# Patent

## Title
Marker for identifying variety/line of plant of the genus Saccharum and the use thereof

## Abstract

It is an object of the present invention to provide a method for identifying the variety/line of a plant of the genus Saccharum with the use of novel DNA markers that allow high-precision identification of a wide range of varieties/lines of plants of the genus Saccharum. A method for identifying the variety/line of a plant of the genus Saccharum, comprising using a simple sequence repeat polymorphism in at least one DNA sequence selected from SEQ ID NOS: 1 to 12 is provided.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a National Stage of International Application No. PCT/JP2010/004425, filed on Jul. 7, 2010, which claims priority from Japanese Patent Application No. 2009-160620, filed on Jul. 7, 2009, the contents of all of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The present invention relates to a characteristic DNA sequence containing an SSR sequence developed from the genome of the genus Saccharum and a method for identifying the variety/line of a plant of the genus Saccharum with the use of the sequence.
BACKGROUND ART
Sugarcane has been cultivated as a raw material for sugar or for alcoholic drink. In addition, sugarcane has been used in a variety of industrial fields, including the use as a raw material for biofuel. Under such circumstances, there is a need to develop a method for identifying a sugarcane variety/line in a convenient manner in order to breed sugarcane varieties having desirable characteristics (e.g., sugar content, enhanced vegetative capacity, sprouting capacity, disease resistance, insect resistance, and cold resistance).
The following three ways may be used for identification of a plant variety/line: “characteristics comparison” for comparison of characteristics data, “comparison of cultivation” for comparison of plants cultivated under the same conditions, and “DNA assay” for DNA analysis. There are many problems in line identification with the use of characteristics comparison or comparison of 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.
Meanwhile, in the case of sugarcane, the development of genetic marker technology has been delayed because of genomic complexity. 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 cannot be used for line identification of the major varieties cultivated in Japan, Taiwan, India, and other countries and of useful genetic resources.
CITATION LIST
Non Patent Literature


    NPL 1: Maydica 48 (2003) 319-329 “Molecular genotyping of sugarcane clones with microsatellite DNA markers”


SUMMARY OF INVENTION
Technical Problem
It is an object of the present invention to provide a method for identifying the variety/line of a plant of the genus Saccharum with the use of novel DNA markers that allow high-precision identification of a wide range of varieties/lines of plants of the genus Saccharum. 
Solution to Problem
As a result of intensive studies in order to achieve the above object, the present inventors have found characteristic DNA sequences from a number of DNA fragments derived from the genome of the genus Saccharum, such DNA sequences enabling identification of a wide range of varieties/lines of plants of the genus Saccharum. This has led to the completion of the present invention.
Specifically, the present invention encompasses the following features.
(1) A method for identifying the variety/line of a plant of the genus Saccharum, comprising using a simple sequence repeat polymorphism in at least one DNA sequence selected from SEQ ID NOS: 1 to 12.
(2) The identification method according to (1), comprising using simple sequence repeats in three types of DNA sequences selected from SEQ ID NOS: 1, 2, and 6.
(3) The identification method according to (1), comprising using simple sequence repeats in three types of DNA sequences selected from SEQ ID NOS: 2, 6, and 12.
(4) The identification method according to (1), comprising using a simple sequence repeat polymorphism in any one of DNA sequences of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 10, and 12, wherein a plant of the genus Saccharum to be identified is of the variety NiF8 or Ni9.
(5) The identification method according to (1), comprising using a simple sequence repeat polymorphism in any one of DNA sequences of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 11, and 12, wherein a plant of the genus Saccharum to be identified is of the variety F177 or Nco310.
(6) The identification method according to (1), comprising using a simple sequence repeat polymorphism in the DNA sequence represented by SEQ ID NO: 12, wherein a plant of the genus Saccharum to be identified is a variety cultivated in Japan.
(7) The identification method according to (1), further comprising using simple sequence repeat polymorphisms in the DNA sequence(s) represented by SEQ ID NO(S): 13 and/or 14.
(8) The method according to any one of (1) to (7), wherein identification is carried out by the steps of:
(a) carrying out PCR amplification using DNA extracted from a sugarcane to be identified as a template and a primer set consisting of a forward and reverse primers that specifically amplify a region containing a simple sequence repeat in the selected DNA sequence(s);

(b) determining the molecular weights of amplified DNA fragments; and

(c) performing genotyping of the region containing a simple sequence repeat based on distribution of molecular weights.

(9) The method according to (8), wherein the determination of the molecular weights of the amplified DNA fragments in step (b) is performed by capillary electrophoresis.
(10) The method according to (8), further comprising comparing the genotype determined with that obtained from a known sugarcane variety/line in step (c).
(11) A kit for conducting the method according to any one of (1) to (10), comprising a primer set consisting of a forward and reverse primers that specifically amplify a region containing a simple sequence repeat in the selected DNA sequence(s).
(12) The kit according to (11), further comprising a correspondence table regarding the genotype of a region containing a simple sequence repeat in the selected DNA sequence(s), obtained from known sugarcane varieties/lines.
Advantageous Effects of Invention
The present invention provides a method for identifying the variety/line of a plant of the genus Saccharum with the use of novel DNA markers that allow high-precision identification of a wide range of varieties/lines of plants of the genus Saccharum. Identification of a wider range of sugarcane varieties with improved precision can be achieved by using the DNA markers of the present invention, if necessary, in combination. In addition, the reproducibility of the method is high.
In addition, the present invention provides a kit for conducting the method of the present invention.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a capillary electrophoresis image obtained as a result of PCR amplification of an SSR marker STY126.
FIG. 2 shows changes in the electrophoresis image for SSR markers STY120 and STY162 due to different DNA contents: lanes 1 to 5: SSR marker STY120 (DNA contents: 3-fold, 2-fold, standard (24 ng), 1⁄2, and 1⁄3 from the left); and lanes 6 to 10: SSR marker STY162 (DNA contents: 3-fold, 2-fold, standard (24 ng), 1⁄2, and 1⁄3 from the left).
FIG. 3-1 shows partial charts of polymorphism detection results for two sugarcane lines (NiF8 and Ni9) with the use of an SSR marker STY133.
FIG. 3-2 shows partial charts of polymorphism detection results for two sugarcane lines (NCO310 and F177) with the use of an SSR marker STY133.
FIG. 4 shows partial charts indicating the reproducibility of polymorphism detection with the use of an SSR marker STY133.



DESCRIPTION OF EMBODIMENTS
Hereinafter, the method for identifying the variety/line of a plant of the genus Saccharum of the present invention (hereinafter simply referred to as the method of the present invention) is described.
The term “variety/line” used in the present invention refers to a group of plants that can be distinguished from other groups of plants by all or some of the morphological/ecological characteristics based on their genotype.
According to the present invention, identification of the variety/line of a plant of the genus Saccharum is intended to include identification of the variety/line of a plant of the genus Saccharum that is unknown in terms of variety/line, and determination of whether or not a plant of the genus Saccharum plant that is unknown in terms of the variety/line corresponds to a specific variety/line. For instance, it includes identification as to which variety/line a plant of the genus Saccharum that is completely unknown in terms of variety/line corresponds to, determination as to whether or not the plant is a variety cultivated in Japan, and determination as to whether or not a plant of the genus Saccharum that is known as a Japanese domestic variety is a main variety in Japan.
According to the present invention, identification of the variety/line of a plant of the genus Saccharum further includes determination of the degree of affinity between a plant of the genus Saccharum to be tested and a given variety/line.
The method of the present invention is characterized in that a simple sequence repeat (hereinafter also referred to as “SSR”) in a characteristic DNA sequence developed from the genome of a plant of the genus Saccharum is used. SSR is a repeat of a specific nucleotide sequence having two to several base pairs (bp), which is present in a sporadic pattern in the genomic DNA of an organism. SSR can be represented by (AC)n or (GT)n (wherein “A” denotes adenine, “C” denotes cytosine, “G” denotes guanine, “T” denotes thymine, and “n” denotes an integer of 2 or more) depending on the type of nucleotides of which a repeat consists. Differences in the repeat number “n” among varieties/lines form polymorphisms. In general, an SSR can be identified with a highly conserved sequence region adjacent to the SSR. Therefore, each characteristic DNA sequence described herein contains an SSR forming a polymorphism, and sequences adjacent to such SSR.
Characteristic DNA sequences that can be used for the present invention are provided below. Herein, each underlined portion represents an SSR in a characteristic DNA sequence.








STY099



(SEQ ID NO: 1)



ctcacgaaacgatcaagagatgtacgtcctgaatcctctcccatcctccaaaaggaaaatcattTCTCTCTCTCTCTCTCTC



 


TCTCTCTCTCTCTCTCTCTCTCatcaaaagtaaacaagagaattctattcgtggcctacacaaaacccttccttttctatct


 


cccacggaaagaatggttttttcctcaaaggaaaaaaaagggaaaagataaatattttctcctgacgcacaaataaagccat


 


gtagtag


 


STY117


(SEQ ID NO: 2)



acatcaaaactctcaatcgattaaaatatagatcatgaacaatgtaactagaaaaatacctgacagtcatctgttgataca



 


ttccaggacctatttgtcgatggtaaacttcgttcctgcagttgcagcgcctctttcctggtgctggctttatgacgttttT


 


CTCTCTCcaaacctatgacacattgaaaattgaaaagtaaataaCAACAACAACAACAACAACAACAACAACAACAACAACA


 


Acatagccttttgtcccaagcaagttggggtaggctagagatgaaaagtaaatacaaatactatacttttcccttgtgatca


 


acacagt


 


STY120


(SEQ ID NO: 3)



cctagcaacataagagggcttatctatcTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTtcgctt



 


atgtccaaaacttgtggtagaaacttagagggactccgttgtgttcatgttggtttggggggctggagtccactctacaccc


 


cctaaatccgcctctcattgtaggcaaagttatacacctaccccaaacaagtgactccatgttgcttctattaagatttgaa


 


tttactataaacgtaatgacatcataaaaaaaggatcgagctaaaatgtaattcgaatg


 


STY123


(SEQ ID NO: 4)



actatcgtcaagattccttaaatctcctccatacatgagaggtgatttagccattgaccaaagtgccatctgtaaggtcatg



 


tcaaagaaaaaagagttcattactacagaaaaatagataCAACAACAACAACAACAACAACAACAACAAcatagccttttgt


 


cccaagcaagttggggtaggctacagaaaaaatagatacagaacacttaaatttgaagtgatcacCAACAACAAcctgtgct


 


ttctgttcatcaagagtaaggttacatttcctgtgaggaccctgatttacacctgcattaacagaagaaaaagggtctgttt


 


ctgatacagctgcatgtatattatttcattcaagatattcttggaaaaagaataagggaagggaatggttatgaatttttac


 


gtgttataagtaatttatcgcggatagatatttactctaTGTGTGTGctttactcgatctgatctgaatatcaacagaacag


 


actaaaattaagt


 


STYI33


(SEQ ID NO: 5)



CccttagcctctaggtgaagtgatgaagcgtgaagttgatgctggtagcaacagttctttTCTTCTTCTTCTTCTTCTTCTT



 


CTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTICTTCTTCTTCTTCTcctcctcttttgaccaa


 


cctacggcaagcaatcgccgggttaatatccgtagaatgtgtttacgtgcatgttttagccaatattcaatctgtctgttcc


 


ttctcttgt


 


STY137


(SEQ ID NO: 6)



ctacccattttgctgccatcatccccttcttattctcatgctatatctcttccattcaggttcctaacgacgagagtgtcta



 


attcgaaaaaagaattatatccatcctccagaaatcctcctcCCACCACCACCACCAcccagtctgtttttagcctgctag


 


STY144


(SEQ ID NO: 7)



acctgtctatattgatccctggaaaaaccttccgcTCTCTCTCttaCTCTCTttctccCTCTCTggatttgtgaactaccac



 


tgtttataccaaataaacaagaacttaccgactgatctacacctagaacggtcctaaggatctaacattggt


 


STY145


(SEQ ID NO: 8)



acacaaccatccctagattatactccaacccctcatcttcagactctacctccccattgcCCTCCTCCTcttcctcatctTC



 


ATCATCActttcatccctttcttgttcctcctcatcctcgtcgctctcatcaCCACCACCAcctccAGAAGAAGAcaaagac


 


tatgaagtcaaactcgaggatggtggcaaagatgaaggctcatgatgacatttctatgaggttgactgatggcttattggag


 


catttaaggcattcttgaacctcctcttgt


 


STY166


(SEQ ID NO: 9)



ccaccttttttgtttttctctttcttcttttccccatttcacctttttaccacaaatgtaacccgagccctccccatggaaa



 


ctataaaaggggaggcagggtccacatctaggacaagacataaagacgcataagatttcacaatgaccaatcgaaACACACA


 


CctcACACACaaatcccctgaagagacctgggatccgcacccTCTCTCgaaagcttgtaacccctactatgagt


 


STY168


(SEQ ID NO: 10)



acggacccgactaacctgtgctgttagtgtTGTGTGTGTGTGTGTGTGTGTATATATATATATATATATATATAccttgcag



 


gagaagaggattattgtaacatctgatcccatcccatgg


 


STY173


(SEQ ID NO: 11)



acatcattggacgctgttacctTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGaa



 


caaaggcagTGTGTGttttcttgcagttccagtcttgtatgtctgactgcaatttattttcgg


 


STY200


(SEQ ID NO: 12)



acaagcctactcccatactccatagacacccTCTCTCTCTCtagaTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT



 


CTCTCTCTCccaaccctcttcaactttgtgcatttccttccctagttgggacacctaattctgacatTCTCTCgttcaagtg


 


agaatctacctaggtgaagcaccttctatgagctcgtctgt






In the method of the present invention, the above characteristic DNA sequences can be used alone or in combination depending on a plant of the genus Saccharum to be tested.

For instance, if the variety/line of a plant of the genus Saccharum to be tested is completely unknown, a wide range of varieties/lines cultivated in Japan and foreign countries can be identified by selecting three different characteristic DNA sequences STY099, STY117, and STY137 and using SSR polymorphisms in these DNA sequences in combination.
If it is highly probable that the variety/line of a plant of the genus Saccharum to be tested is a variety cultivated in Japan, the variety/line can be identified by selecting three different characteristic DNA sequences STY117, STY137, and STY200 and using SSR polymorphisms in these DNA sequences in combination.
If it is known that the variety/line of a plant of the genus Saccharum to be tested is either NiF8 or Ni9, which are the main varieties in Japan, the variety/line can be identified as NiF8 or Ni9 by selecting one characteristic DNA sequence from the group consisting of STY099, STY117, STY120, STY123, STY133, STY137, STY144, STY145, STY168, and STY200 and using the SSR polymorphism in the DNA sequence.
If it is known that the variety/line of a plant of the genus Saccharum to be tested is either F177 or Nco310, which are the main varieties that have been introduced into Japan, the variety/line can be identified as F177 or Nco310 by selecting one characteristic DNA sequence from the group consisting of STY099, STY117, STY120, STY123, STY133, STY137, STY144, STY145, STY173, and STY200 and using the SSR polymorphism in the DNA sequence.
If it is known that the variety/line of a plant of the genus Saccharum to be tested is a variety cultivated in Japan, a specific variety/line can be identified by selecting STY200 as the characteristic DNA sequence and using the SSR polymorphism in the DNA sequence.
Examples of varieties cultivated in Japan described herein include, but are not limited to, Ni1, NiN2, NiF3, NiF4, NiF5, Ni6, NiN7, NiF8, Ni9, NiTn10, Ni11, Ni12, Ni14, Ni15, Ni16, Ni17, NiTn19, NiTn20, Ni22, and Ni23. Examples of main varieties in Japan described herein include, but are not limited to, NiF8, Ni9, NiTn10, and Ni15. Examples of main varieties that have been introduced into Japan described herein include, but are not limited to, F177, Nco310, and F172.
Persons skilled in the art can clearly understand which characteristic DNA sequence should be selected depending on a plant of the genus Saccharum to be tested, with reference to table 4 shown herein below.
Further, in the method of the present invention, either one of or both of the characteristic DNA sequence STY127 and STY162 given below can also be used.








STY127



(SEQ ID NO: 13)



actccggacaaggtttatgagtttgataagagccctactatagcaaacaaactagtttcTCTTCTTCTTCTTCTTCTTCTTC



 


TTCTTCTTCTTCTTCTTCTTCTTCTtccttccttccttccttctttcctagccagagccccacacctctttgagtgattacc


 


atgatatgggcttgtttcattaaaaactctatccaaaaacctagtgaaaaaaattaaaatgattgttagagaaaagATATAT


 


cacatttat


 


STY162


(SEQ ID NO: 14)



ctgaaatattgaaaatactcctaaaaatttcttcataggaaacatgacagtggtaataaaacatctcaatgaacagaACA



 


CACctttctactgtggtacttgaattgaaagcacctatccaattgaatgcaagaaacaatagatttgtcatattcatagt


 


tgcaagacatagataaacagagtgctgaacagccaacatgaatatacgattgctctccagtctggcagttctgaaacaca


 


agccagtttcagaaa






In the method of the present invention, identification of a plant of the genus Saccharum with the use of SSR polymorphisms in the above characteristic DNA sequences can be performed by the following steps (a) to (c):
(a) carrying out PCR amplification using DNA extracted from a target sugarcane to be identified as a template and a primer set consisting of a forward and reverse primers that specifically amplify a region containing SSR (hereinafter also referred to as an “SSR marker”) in the above selected characteristic DNA sequence;

(b) determining the molecular weights of amplified DNA fragments; and

(c) performing genotyping for the SSR marker based on distribution of molecular weights.

A DNA sample of a plant of the genus Saccharum to be tested which is used in the present invention can be obtained by extraction from tissues, such as seeds, leaves, roots, and stems, of the plant. DNA extraction can be carried out in accordance with a method generally known to persons skilled in the art. For instance, tissue of the plant is minced and homogenized in an appropriate buffer, followed by total DNA extraction by a known DNA extraction method such as phenol extraction. A DNA extraction kit used for this purpose may be a commercially available DNA extraction kit. For example, a Plant Genomics DNA Mini kit (BioGene) can be used.
A primer set used in the step (a) can be designed based on sequence information of the above characteristic DNA sequence such that a region containing an SSR that is a target for polymorphism detection is specifically amplified. In addition, if the above characteristic DNA sequence contains several SSRs, a primer set can be designed such that a region containing a single SSR or several SSRs is amplified. The lengths of a forward primer and a reverse primer used in the present invention are not particularly limited as long as a target region can be specifically amplified. For instance, such length can fall within the range of 15 to 50 nucleotides and preferably 17 to 25 nucleotides.
Examples of a set of primers that can specifically amplify SSR marker in the above characteristic DNA sequence are provided in table 5 shown below.
The primer set used in the present invention is preferably labeled at 5′-end for the convenience of subsequent determination of the molecular weights of amplified DNA fragments. Labeling can be carried out by a means known to persons skilled in the art, and including, but are not limited to, FITC, 32P, alkaline phosphatase, rhodamine, fluorescamine, dansyl, and derivatives thereof.
PCR conditions for step (a) are not limited as long as a target region can specifically be amplified. For example, such conditions can include 20 to 50 cycles of denaturation at 94 degree C. to 95 degree C. for 10 seconds to 1 minute, annealing at 50 degree C. to 65 degree C. for 10 seconds to 1 minute, and elongation at 72 degree C. for 30 seconds to 10 minutes.
DNA fragments subjected to PCR amplification as described above include a plurality of DNA fragments, which are only different from each other in terms of the repeat number of a target SSR due to the fact that a cultivar of sugarcane is an aneupolyploid.
Next, in step (b), the molecular weights of DNA fragments amplified in step (a) are determined. For molecular weight determination, techniques known to persons skilled in the art such as electrophoresis, mass spectrometry, and sequencing can be used, but are not limited thereto. In the method of the present invention, electrophoresis is preferably used.
Examples of electrophoresis include agarose gel electrophoresis, denatured or non-denatured acrylamide gel electrophoresis, capillary electrophoresis, and the like. As described above, in the present invention, target DNA fragments for molecular weight determination include a plurality of DNA fragments having different molecular weights only in terms of the repeat numbers of an SSR. Therefore, for instance, it is necessary to accurately detect a minute molecular weight difference derived from a very short nucleotide sequence difference of 2 nucleotides. Hence, in the method of the present invention, it is preferable to use high-resolution polyacrylamide gel electrophoresis or capillary electrophoresis, and it is particularly preferable to use capillary electrophoresis.
Subsequently, in step (c), genotyping of a target SSR marker is carried out based on the distribution of determined molecular weights of DNA fragments. The thus determined genotype is optionally compared with the genotype of a relevant SSR marker of a known variety/line which has previously been known or has been obtained as above, to determine what kind of variety/line the determined genotype belongs to, or to determine whether the determined genotype corresponds to none of known varieties/lines, or to determine the degree of affinity with a known specific variety/line.
The method of the present invention is advantageous in that a wider range of varieties/lines can be identified and reproducibility is higher, compared with, for example, sugarcane SSR markers disclosed in Non-Patent Document 1, based on the number of characteristic DNA sequences that can be used and SSR diversity in the sequences.
Further, the present invention encompasses a kit for carrying out the method of the present invention. The kit of the present invention comprises at least one primer set consisting of a forward and reverse primers that specifically amplify an SSR marker in the above characteristic DNA sequence.
The lengths of a forward primer and a reverse primer comprised in the kit of the present invention are not particularly limited as long as a target region can specifically be amplified. For instance, such length can fall within the range of 15 to 50 nucleotides and preferably 17 to 25 nucleotides.
Examples of such primer set are provided in table 5 shown below.
The primer set comprised in the kit of the present invention is preferably labeled at 5′-end for the convenience of subsequent determination of the molecular weights of amplified DNA fragments. Labeling can be carried out by a means known to persons skilled in the art, including, but are not limited to, FITC, 32P, alkaline phosphatase, rhodamine, fluorescamine, dansyl, and derivatives thereof.
Preferably, the kit of the present invention further comprises a correspondence table regarding the genotype of an SSR marker(s) in the above characteristic DNA sequence(s), obtained from known sugarcane varieties/lines. Based on the genotyping results obtained from a plant of the genus Saccharum to be tested with the use of the kit, it can be readily determined what kind of variety/line the plant of the genus Saccharum to be tested belongs to, or whether the plant of the genus Saccharum to be tested belongs to no known varieties/lines, or the degree of affinity with a known specific variety/line.
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.
EXAMPLES
Example 1
Primary Selection of SSR Markers for Variety/Line Identification
The degree of amplification of an SSR region in an SSR marker was evaluated with the use of 96 lines of the genus Saccharum. As a result of PCR reaction and capillary electrophoresis, amplified fragments (bands) of the SSR region were confirmed for all examined SSR markers. Clear band patterns were obtained from 14 SSR markers among the same. However, unclear band patterns were obtained from 4 SSR markers (STY050, STY126, STY149, and STY167) (FIG. 1, table 1). The presence of unclear band patterns makes it difficult to analyze a plurality of samples. Therefore, 14 SSR markers, excluding 4 such SSR markers, were selected by primary selection.







TABLE 1







SSR markers examined for development of line identification


technology and primary selection results











SSR marker
Band pattern
Primary selection






STY050
Unclear
x



STY099
Clear
∘



STY117
Clear
∘



STY120
Clear
∘



STY123
Clear
∘



STY126
Unclear
x



STY127
Clear
∘



STY133
Clear
∘



STY137
Clear
∘



STY144
Clear
∘



STY145
Clear
∘



STY149
Unclear
x



STY162
Clear
∘



STY166
Clear
∘



STY167
Unclear
x



STY168
Clear
∘



STY173
Clear
∘



STY200
Clear
∘









Example 2
Stability Test of SSR Markers for Variety/Line Identification
With the use of B3439 DNA, 14 SSR markers selected by primary selection were evaluated in terms of stability in response to changes in the DNA content upon PCR reaction. As a result, in the case of a PCR reaction at a DNA content of 12 ng (half amount of that used for a general PCR reaction) or more, changes in the band pattern in response to changes in the DNA content were not observed for any examined SSR marker. However, in the case of a PCR reaction at a DNA content of 8 ng (one-third amount of that used for a general PCR reaction), loss of band was found in 2 SSR markers, compared with the band patterns obtained at a DNA content of 12 ng (half amount of that used for a general PCR reaction) or more (FIG. 2, table 2).
The 2 above SSR markers were assumed to have low stability in response to changes in the DNA content. In line identification analysis, errors in terms of the DNA content are more likely to be caused in samples than by an apparatus, reagent, or the like. Therefore, it was thought to be difficult to use these 2 SSR markers for line identification. Based on the above results, 12 SSR markers having excellent stability in response to changes in the DNA content were selected for an SSR marker set for line identification. Meanwhile, at a decreased DNA content, an SSR marker generating a band below 60 bp was observed (FIG. 2). The SSR markers used herein were designed for amplification of 60-bp regions or larger regions. Therefore, it was thought that such a band below 60 bp had not been derived from an SSR region amplification fragment, and thus that it was a non-specific band.







TABLE 2







SSR marker amplification results based on the genomic DNA content








SSR
DNA content (standard: 24 ng)*












marker
3-fold
2-fold
Standard
1⁄2
1⁄3





STY099
◯
◯
◯
◯
◯


STY117
◯
◯
◯
◯
◯


STY120
◯
◯
◯
◯
◯


STY123
◯
◯
◯
◯
◯


STY127
◯
◯
◯
◯
X


STY133
◯
◯
◯
◯
◯


STY137
◯
◯
◯
◯
◯


STY144
◯
◯
◯
◯
◯


STY145
◯
◯
◯
◯
◯


STY162
◯
◯
◯
◯
X


STY166
◯
◯
◯
◯
◯


STY168
◯
◯
◯
◯
◯


STY173
◯
◯
◯
◯
◯


STY200
◯
◯
◯
◯
◯





*“X”: Loss of band






Example 3
Identification Ability of SSR Markers for Variety/Line Identification
In order to evaluate the identification ability of an SSR marker STY133 for variety/line identification, 4 lines (NiF8, Ni9, NCO310, and F177) were examined in terms of band differences. The numbers of obtained bands were 43 bands, 35 bands, 39 bands, and 45 bands, respectively. The average number of common bands was 27.2 for all lines. Meanwhile, the average number of non-common bands was 26.7. Accordingly, the individual lines could be separately identified (FIG. 3).
In addition, as a result of evaluation of reproducibility of the STY133 band with the use of 4 lines (NiF8, Ni14, F160, and NCO310), reproducibility was confirmed for each line (FIG. 4).
Further, based on the results of the numbers of bands and the appearance rates for 125 lines of the genus Saccharum, the SSR marker set for line identification was evaluated in terms of the identification ability. As a result of analysis, the maximum, minimum, and average numbers of bands per SSR marker were 51, 15, and 29, respectively. In total, 348 bands were obtained (table 3). The maximum, minimum, and average appearance rates of the individual bands were 96.9%, 1.0%, and 10.8%, respectively. The maximum, minimum, and average numbers of bands for each line detected with an SSR marker set were 52, 24, and 39, respectively. The probability of accidental coincidence of all bands for two lines, which was obtained from the appearance rates of the individual bands and the number of bands for each line, was found to be very low (6.0E-6 or less).







TABLE 3







Number of polymorphisms in SSR markers










SSR marker
Number of polymorphisms






STY099
27



STY117
51



STY120
27



STY123
15



STY127
No data



STY133
36



STY137
27



STY144
25



STY145
18



STY162
No data



STY166
15



STY168
50



STY173
15



STY200
42




Total 348




Average 29









Example 4
For 125 varieties/lines of the genus Saccharum, the genotype of each SSR marker was determined with the use of SSR markers for variety/line identification.
Table 4 shown below lists genotyping results for each SSR marker obtained from 125 varieties/lines of plants of the genus Saccharum examined in this Example.







TABLE 4







SSR marker band patterns of sugarcane main varieties/lines



















Variety/Line name
STY099
STY117
STY120
STY123
STY133
STY137
STY144
STY145
STY166
STY168
STY173
STY200





Ni1
A001
B001
C001
D002
E096
F001
G001
H001
I001
J001
K020
L001


NiN2
A002
B002
C002
D007
E001
F002
G002
H002
I001
J002
K001
L002


NiF3
A003
B003
C002
D002
E002
F003
G003
H002
I001
J003
K020
L003


NiF4
A004
B004
C002
D004
E003
F002
G004
H003
I001
J004
K001
L004


NiF5
A005
B002
C003
D001
E004
F004
G003
H001
I002
J004
K020
L005


Ni6
A006
B005
C004
D001
E005
F005
G002
H004
I002
J005
K001
L006


NiN7
A007
B002
C005
D007
E006
F006
G005
H001
I002
J005
K001
L007


NiF8
A008
B002
C006
D007
E007
F007
G006
H001
I001
J006
K001
L008


Ni9
A009
B004
C002
D004
E008
F002
G004
H003
I001
J004
K001
L009


NiTn10
A010
B006
C007
D015
E009
F008
G004
H003
I001
J004
K001
L010


Ni11
A011
B074
C008
D007
E010
F009
G007
H001
I001
J007
K002
L011


Ni12
A012
B007
C009
D002
E011
F010
G007
H004
I001
J008
K020
L012


Ni14
A013
B008
C010
D015
E010
F011
G008
H005
I001
J005
K001
L013


Ni15
A014
B009
C011
D015
E012
F002
G009
H006
I001
J004
K020
L014


Ni16
A015
B010
C012
D003
E013
F012
G008
H001
I002
J005
K020
L015


Ni17
A016
B002
C011
D007
E014
F006
G010
H005
I001
J004
K001
L016


NiTn19
A017
B011
C013
D002
E015
F013
G007
H001
I002
J009
K002
L017


NiTn20
A018
B012
C013
D002
E016
F014
G007
H002
I002
J010
K020
L018


Ni22
A019
B002
C014
D004
E017
F009
G004
H003
I001
J006
K020
L019


Ni23
A019
B006
C014
D004
E007
F002
G004
H001
I001
J005
K001
L020


F135
A020
B013
C002
D007
E018
F002
G011
H001
I002
J002
K020
L021


F141
A021
B014
C014
D007
E019
F015
G008
H004
I001
J005
K020
L022


F146
A022
B002
C015
D007
E020
F016
G008
H007
I003
J003
K020
L023


F149A
A023
B015
C014
D007
E021
F017
G007
H001
I001
J011
K020
L024


F160
A024
B002
C014
D007
E010
F018
G003
H002
I001
J005
K001
L025


F172
A025
B002
C013
D002
E022
F019
G008
H004
I002
J010
K002
L026


F175
A026
B002
C002
D004
E023
F020
G007
H005
I001
J012
K020
L027


F177
A027
B016
C016
D004
E024
F012
G007
H001
I002
J004
K002
L028


Nco310
A028
B002
C002
D015
E025
F021
G003
H005
I002
J004
K001
L009


NCo310xSorghumSart
A029
B017
C017
D005
E026
F022
G012
H004
I001
J002
K003
L029


NCo376
A030
B002
C002
D007
E027
F002
G011
H001
I002
J002
K020
L030


P0J2725
A031
B018
C018
D004
E028
F023
G013
H004
I004
J013
K004
L031


P0J0143
A028
B017
C019
D010
E029
F024
G005
H005
I002
J014
K002
L032


P0J2878
A032
B019
C020
D015
E030
F025
G009
H008
I001
J015
K020
L033


Badila
A033
B020
C002
D015
E031
F026
G008
H004
I002
J004
K001
L034


Chittan
A034
B021
C021
D007
E019
F006
G008
H004
I005
J016
K005
L035


GlagahKloet
A035
B022
C022
D021
E032
F027
G014
H009
I006
J017
K020
L036


robustum5
A036
B023
C023
D006
E033
F028
G015
H005
I002
J010
K006
L037


IJ-76-349
A037
B002
C006
D007
E034
F029
G003
H001
I001
J006
K001
L008


Q117
A038
B004
C002
D007
E023
F030
G007
H010
I001
J012
K020
L038


Q124
A039
B024
C024
D005
E035
F031
G016
H011
I002
J004
K001
L039


B3439
A040
B002
C025
D015
E036
F032
G017
H005
I002
J004
K001
L009


CAC57-02
A041
B014
C014
D007
E037
F002
G009
H004
I001
J008
K002
L040


CB40-77
A042
B025
C026
D002
E038
F033
G018
H004
I001
J004
K020
L041


Co0290
A043
B017
C027
D002
E039
F034
G019
H001
I005
J004
K002
L042


Co0312
A044
B017
C028
D007
E040
F035
G020
H012
I007
J004
K020
L043


Co0331
A012
B002
C029
D002
E041
F009
G018
H004
I001
J005
K020
L044


Co0356
A045
B024
C030
D002
E042
F036
G008
H001
I001
J018
K020
L045


Co0421
A046
B026
C078
D015
E043
F009
G018
H004
I001
J019
K020
L046


Co0453
A047
B024
C031
D015
E043
F009
G018
H004
I001
J019
K020
L047


Co0740B
A048
B002
C032
D007
E044
F037
G021
H001
I002
J020
K001
L024


Co0798
A033
B002
C033
D007
E045
F038
G005
H004
I002
J002
K007
L048


Co1001B
A028
B023
C002
D012
E046
F002
G008
H004
I002
J003
K020
L049


CP33-224
A049
B002
C034
D007
E047
F039
G005
H001
I001
J006
K001
L050


CP34-079
A050
B027
C003
D007
E048
F040
G008
H012
I001
J004
K002
L051


CP36-013
A028
B028
C002
D002
E025
F002
G008
H004
I001
J006
K020
L052


CP36-211
A028
B026
C035
D010
E029
F041
G022
H005
I002
J002
K002
L053


CP43-033
A028
B026
C035
D010
E029
F042
G018
H005
I001
J010
K002
L053


CP44-101
A028
B017
C035
D010
E029
F043
G018
H005
I001
J002
K002
L053


CP44-155
A050
B029
C035
D008
E049
F044
G001
H001
I001
J006
K002
L054


CP45-150
A028
B017
C035
D010
E029
F041
G018
H005
I001
J010
K002
L053


CP52-068D
A028
B030
C036
D009
E050
F045
G021
H013
I001
J005
K001
L055


CP57-614A
A051
B028
C027
D007
E051
F046
G008
H001
I001
J006
K020
L002


CP57-621
A052
B017
C002
D009
E052
F046
G005
H004
I001
J021
K020
L056


CP77-0415
A028
B031
C037
D010
E050
F047
G019
H013
I008
J005
K001
L055


H32-8560
A053
B026
C038
D002
E053
F048
G023
H004
I001
J022
K002
L057


M202x46
A054
B032
C039
D011
E054
F049
G009
H004
I001
J004
K008
L058


PT43-52
A055
B029
C040
D012
E055
F009
G009
H001
I001
J002
K020
L059


Q068
A056
B002
C041
D007
E056
F009
G008
H001
I001
J023
K020
L060


Q073
A057
B033
C010
D015
E011
F040
G009
H004
I001
J001
K009
L061


Eros
A058
B034
C002
D015
E023
F022
G024
H005
I002
J003
K020
L062


R397
A059
B002
C042
D002
E057
F017
G001
H004
I001
J024
K020
L063


Trojan
A060
B002
C001
D013
E058
F040
G009
H004
I001
J025
K020
L064


US72-1288
A061
B035
C043
D015
E059
F050
G018
H001
I002
J026
K020
L064


US56-15-2
A062
B036
C044
D014
E060
F051
G025
H014
I009
J027
K020
L065


US56-15-8
A063
B037
C045
D015
E061
F052
G026
H015
I002
J028
K020
L066


SES182
A064
B038
C046
D021
E062
F002
G027
H016
I005
J029
K020
L067


SES205A
A065
B039
C047
D021
E063
F039
G028
H017
I005
J030
K020
L068


SES231
A066
B040
C048
D021
E064
F053
G029
H018
I010
J031
K020
L069


IJ76-514
A067
B017
C078
D015
E065
F002
G026
H004
I001
J032
K020
L070


IN84-111
A068
B041
C049
D021
E066
F054
G029
H017
I011
J033
K020
L071


IS76-174
A069
B042
C050
D021
E067
F055
G030
H019
I011
J034
K020
L072


JW19
A069
B043
C051
D021
E068
F055
G030
H019
I011
J034
K020
L072


JW28
A070
B044
C052
D016
E069
F056
G029
H001
I012
J035
K020
L073


JW49
A071
B045
C053
D004
E070
F057
G031
H001
I011
J036
K020
L074


JW50
A072
B046
C054
D016
E071
F058
G042
H020
I012
J035
K020
L073


JW69
A073
B047
C055
D021
E072
F059
G025
H021
I011
J037
K020
L075


JW97
A074
B048
C056
D021
E073
F060
G032
H022
I011
J038
K020
L076


JW111
A074
B049
C056
D016
E073
F060
G032
H022
I011
J039
K020
L076


JW233
A075
B050
C057
D021
E074
F006
G029
H023
I012
J040
K020
L020


JW516
A076
B051
C058
D021
E075
F060
G033
H022
I013
J041
K020
L077


JW544
A077
B052
C059
D016
E071
F061
G042
H001
I012
J035
K020
L078


06JW-1
A078
B044
C060
D016
E074
F062
G042
H001
I006
J042
K020
L073


Yomitanzan
A079
B053
C061
D004
E076
F063
G008
H010
I002
J043
K010
L079


Maneria
A079
B054
C061
D004
E076
F063
G008
H010
I002
J043
K011
L079


Chunee
A080
B055
C062
D021
E076
F064
G008
H002
I002
J044
K012
L080


KRF093-1
A081
B056
C063
D004
E077
F065
G042
H001
I002
J012
K001
L081


Burma
A082
B057
C064
D004
E078
F021
G008
H024
I014
J045
K013
L082


S. spont. Glagah
A083
B058
C065
D021
E079
F066
G042
H025
I010
J046
K020
L083


PonapeW. T
A083
B059
C066
D021
E079
F067
G042
H025
I010
J046
K020
L084


Tainan
A084
B060
C067
D021
E080
F068
G029
H026
I015
J059
K020
L085


PonapeW. S
A085
B002
C005
D007
E081
F016
G034
H001
I001
J032
K014
L086


Marga
A086
B074
C068
D021
E082
F069
G042
H027
I012
J029
K015
L087


Pana
A087
B061
C069
D021
E083
F070
G035
H028
I011
J047
K020
L088


Cavengerie
A056
B013
C027
D015
E035
F006
G008
H001
I001
J002
K001
L089


RoseBamboo
A088
B062
C070
D019
E084
F071
G036
H001
I001
J048
K016
L100


BoisRouge
A089
B063
C016
D010
E085
F072
G037
H004
I016
J049
K011
L100


BambooCane
A090
B064
C071
D017
E086
F073
G008
H029
I002
J050
K011
L090


BlackTanna
A091
B024
C016
D018
E087
F006
G034
H001
I002
J006
K001
L091


Wakayama
A092
B065
C071
D004
E088
F073
G008
H029
I001
J050
K011
L092


NatalUba
A093
B066
C071
D004
E088
F073
G008
H029
I001
J050
K011
L093


Tekcha
A094
B067
C072
D004
E088
F074
G038
H029
I001
J051
K017
L092


Kouchi
A002
B002
C016
D015
E089
F075
G008
H001
I001
J002
K014
L094


Mungo254
A095
B068
C016
D019
E090
F076
G036
H001
I001
J052
K010
L100


Nargori
A096
B069
C016
D019
E090
F076
G036
H001
I001
J052
K003
L100


PonapeHuruki-mura
A083
B070
C073
D021
E079
F077
G042
H030
I017
J053
K020
L095


Molokai1032
A097
B071
C074
D021
E091
F078
G039
H031
I012
J054
K020
L024


Robustum6
A098
B072
C001
D020
E092
F079
G040
H004
I002
J055
K018
L096


Robustum9
A099
B073
C075
D021
E093
F011
G008
H001
I018
J056
K020
L097


Robustum16
A100
B002
C076
D021
E093
F011
G020
H001
I002
J056
K020
L098


Robustum21
A101
B054
C077
D007
E094
F080
G041
H032
I002
J057
K019
L099


IK76-126
A102
B074
C078
D021
E096
F002
G042
H033
I019
J034
K020
L100


JW630
A102
B074
C078
D021
E096
F081
G042
H033
I019
J058
K020
L100


IS76-156
A102
B074
C078
D021
E096
F082
G042
H033
I019
J034
K020
L100


R570
A103
B074
C078
D021
E095
F083
G008
H004
I019
J059
K020
L100









In the table, the individual SSR markers are represented by letters “A” to “L,” with numbers representing the allelic genotypes thereof. For instance, “STY099” indicates the presence of 103 types of genotypes among 125 tested varieties/lines.

As is apparent from table 4, all 125 tested sugarcane varieties/lines could be identified using the above SSR markers, if necessary, in combination.
Table 5 shown below lists PCR primer sequences for SSR marker amplification used in the above Examples.







TABLE 5







SSR marker primer sequence








SSR
Primer sequence









marker name
Forward
Reverse





STY099
CCTGAATCCTCTCCCATCCT (SEQ ID NO: 15)
GAAGGGTTTTGTGTAGGCCA (SEQ ID NO: 16)


 


STY117
GGTGCTGGCTTTATGACGTT (SEQ ID NO: 17)
TGCTTGGGACAAAAGGCTAT (SEQ ID NO: 18)


 


STY120
CCTAGCAACATAAGAGGGCTT (SEQ ID NO: 19)
CCAACATGAACACAACGGAG (SEQ ID NO: 20)


 


STY123
TTGACCAAAGTGCCATCTGT (SEQ ID NO: 21)
TGTAGCCTACCCCAACTTGC (SEQ ID NO: 22)


 


STY127
TCCGGACAAGGTTTATGAGTTT (SEQ ID NO: 23)
GGGCTCTGGCTAGGAAAGAA (SEQ ID NO: 24)


 


STY133
GCGTGAAGTTGATGCTGGTA (SEQ ID NO: 25)
GCCGTAGGTTGGTCAAAAGA (SEQ ID NO: 26)


 


STY137
CTGCCATCATCCCCTTCTTA (SEQ ID NO: 27)
AGCAGGCTAAAAACAGACTGG (SEQ ID NO: 28)


 


STY144
TGCTTGGGACAAAAGGCTAT (SEQ ID NO: 29)
TGGACCAAAACGTCAGGAAT (SEQ ID NO: 30)


 


STY145
CCAACCCCTCATCTTCAGAC (SEQ ID NO: 31)
GACGAGGATGAGGAGGAACA (SEQ ID NO: 32)


 


STY162
GGGAAACAATTTCCGTCAGA (SEQ ID NO: 33)
TGCTTGGGACAAAAGGCTAT (SEQ ID NO: 34)


 


STY166
CTATAAAAGGGGAGGCAGGG (SEQ ID NO: 35)
CCAGGTCTCTTCAGGGGATT (SEQ ID NO: 36)


 


STY168
CCCGACTAACCTGTGCTGTT (SEQ ID NO: 37)
CATGGGATGGGATCAGATGT (SEQ ID NO: 38)


 


STY173
CATCATTGGACGCTGTTACC (SEQ ID NO: 39)
CAAGACTGGAACTGCAAGAAA (SEQ ID NO: 40)


 


STY200
CAAGCCTACTCCCATACTCCA (SEQ ID NO: 41)
TGTCCCAACTAGGGAAGGAA (SEQ ID NO: 42)









INDUSTRIAL APPLICABILITY
According to the method and the kit of the present invention, a wide range of varieties/lines of plants of the genus Saccharum can easily be identified with high accuracy and high reproducibility.
In view of the above, it is expected that industrial advantages described below, for example, can be obtained.
(1) It is possible to confirm the original line/variety of a plant of the genus Saccharum and the occurrence or nonoccurrence of outcrossing or mixing up of varieties/lines during production or within the marketing or distribution chain.
(2) Plants of the genus Saccharum having excellent characteristics can be clearly identified, and thus improvement of next-generation energy crops can be significantly promoted.
(3) The development of varieties having excellent heterosis can be promoted.
(4) Degrees of affinity among varieties/lines of the genus Saccharum can be obtained, and thus affinity-related information can be provided.

## Claims

1. A method for identifying the variety/line of a plant of the genus Saccharum, comprising a step of genotyping at least one simple sequence repeat selected from simple sequence repeat polymorphisms in DNA sequences of SEQ ID NOs: 1, 2, 3, 5, 6, 10, and 12, wherein the step of genotyping is carried out by the steps of:
(a) carrying out PCR amplification using DNA extracted from a sugarcane to be identified as a template and a primer set consisting of a forward and a reverse primer that specifically amplify a region containing a simple sequence repeat in a DNA sequence of SEQ ID NOs: 1, 2, 3, 5, 6, 10, or 12;
(b) determining the molecular weights of amplified DNA fragments; and
(c) performing genotyping of the region containing a simple sequence repeat based on distribution of molecular weights.

2. The method according to claim 1, further comprising a step of genotyping simple sequence repeat polymorphisms in the DNA sequence(s) represented by SEQ ID NO(S): 13 and/or 14.

3. The method according to claim 1, wherein the determination of the molecular weights of the amplified DNA fragments in step (b) is performed by capillary electrophoresis.

4. The method according to claim 1, further comprising comparing the genotype determined with that obtained from a known sugarcane variety/line in step (c).