map Datasheet
Genotype data of possible 3004 markers and 4569 markers were obtained based on the signal data detected in 2 above of the NiF8 and Ni9 sugarcane varieties and the progeny line (line 191). Based on the obtained genotype data, chromosomal marker position information was obtained by calculation using the gene distance function (Kosambi) and the AntMap genetic map creation software (Iwata H, Ninomiya S (2006) AntMap: constructing genetic linkage maps using an ant colony optimization algorithm, Breed Sci 56: 371-378). Further, a genetic map datasheet was created based on the obtained marker position information using Mapmaker/EXP ver. 3.0 (A Whitehead Institute for Biomedical Research Technical Report, Third Edition, January, 1993).
(2) Acquisition of Smut Resistance Data
From October 26 to 28, 2009, stalks of NiF8, Ni9, and the 191 hybrid progeny line were harvested. They were subjected to treatment for stimulating germination at room temperature and high humidity for 2 to 3 days, followed by wound inoculation with smut spores. For wound inoculation, wounds were made on both sides of buds (6 wounds in total; approximately 4.0 mm in depth), and then a spore suspension (107 to 108 spores/ml) was applied to the wounds using a brush. Smut spores in the spore suspension were collected from smut whips of Ni9 stocks naturally infected with smut, which were cultivated in Okinawa in 2009. Seedlings subjected to wound inoculation were cultivated for 2 to 3 days at room temperature and high humidity and planted in nursery boxes from Oct. 30 to Nov. 1, 2009 (40 buds/box, 2 boxes/line). The planted seedlings were cultivated at high humidity in a greenhouse until Sep. 2, 2010. The degree of the development of smut was investigated by counting, as the number of affected seedlings, the number of seedlings showing a symptom of smut, which is the outgrowth of a smut whip from the apex of a stalk. After the count of the affected seedlings, the plant bodies of affected seedlings were harvested at the ground level so that they could be removed. The number of seedlings affected with smut was investigated on June 23, July 21, August 18, and Sep. 2, 2010 for a total of four instances. The incidence of smut was calculated as a percentage of the number of germinating stocks (excluding stocks killed by non-smut causes) accounted for by the number of affected stocks.
(3) Quantitative Trait (Quantitative Trait Loci: QTL) Analysis
Based on the genetic map datasheet obtained in (1) above and the smut resistance data obtained in (2) above, QTL analysis was carried out by the composite interval mapping (CIM) method using the QTL Cartographer gene analysis software (Wang S., C. J. Basten, and Z.-B. Zeng (2010). Windows QTL Cartographer 2.5. Department of Statistics, North Carolina State University, Raleigh, N.C.). Upon analysis, the LOD threshold was determined to be 2.5. As a result, as shown in
As shown in
In addition, as examples of signals detected in 2 (6) above, table 9 shows signal levels of 14 types of markers among markers N827337 to N821515 present in the 5th linkage group of NiF8 for NiF8 and Ni9 and their progeny lines. In particular, the signal levels of N802870 are shown in
Signal levels of 14 types of markers were found to be remarkably high for progeny lines exhibiting reduction of smut resistance among the linkage groups present in NiF8. These results also revealed that 14 types of markers among markers N827337 to N821515 present in the 5th linkage group can be used as markers associated with resistance to sugarcane smut.
Similarly, table 10 lists signal levels of 8 types of markers among markers N826561 to N826906 present in the 17th linkage group of NiF8 in NiF8 and Ni9 and the progeny lines. In particular, the signal levels of N827136 are shown in
Signal levels of 8 types of markers were found to be remarkably high for progeny lines exhibiting excellent smut resistance among the linkage groups present in NiF8. These results also revealed that 8 types of markers among markers N826561 to N826906 present in the 17th linkage group can be used as markers associated with resistance to sugarcane smut.
Similarly, table 11 lists signal levels of 10 types of markers among markers N816552 to N821999 present in the 40th linkage group of NiF8 in NiF8 and Ni9 and the progeny lines. In particular, the signal levels of N812680 are shown in
Signal levels of 10 types of markers were found to be remarkably high for progeny lines exhibiting reduction of smut resistance among the linkage groups present in NiF8. These results also revealed that 10 types of markers among markers N816552 to N821999 present in the 40th linkage group can be used as markers associated with resistance to sugarcane smut.
Similarly, table 12 lists signal levels of 19 types of markers among markers N915070 to N920207 present in the 1st linkage group of Ni9 in NiF8 and Ni9 and the progeny lines. In particular, the signal levels of N916081 are shown in
Signal levels of 19 types of markers were found to be remarkably high for progeny lines exhibiting excellent smut resistance among the linkage groups present in Ni9. These results also revealed that 19 types of markers among markers N915070 to N920207 present in the 1st linkage group can be used as markers associated with resistance to sugarcane smut.
Similarly, table 13 lists signal levels of 11 types of markers among markers N914284 to N916129 present in the 13th linkage group of Ni9 in NiF8 and Ni9 and the progeny lines. In particular, the signal levels of N919839 are shown in
Signal levels of 11 types of markers were found to be remarkably high for progeny lines exhibiting reduction of smut resistance among the linkage groups present in Ni9. These results also revealed that 11 types of markers among markers N914284 to N916129 present in the 13th linkage group can be used as markers associated with resistance to sugarcane smut.
Similarly, table 14 lists signal levels of 10 types of markers among markers N901178 to N900802 present in the 14th linkage group of Ni9 in NiF8 and Ni9 and the progeny lines. In particular, the signal levels of N918761 are shown in
Signal levels of 10 types of markers were found to be remarkably high for progeny lines exhibiting reduction of smut resistance among the linkage groups present in Ni9. These results also revealed that 10 types of markers among markers N901178 to N900802 present in the 14th linkage group can be used as markers associated with resistance to sugarcane smut.
Similarly, table 15 lists signal levels of 13 types of markers among markers N901524 to N918080 present in the 14th linkage group of Ni9 in NiF8 and Ni9 and the progeny lines. In particular, the signal levels of N901160 are shown in
Signal levels of 13 types of markers were found to be remarkably high for progeny lines exhibiting excellent smut resistance among the linkage groups present in Ni9. These results also revealed that 13 types of markers among markers N901524 to N918080 present in the 14th linkage group can be used as markers associated with resistance to sugarcane smut.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011-101050 | Apr 2011 | JP | national |
| 2012-094995 | Apr 2012 | JP | national |
This application is a Divisional of U.S. application Ser. No. 14/113,539 filed Oct. 23, 2013, which is a National Stage of International Application No. PCT/JP2012/060671, filed Apr. 20, 2012, which claims priority to Japanese Patent Application No. 2011-101050, filed Apr. 28, 2011, and to Japanese Patent Application No. 2012-094995, filed Apr. 18, 2012, the contents of all of which are incorporated herein by reference in their entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20090222941 | Taguchi et al. | Sep 2009 | A1 |
| 20100138950 | Ragot | Jun 2010 | A1 |
| 20110154528 | Ragot et al. | Jun 2011 | A1 |
| Number | Date | Country |
|---|---|---|
| 2010-516236 | May 2010 | JP |
| 2007125958 | Nov 2007 | WO |
| Entry |
|---|
| Butterfield, 2007, PhD thesis “Marker Assisted Breeding in Sugarcane: A Complex Polyploid”, University of Stellenbosch, pp. 1-75. |
| Ji et al., “Comparative QTL Mapping of Resistance to Sporisorium reiliana in Maize Based on Meta-analysis of QTL Locations” 2007, vol. 8, No. 2, pp. 132-139. |
| Xu et al., “Identification of RAPD Marker Linked to Smut Resistance Gene in Sugarcane”, Chin J Appl Environ Biol, Jun. 25, 2004, vol. 10, No. 3, pp. 263-267, ISSN 1006-687X. |
| Piperidis et al., “Comparative genetics in sugarcane enables structured map enhancement and validation of marker-trait associations”, Molecular Breeding, 2008, vol. 21, 233-247. |
| Pan, et al., “Molecular Genotyping of Sugarcane Clones with Microsatellite DNA Markers”, Maydica, 2003, pp. 319-329, vol. 48. |
| de Setta et al., “Building the sugarcane genome for biotechnology and identifying evolutionary trends,” BMC Genomics 15:540, 17pps. (2014). |
| Butterfield, “Marker Assisted Breeding in Sugarcane: a Complex Polyploid,” Ph.D. Thesis, University of Stellenbosch, pp. 1-75 (2007). |
| Aitken et al., “A combination of AFLP and SSR markers provides extensive map coverage and identification of homo(eo)logous linkage groups in a sugarcane cultivar,” Theor. Appl. Genet. 110:789-801 (2005). |
| Gupta et al., “Array-based high-throughput DNA markers for crop improvement,” Heredity 101:5-18 (2008). |
| Aitken et al., “A comprehensive genetic map of sugarcane that provides enhanced map coverage and integrates high-throughput Diversity Array Technology (DArT) markers,” BMC Genomics 15:152 (2014). |
| Ji et al., “Comparative QTL Mapping of Resistance to Sporisorium reiliana in Maize Based on Meta-analysis of QTL Locations,” Journal of Plant Genetic Resources, 2007, vol. 8, No. 2, pp. 132-139. |
| Restriction Requirement, dated Aug. 18, 2015, issued by the United States Patent and Trademark Office in U.S. Appl. No. 14/113,539. |
| Non-Final Office Action, dated Nov. 19, 2015, issued by the United States Patent and Trademark Office in U.S. Appl. No. 14/113,539. |
| Final Office Action, dated May 3, 2016, issued by the United States Patent and Trademark Office in U.S. Appl. No. 14/113,539. |
| Advisory Action, dated Aug. 9, 2016, issued by the United States Patent and Trademark Office in U.S. Appl. No. 14/113,539. |
| Non-Final Office Action, dated Sep. 28, 2016, issued by the United States Patent and Trademark Office in U.S. Appl. No. 14/113,539. |
| Final Office Action, dated Jan. 27, 2017, issued by the United States Patent and Trademark Office in U.S. Appl. No. 14/113,539. |
| Notice of Allowance, dated May 19, 2017, issued by the United States Patent and Trademark Office in U.S. Appl. No. 14/113,539. |
| Communication, dated Nov. 29, 2018, issued by the United States Patent and Trademark Office in U.S. Appl. No. 15/664,093. |
| Communication, dated Dec. 10, 2018, issued by the United States Patent and Trademark Office in U.S. Appl. No. 15/664,139. |
| Communication, dated Sep. 10, 2018, issued by the United States Patent and Trademark Office in U.S. Appl. No. 15/664,093. |
| Communication, dated Sep. 4, 2018, issued by the United States Patent and Trademark Office in U.S. Appl. No. 15/664,139. |
| Communication, dated Mar. 15, 2019, issued by the United States Patent and Trademark Office in U.S. Appl. No. 15/664,093. |
| Communication, dated Mar. 26, 2019, issued by the United States Patent and Trademark Office in U.S. Appl. No. 15/664,139. |
| Communication, dated Aug. 08, 2019, issued in U.S. Appl. No. 15/664,093. |
| Communication, dated Aug. 20, 2019, issued in U.S. Appl. No. 15/664,139. |
| Number | Date | Country | |
|---|---|---|---|
| 20170327908 A1 | Nov 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14113539 | US | |
| Child | 15664114 | US |
