JP2005027566A - Method of individual identification for zoysia plant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple method for identifying the uniformity of a Zoysia plant on a sod utilizing a genetic technique without the observation of its morphological characteristics by the use of a simple sequence-reiterated marker(SSR) enabling its individual identification. <P>SOLUTION: The method for identifying the variety, strain or individual of a Zoysia plant comprises the following practice: With either one of simple reiterated sequences on the chromosome of the plant represented in a specific sequence as a template, a PCR amplification is carried out, and the molecular weight of the resulting amplified product is confirmed. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a simple repetitive sequence that can be used for easy individual identification, a method using the same, and a method for providing information on a fertile and genetic background that varies from individual to individual and is vegetatively breeding.

  Plants belonging to the genus Shiba, especially Noshiba, Kushunshiba and Koraishiba, are plants with extremely high commercial demand, which are used over a wide area in general households, parks, athletic fields and the like. This classification among the plants of the genus Shiba has so far been performed only from morphological characteristics.

  However, since the plant of the genus Shiba belongs to a small plant body and classification and individual identification using a slight difference in form are compelled, individual identification by form is generally difficult. In particular, the diversification of social needs in the future is expected to increase the number of new registered varieties of the genus Shiba, but it may be difficult to distinguish and identify based only on conventional morphological features. Is done.

  As an identification method different from morphological observation, there is a linkage analysis method using RFLP (restriction fragment length polymorphism). Studies are also underway to identify useful genes of crops based on RFLP and to improve various properties such as disease resistance, insect resistance, and high yield using them as markers.

  However, the operation of RFLP is cumbersome and requires a large amount of DNA. It takes a lot of time and labor to identify a large number of samples with a plurality of markers, and sod (flat plate-like) mixed with varieties. It is impossible to certify turf with soil punched in

  In addition, an individual identification method using a simple repetitive sequence of DNA, which is an alternative method, has been developed for animals or a plurality of plants, and is a reliable and simple method used for paternity testing in humans. However, it is difficult to use sequences across genera, and in particular, there is no information about simple repeat sequences in plants of the genus Shiba. Has not reached.

  In view of the actual situation as described above, the present invention efficiently utilizes a gene region containing a repetitive sequence in order to make the best use of the characteristics of a plant of the genus Shiba, which is proliferated by vegetative propagation and a single sod is the main distribution form. The object is to develop a technique that can be isolated from a plant belonging to the genus Psyllium and individual identification of the plant belonging to the genus Psyllium using a genetic method without using morphological features.

  As a result of diligent research, the present inventors have succeeded in isolating simple repetitive sequences effective for individual identification of plants belonging to the genus Shiba, and based on this, a method enabling efficient and reliable individual identification. developed.

  That is, the present invention is characterized in that PCR amplification is carried out using a chromosomal region corresponding to any of the simple repetitive sequences of Shiba plants represented by SEQ ID NOs: 1 to 137 as templates, and the molecular weight of the amplified product is confirmed. A method for identifying the variety, line, or individual of a genus plant, and a method for verifying the uniformity of a fern plant on a sod, wherein the fern plant shown in SEQ ID NOs: 1 to 137 on a fern plant chromosome collected from the sod PCR amplification using a chromosomal region corresponding to any of the simple repetitive sequences as a template and confirming the molecular weight of the amplified product, and a set of PCR primer sets for use in the method From the group consisting of the following primer sets 1 to 102 consisting of forward primer and reverse primer 1 or reverse primer 2 Is-option relates to at least one of the PCR primer sets.

  According to the present invention, it is possible to identify an individual, and further, to acquire a method that enables identification of existing and native plant of the genus Shiba and its progeny, and based on this, is a major form of distribution of the genus Shiba. The purpose is to develop and provide a method to verify the uniformity of the sod. Even if it is a completely unknown individual, if it is the same as an existing variety, it can be identified by using the present invention, and further, it can be confirmed whether it is a parent-child relationship or a derivative line . In addition, even when the varieties are mixed, the frequency of mixing can be accurately determined up to about 40%, so the quality of sod, which is the main distribution form of Shiba, can be confirmed, so the quality display It is also an excellent method. By using the present invention, an epoch-making effect that protects the breeder's rights and enables the provision of stable quality sods can be obtained.

  The polymorphism of the PCR amplification product is confirmed by, for example, acrylamide gel electrophoresis for each breed, strain and individual, to identify the breed, strain and individual, and to further apply this technical means, which is the main distribution form It is characterized in that the uniformity of the sod is detected qualitatively.

  In the present invention, the genus plant refers to all plants included in the genus Zoysia. Specific examples include Zoysia japonica, Zoysia matrella, Zoysia tenifolia, and Zoysia macrostachya.

  The present invention is based on the successful cloning of a simple repetitive sequence that has been thought to exist in the genomic DNA of the genus Pleuro. The specific nucleic acid sequence of this simple repetitive sequence is as shown in SEQ ID NOs: 1 to 137. This sequence was isolated and confirmed from the genomic DNA of the cultivar “Asaka”. Among these AG single repeat sequences, an SSR region consisting of 6 to 28 AG repeat sequences and having an “interrupted” structure in which another base (AA) is mixed in the repeat sequence is also observed. .

  Many of these simple repetitive sequences are scattered on the genome of the plant of the genus Shiba, and it is confirmed that different characteristic nucleic acid sequences are located at both ends of the repetitive portion of a certain base sequence. It was.

  In general, it is known that the length (the number of repetitions) of a repetitive sequence itself on a genome varies depending on the species. Therefore, it is expected that these simple repetitive sequences confirmed in the genus plants also differ in the number of repetitions, that is, the length of the repetitive sequences, for each species or individual of the genus plants. Therefore, paying attention to the first simple repetitive sequence, set No. as a PCR primer set having a nucleic acid sequence capable of specifically amplifying it. A primer set of 1 (ZjAG133) was designed, and PCR was performed using this primer as a template with the genomic DNA of multiple genus plants to confirm the difference in size of the amplified fragment depending on the plant species. Was made. From this, it became possible to identify the variety, strain, or individual of the genus plant by visually selecting each simple repeat sequence as a marker and using it.

  The primer set of the present invention is a primer set that can be used when a gene region containing the above simple repetitive sequence is specifically amplified by the PCR method.

  The nucleic acid sequence can be designed by confirming a specific genomic sequence located on both sides of a simple repetitive sequence and then hybridizing to such a sequence, but also the secondary structure in which the primers themselves or the primers are not desirable. It is preferable to design the arrangement appropriately so as not to constitute the above.

  Specifically, the Tm value of the primer is preferably about 65 ° C., and the difference between the Tm values of the forward and reverse primers is at most 5 ° C., preferably within 2 ° C. The primer sequence is preferably a sequence that has no homology within its own sequence and does not form a hairpin structure, and should be designed in consideration of the sequence homology between primers. However, when it is not possible to overcome these difficulties from the information of repetitive sequences, it is desirable that the binding derived from the self-homology of the primer sequences be δG <−5 kcal, and in the case of two different primer sequences, −7 kcal or less. .

  These primer design conditions indicate optimum conditions, and the primer nucleic acid sequence can be a specific restriction enzyme cleavage site, for example, as long as the target simple repetitive sequence can be amplified by PCR. It can be freely designed to have a specific nucleic acid sequence that hybridizes to a specific gene sequence.

  A DNA fragment containing a repetitive sequence from a plant of the genus Shiba can be prepared as follows.

  Genomic DNA of Shiba spp. Plants is extracted by, for example, the CTAB method, and a known primer sequence is ligated to a specific restriction enzyme fragment site by using, for example, the AFLP method to obtain a genomic DNA fragment that enables amplification based on the PCR method . Further, genomic DNA is amplified by a PCR method using a known primer sequence, and a fragment of 100 bp to 500 bp effective for the subsequent analysis is selected from the obtained amplification product using a filter using a molecular weight sieve.

  Next, a biotinylated simple repeat sequence (for example, AG repeated 30 times) synthesized using an anti-streptavidin-conjugated iron bead is preferably used for the PCR product having the desired simple repeat sequence, for example, (AG) n. The sequence and the number of repetitions are arbitrary) and are selected using hybridization of the amplified PCR product and biotin-streptavidin binding. Simple repeat sequences are efficient if they are selected using AG repeats, but any other simple repeat sequences may be used as long as they are simple repeat sequences.

  A PCR product containing the selected repetitive sequence, for example, an AG repetitive sequence, is further amplified by the PCR method and inserted into an E. coli plasmid using, for example, TA cloning. The obtained plasmid is transformed into E. coli by electroporation or the like, and only E. coli having a plasmid having an insertion region is cloned by, for example, a colony identification method by disrupting the β-galactosidase gene of the inserted fragment. A large amount of Escherichia coli plasmid is obtained by propagation of the obtained clone, and after secondary selection by plasmid dot blotting or the like, a clone having an insert fragment having a simple repetitive sequence is isolated and its nucleic acid sequence is determined. Thus, genetic information of repetitive sequences can be obtained.

  The operation for obtaining the sequence information of the gene region having the above simple repetitive sequence is performed by a known method known as a genomic DNA enrichment method (genome region enrichment method, enriched genomic library method, Yamamoto, T. et al., However, it is particularly effective as a technique for obtaining a gene region containing a simple repetitive sequence in plants of the genus Shiba. However, for the purpose of obtaining simple repetitive sequence information, the RAHM method (random amplified hybridization microsatellites, Cifarelli et al., 1995), the 5 ′ uncarded PCR method (Fisher et al., 1996), etc. may be used. .

  Primers are designed as described below from gene information including simple repetitive sequences obtained in this manner, and used for identification of cultivars, strains and individuals.

  Based on the obtained genetic information, any PCR amplification primer of 16-30, preferably about 20 mer, is designed on both sides of the simple repetitive sequence. The primer design should have a Tm value of about 65 ° C., and the difference between the Tm values of the forward and reverse primers should be at most 5 ° C., preferably within 2 ° C. The primer sequence is preferably a sequence that has no homology within its own sequence and does not form a hairpin structure, and should be designed in consideration of the sequence homology between primers. However, when it is not possible to overcome these difficulties from the information of repetitive sequences, it is desirable that the binding derived from the self-homology of the primer sequences be δG <−5 kcal, and in the case of two different primer sequences, −7 kcal or less. .

  These primer design conditions indicate optimum conditions. If a simple repetitive sequence is included at the 3 ′ end, the upstream nucleic acid sequence can be expressed by, for example, a specific restriction enzyme cleavage site or a specific restriction enzyme. It can be freely designed so as to have a specific nucleic acid sequence or the like that hybridizes to the gene sequence.

  About the primer designed and synthesize | combined as mentioned above, in order to improve the detection sensitivity of the amplified band, it is desirable to attach | subject fluorescent labels, such as FAM, and to use for PCR reaction.

Using such primers, for example, about 20 μL of PCR reaction solution (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl ₂ , 0.01% gelatin, 0.4 mM each of dNTPs, 50 ng of each forward primer labelled After PCR reaction (dissociation reaction at 95 ° C for 1 minute, annealing reaction at 65 ° C for 1 minute, extension reaction at 72 ° C for 1 minute and 30 seconds) with fluorescent chemical and unlabelled reverse primer, 50 ng genomic DNA and 0.5 unit Taq polymerase) The annealing temperature was lowered by 1 ° C., and these repetitions were repeated 10 times until the annealing temperature reached 56 ° C., then 30 cycles of 95 ° C. for 1 minute dissociation reaction, 55 ° C. for 1 minute annealing reaction, 72 ° C. for 1 minute Then, the extension reaction is performed for 30 seconds, and finally the final extension reaction of the amplified product is performed at 72 ° C. for about 6 minutes, and the reaction is terminated and stored at 4 ° C.).

  When fluorescently labeled primers are used, detection of amplification products by PCR reaction can be detected using fluorescence, but when primers that are not fluorescently labeled are used, ethidium bromide, SYBER GREEN, etc. It can also be detected using a staining method.

  The base length (bp) of the amplification product obtained by the PCR reaction can be confirmed by, for example, a sequencer having a fragment analysis function such as ABI3100 or by electrophoresis using a modified acrylamide containing about 4% 6M urea. it can.

  The amplification product identified in this way has a band size that varies from cultivar to cultivar for each genus. If the band size of the breed is confirmed in advance, not only can the breed of the unknown individual be estimated, but by using the fact that other than the band size of the parental line, it will not be inherited by the offspring, It can be estimated whether it is a derived individual. Furthermore, since the band obtained from one primer combination originates from one common locus in the genus Shiba, it is not necessary to discuss the certainty of breed identification. If described by the length of the band amplified by each primer combination, an infinite number of combinations depending on the number of loci and the type of allele is accurately described, and the possibility of discrimination is infinite.

  Hereinafter, examples will be described in detail, but the present invention is not limited to these examples.

Creation of SSR markers for Shiba genus plants 1) Creation of genomic region enriched library Genomic DNA was extracted from 5 g of leaves of Shiba variety "Asaka" by the CATB method (Murray and Thompson 1980). After measuring the concentration of the obtained genomic DNA with a spectrophotometer, according to the method of Yamamoto et al. (2001), 600 ng of genomic DNA was converted with restriction enzymes EcoR1 and Mse1 using a commercially available kit AFLP Analysis System (Life Technologies). After digestion for 2.5 hours, the mixture was treated at 70 ° C. for 15 minutes, immediately cooled to inactivate the restriction enzyme, and adapter sequences were added to both ends of the DNA fragment by T4 DNA ligase. The obtained DNA fragment was concentrated and recovered by Suprec 2-PCR (Takara).
2) Concentration of SSR region In order to make the DNA fragment to which the adapter sequence was added into a complete double strand, using the recovered DNA fragment as a template and primers designed on the adapter sequence (Sequence Listing 1 and Sequence Listing 2) PCR reaction was performed. The reaction composition and reaction conditions in the PCR reaction are as follows. Note that Ampli Taq GOLD manufactured by ABI was used as the thermostable DNA polymerase.

Composition of reaction solution Template DNA 10 μl
10 × Ampli Taq GOLD buffer (15 mM MgCl ₂ ) 20 μl
50 mM MgCl ₂ 6 μl
dNTPs (2 μM each) 16 μl
10 pmol / μμl Primer 1 5 μl
100 pmol / μl primer 2 3 μl
Thermostable DNA polymerase 2μl
138 μl of water
Total volume 200μl
Reaction conditions
72 ° C for 15 minutes
10 cycles of 90 ° C for 30 seconds, 56 ° C for 1 minute, 72 ° C for 1 minute.

After completion of the PCR reaction at 72 ° C. for 3 minutes, 5 μl of the reaction solution was electrophoresed on a 2% TAE-agarose gel to confirm the amplified product. As a result, a smear migration image was observed in the region of about 200-500 bp, and it was judged that each DNA fragment was amplified.

Next, to the total amount of the obtained PCR product, 20 single repeats of AG labeled with biotin at the 3 ′ end were added, treated at 100 ° C., and left at room temperature to obtain (AG) 20 single repeats. Re-association with the SSR region was performed, and the SSR region was concentrated using this mixed solution. The SSR region was concentrated by adding magnetic beads (DYNABEADs M-280) to the mixed solution, stirring and mixing for 30 minutes, and then collecting only the beads with a magnet. The obtained concentrated solution was washed with STEX buffer (100 mM NaCl, 10 mM Tris-Hcl (pH 8.0), 1 mM EDTA, 0.1% Triton X-100), treated again at 100 ° C., and a magnet-containing fraction. Only minutes were collected. The washing operation with the STEX buffer was performed 3 times.
3) Cloning of SSR region Using the DNA fragment concentrated in 2) as a template, PCR reaction was performed using the primers shown in Sequence Listing 1 and 2, and each amplified fragment was cloned. The reaction composition and reaction conditions in the PCR reaction are as follows. As the thermostable DNA polymerase, Ampli Taq GOLD manufactured by ABI was used.

Composition of reaction solution Template DNA 2.5 μl
10 × Ampli Taq GOLD buffer (15mM MgCl ₂ ) 5.0μl
25 mM MgCl ₂ 6.0 μl
dNTPs (2 μM each) 4.0 μl
10 pmol / μl primer 1 1.25 μl
100 pmol / μl Primer 2 0.75 μl
Thermostable DNA polymerase 0.5 μl
SDW 30μl
Total 50μl
Reaction conditions
25 cycles of 90 ° C for 30 seconds, 56 ° C for 1 minute, 72 ° C for 1 minute.

72 ° C. for 3 minutes After completion of the PCR reaction, the amplified fragment was inserted into a plasmid vector using TOPO TA cloning kit (Invitrogen) and introduced into E. coli attached to the kit. The obtained clone was smeared on a 1.5% LB selection medium containing ampicillin, X-gal and IPTG, cultured overnight at 37 ° C. under dark conditions, and then a color reaction of β-galactosidase gene incorporated into the plasmid vector. Only white strains were selected by the blue-white selection method using as an index. In this experiment, 1920 white clones were finally selected from the enriched library derived from the Shiba variety “Asaka” obtained in Example 1.

  The selected E. coli was used to amplify the insert by colony-PCR method. The reaction composition and reaction conditions in the PCR reaction are as follows. As the thermostable DNA polymerase, Ampli Taq GOLD manufactured by ABI was used.

Composition of reaction solution
10 × Ampli Taq GOLD buffer (15mM MgCl ₂ ) 1.0μl
dNTPs (2 μM each) 1.0 μl
10 pmol / μl M13 Reverse primer 0.5 μl
10 pmol/μl M13 Forwerd primer 0.5μl
Thermostable DNA polymerase 0.05μl
SDW 7.45 μl
Total 10μl
Reaction conditions
30 cycles of 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 2 minutes.

After completion of the PCR reaction at 72 ° C. for 3 minutes, the clone containing the SSR region was screened using the amplification product. For the screening, a dot blot hybridization method was applied. That is, the amplification product was made into a single strand by treatment at 100 ° C., and then 1 μl was immobilized on Hybond N + nylon membrane (AmershamPharmacia Biotech.). The membrane was pre-hybridized in a hybridization buffer (5 × SSC, 0.1% sarkosyl, 0.02% SSC, 1% Blocking Regent) at 50 ° C. for 30 minutes, and then 50 pmol of probe DNA was added at 50 ° C. Hybridization was performed for 2 hours. The probe used was a 20-times repeat sequence of AG labeled with biotin at the 5 end.

After hybridization, the membrane was washed and detected with BrightStar BioDetect Kit (Ambion), exposed to X-ray film (Fuji Film) and developed. As a result of identifying positive clones based on the obtained film, 457 clones that strongly hybridized with the probe were identified from among 1920 clones.
4) Determination of nucleotide sequence of SSR region Among 457 positive clones obtained by screening, the nucleotide sequence of 162 clones was determined. Plasmid DNA was cultured by shaking overnight at 37 ° C. in an LB liquid medium containing ampicillin, X-gal and IPTG, and extracted and purified using a plasmid DNA purification kit (Quiagen). The obtained plasmid DNA was subjected to a sequencing reaction using a Big dye terminator cycle sequencing kit (Perkin-Elmer, Applied Biosystems), and the sequence information of the insert DNA was obtained using an ABI373 sequencer (Perkin-Elmer, Applied Biosystems). These DNA sequences are shown in FIG. 1 (SEQ ID NOs: 1 to 137).

  As a whole, no SSR region was observed in 4 out of 162 clones. It was also found that 2 clones were duplicate clones, and the remaining 154 clones were all single clones. Further analysis of these 156 clones revealed that 37 clones, 23.7% of which had short repeat sequences of 3-7 repeats. On the other hand, 119 clones corresponding to 76.3% contained 8 or more SSR regions.

Design of specific amplification primer Based on the sequence information of the insert, a primer that specifically amplifies a region containing a single repetitive sequence was designed. Primer design was performed using software OLIGO ver.6.0 (TAKARA). The primers were designed so that the amplification product had a chain length of 150 to 250 bp with 19-21 mer bases on both sides of the simple repeat sequence. In addition, a region having a Tm value of 65 to 70 ° C. and not taking secondary structure between primers such as binding due to self-homology and a hairpin structure was selected as the primer sequence. In addition, about the primer of one side, the 5 terminal side was fluorescent-labeled (FAM). Although there was sequence information that was difficult for primer design due to the sequence accuracy and the position of the SSR region in each clone, 102 sets of SSR primers could be finally designed (FIG. 2).

Estimation of the number of loci using SSR markers SSR analysis was performed using the primers designed in Example 2 to estimate the number of loci (FIG. 3). Genomic DNA was extracted by CATB method from 11 individuals of the F1 progeny having the ecotype “Unosaki” of Zoysia macrostachya as the pollen parent and “Kaito Taketomi” as the mating parent, and PCR reaction was performed. The reaction composition in the PCR reaction is as follows. As the thermostable DNA polymerase, Ampli Taq GOLD manufactured by ABI was used.

Composition of reaction solution Template DNA (25ng / μl) 1μl
10 × Ampli Taq GOLD buffer (15mM MgCl ₂ ) 1μl
25 mM MgCl ₂ 1 μl
dNTPs (2 μM each) 2 μl
10 pmol / μl primer 1 0.5 μl
10 pmol / μl Primer 2 0.5 μl
Thermostable DNA polymerase 0.03μl
SDW 3.97 μl
Total 10μl
Moreover, the reaction conditions applied the touchdown PCR method which reduces annealing temperature 1 degree / cycle according to the method of Maize Mapping Progect (http://www.maizemap.org).

Reaction conditions
95 ° C for 8 minutes
Eleven cycles of 95 ° C for 1 minute, 65-55 ° C for 1 minute, 72 ° C for 1.5 minutes.

          However, the annealing temperature is decreased by 1 ° C / cycle.

    25 cycles of 95 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 1.5 minutes.

72 ° C for 3 minutes After completion of the PCR reaction, the amplified fragment was diluted 10-fold with deionized formamide, treated at 100 ° C, fractionated by 5% denaturing polyacrylamide gel electrophoresis, and Molecular imager FX (Bio-Rad). Was used to detect the FAM fluorescent label. As a result, 1 to 2 distinct amplified fragments were observed in all 11 F1 progenies of the onysh tested, and it was further confirmed that the parents' bands were inherited without any contradiction. This indicates that the SSR marker obtained with this primer is derived from a single locus.

Using the existing varieties and 21 ecotypes from all over Japan, we estimated the number of allelic loci, calculated the observed heterozygosity value _HO based on this, and examined the discriminating ability of the designed primers. . In the 12 primers tested, the estimated number of alleles in the Shiba variety and the Ecotype 21 individuals was 5 (ZjAG125) -17 (ZjAG130), and the average value was 11.9. Moreover, the observed value of the heterozygote degree by these primers was 0.14-0.57, and the average value was 0.36. FIG. 4 shows the results of SSR analysis in 21 test specimens using the primer ZjAG 133 among these 12 primers. The estimated number of alleles obtained with this primer was 13. The observed value H ₀ of the heterozygosity was 0.14, which was the lowest value among the test primers.

Identification method of Shiba genus cultivar SSR analysis was performed on 7 existing varieties using 8 primers among the designed primers. The estimated number of alleles among the seven test varieties in the primers used was 4-7. FIG. 5 shows the results of SSR analysis using the primer ZjAG140. In this primer, polymorphism was recognized among the seven test varieties, and all varieties could be identified. In addition, it was clarified that by combining two different primers (ZjAG116 and ZjAG130), a band pattern specific to each variety can be discriminated, and an effective and sufficient primer set can be provided for variety identification (FIG. 6). .

Uniformity verification of sod SSR analysis of the mixed DNA was performed using primers that were recognized to be effective in variety identification. In the SSR analysis, PCR reactions were carried out using genomic DNAs of two kinds of Shiba cultivars “Asaka” and foreign species “Queensland” having different alleles as template DNAs at different mixing ratios of these two genomic DNAs (FIG. 7). . As a result, it was clarified that the band could be confirmed up to 40% of the mixed sample, and the homogeneity of the sod could be verified with the above-mentioned primer using different kinds of Shiba varieties of two alleles.

The nucleic acid sequence of the SSR marker of the plant of the genus Shiba obtained by the present invention is shown. The nucleic acid sequence of the SSR marker of the plant of the genus Shiba obtained by the present invention is shown. The nucleic acid sequence of the SSR marker of the plant of the genus Shiba obtained by the present invention is shown. The nucleic acid sequence of the SSR marker of the plant of the genus Shiba obtained by the present invention is shown. The nucleic acid sequence of the SSR marker of the plant of the genus Shiba obtained by the present invention is shown. The nucleic acid sequence of the SSR marker of the plant of the genus Shiba obtained by the present invention is shown. The nucleic acid sequence of the SSR marker of the plant of the genus Shiba obtained by the present invention is shown. The nucleic acid sequence of the SSR marker of the plant of the genus Shiba obtained by the present invention is shown. The nucleic acid sequence of the SSR marker of the plant of the genus Shiba obtained by the present invention is shown. The nucleic acid sequence of the SSR marker of the plant of the genus Shiba obtained by the present invention is shown. The nucleic acid sequence of the SSR marker of the plant of the genus Shiba obtained by the present invention is shown. The nucleic acid sequence of 102 primer sets for amplifying the genomic region containing the simple repeat sequence of the genus Shiba obtained by the present invention is shown. The nucleic acid sequence of 102 primer sets for amplifying the genomic region containing the simple repeat sequence of the genus Shiba obtained by the present invention is shown. The electrophoresis result of an amplification product when PCR reaction is performed using primer set ZjAG136 is shown. The results of electrophoresis of amplification products when PCR reaction was performed using the primer set ZjAG02 / 133 using 21 existing varieties and a total of 21 ecotypes in various parts of Japan are shown. The electrophoresis result of an amplification product when PCR reaction is performed using 7 existing varieties using the primer set ZiAG02 / 140 is shown. The test result of the discrimination ability of the region amplified by the primer obtained by the present invention is shown. The electrophoresis result of an amplification product when PCR reaction is performed by combining primer sets ZjAG02 / 116 and ZjAG02 / 130 is shown. Primer set No. The result of having performed SSR analysis of mixed DNA using ZjAG125 is shown.

Claims (3)

  PCR amplification using a chromosomal region corresponding to any of the simple repetitive sequences of the genus Shiba as shown in SEQ ID NOs: 1 to 137 as a template, and confirming the molecular weight of the amplified product, A method for identifying strains or individuals.   A method for verifying the uniformity of a shiba plant on a sod, wherein PCR amplification is performed using a chromosomal region corresponding to any one of the simple repetitive sequences shown in SEQ ID NOs: 1 to 137 of the plant of the genus Shiba collected from the sod as a template, The method as described above, wherein the molecular weight of the amplification product is confirmed. A set of PCR primer sets for use in the method according to claim 1 or 2, which is selected from the group consisting of the following primer sets consisting of a forward primer and a reverse primer 1 or a reverse primer 2; One PCR primer set.