KR100803392B1 - Ssr primer derived from job's tears and use thereof - Google Patents

Abstract

An SSR primer pair is provided to be able to be very usefully used for preparing a DNA profile of Coix lachryma-jobi by effectively detecting a DNA polymorphism and effectively analyze the redundancy with conventional resources and establish the novelty when a novel genetic source is introduced by efficiently evaluating the genetic source of the Coix lachryma-jobi. An SSR primer pair derived from Coix lachryma-jobi is selected from the group consisting of a primer pair described as SEQ ID : NOs. 1 and 2; a primer pair described as SEQ ID : NOs. 3 and 4; a primer pair described as SEQ ID : NOs. 5 and 6; a primer pair described as SEQ ID : NOs. 7 and 8; a primer pair described as SEQ ID : NOs. 9 and 10; a primer pair described as SEQ ID : NOs. 11 and 12; a primer pair described as SEQ ID : NOs. 13 and 14; a primer pair described as SEQ ID : NOs. 15 and 16; a primer pair described as SEQ ID : NOs. 17 and 18; a primer pair described as SEQ ID : NOs. 19 and 20; a primer pair described as SEQ ID : NOs. 21 and 22; a primer pair described as SEQ ID : NOs. 23 and 24; a primer pair described as SEQ ID : NOs. 25 and 26; a primer pair described as SEQ ID : NOs. 27 and 28; a primer pair described as SEQ ID : NOs. 29 and 30; a primer pair described as SEQ ID : NOs. 31 and 32; and a primer pair described as SEQ ID : NOs. 33 and 34. A method for detecting a DNA polymorphism of Coix lachryma-jobi comprises the steps of: (a) extracting a genome DNA from the Coix lachryma-jobi; (b) performing a PCR using the extracted genome DNA as a template and the SSR primer pair; and (c) isolating PCR products by size.

Description

SSR primer derived from job's tears and use else}

The present invention relates to job's tears ( Coix). lachryma - jobi The SSR primer isolated from L.) and its use, and more specifically, SSR primer pair having two base sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 34 and performing PCR using the same It relates to a method for detecting DNA polymorphism of Yulmu containing.

Large quantities of genetic resources are collected annually in most major crops and cultivated crops used for breeding. However, on the other hand, the evaluation of genetic resources has not been sufficiently made. Rapid discrimination between species and subspecies varieties of collected genetic resources is very important for accurate management and protection of genetic resources. In addition to the identification of varieties, the identification and classification of trait traits and genetic relationships of genetic resources are very important for the conservation of genetic resources, the creation of new varieties, and the improvement of crops.

Recent rapid advances in molecular biology have led to the development of nucleic acid fingerprint analysis methods and various DNA markers that enable bio-diversity studies at the nucleic acid (DNA) level. This makes it possible to search useful traits, identify species of species, classify and identify varieties, and analyze flexible relationships of populations in a simple and quick manner with little influence on the external environment. Fingerprinting methods using polymerase chain reaction (PCR) developed so far include randomly amplified polymorphic DNAs (RAPD), amplified fragment length polymorphic DNA (AFLP), and simple sequence repeat (SSR). The RAPD method has a disadvantage of poor reproducibility because the non-specific PCR product is amplified, and the AFLP method is spotlighted by high DNA polymorphism detection, but has a disadvantage of complicated appearance and analysis of a low reproducible band. In contrast, the SSR method is a method of constructing a PCR primer based on nucleotide sequence information of a microsatellite region, which is a DNA repeat sequence. Frequently used. In particular, the SSR method has the advantage that it is not affected by the external environment at all. Due to the superiority of this SSR method, studies are underway to develop SSR markers in several major crops and cultivated crops.

In rice, supersatellite markers have been developed that amplify SSR repeated in the rice genome by PCR technology to identify genotypes of rice varieties (Korean Patent Application No. 2001-34003). Tang's team in the United States developed 879 SSR markers and published a genetic map of sunflowers (Tang et al., Theor . Appl . Genet ., 105: 1124-1136, 2002). In addition, many SSR markers have been developed in barley and soybeans. However, in Yulmu, SSR markers have not been developed at all in Korea, and there is little research related to them in foreign countries.

Accordingly, the present inventors have completed the present invention by developing SSR primer pairs that show a large number of polymorphic variation in various yulmu line, while researching to develop an SSR marker capable of efficiently evaluating the yulmu gene.

It is therefore an object of the present invention to provide an SSR marker and its use capable of efficiently evaluating the genetic resources of yulmu.

In order to achieve the above object, the present invention provides an SSR primer pair having two nucleotide sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 34.

In order to achieve the other object of the present invention, the present invention provides a method for detecting DNA polymorphism of yulmu and varieties of yulmu including the PCR using the SSR primer pair.

In order to achieve another object of the present invention, the present invention provides a kit for detecting DNA polymorphism of Yulmu and a kit for identifying varieties of yulmu including the SSR primer pair, DNA polymerase and PCR reaction buffer.

Hereinafter, the present invention will be described in detail.

The present invention is characterized by providing an SSR marker capable of specifically analyzing the genetic polymorphism of yulmu.

In the present invention, SSR markers were developed from Yulmu by the method of biotin-streptavidin capture (Dixit et al., Mol . Eco.Note, 5: 736-738). The SSR marker isolated from the silkworm is provided for the first time by the present invention.

Specifically, the SSR marker of the present invention was developed by the following method. Genomic DNA extracted from Yulmu was treated with restriction enzymes to prepare DNA fragments and then ligated with two adapters of AP11 and AP12.

Ligation products were hybridized with biotin-labeled SSR probes and then separated using magnetic beads. PCR was performed using the AP11 primer to amplify and analyze the sequence to obtain 500 DNA fragments containing Yulmu supersatellite.

By analyzing the DNA fragments obtained above, 200 pairs of bidirectional primers were designed to select only fragments containing five or more repeating units and to amplify the supersatellite based on the portion containing the repeating unit. 17 pairs of SSR primer pairs showing polymorphic variation were selected by performing DNA profiling through PCR of various cultivated yeast strains and varieties using the primer pairs.

The SSR marker provided by the present invention refers to a pair of PCR primers, and includes a forward primer and a reverse primer. SSR primer pair provided in the present invention has two base sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 34. Preferably, GBJT25 (primary pairs represented by SEQ ID NOs: 1 and 2), GBJT31 (primary pairs represented by SEQ ID NOs: 3 and 4), GBJT32 (primary pairs represented by SEQ ID NOs: 5 and 6), GBJT41 (SEQ ID NO: 7 And primer pairs represented by 8), GBJT68 (primary pairs represented by SEQ ID NOs: 9 and 10), GBJT130 (primary pairs represented by SEQ ID NOs: 11 and 12), and GBJT136 (primer pairs represented by SEQ ID NOs: 13 and 14) , GBJT149 (primary pairs represented by SEQ ID NOs: 15 and 16), GBJT157 (primary pairs represented by SEQ ID NOs: 17 and 18), GBJT161 (primary pairs represented by SEQ ID NOs: 19 and 20), GBJT164 (SEQ ID NOs 21 and 22) Primer pairs represented by SEQ ID NO. (Primary pair represented by SEQ ID NOs: 29 and 30), GBJT185 (SEQ ID NO: Primer pairs represented by Nos. 31 and 32) and GBJT198 (primary pairs represented by SEQ ID NOs: 33 and 34). Information on the primer pairs provided in the present invention and the repeat motifs present in the supersatellites amplified by them, the size of the supersatellites to be amplified, the Tm (° C.) and the chromosomal alleles in which the supersatellites are present are given below. It is shown in Table 1.

Characteristics of SSR Primer Pairs of the Present Invention and Supersatellites Amplified therefrom Primer Name Standing column SEQ ID NO: Repeat motif Range of size of supersatellite to be amplified (bp) Tm (℃) Number of alleles GBJT25 ^a F GCGCTCTGAAGACACCAC One (GCA) ₄ CCA (GCA) ₂ , (TGG) ₅ 262-278 58 3 ^b R CCGATGATCACCCTCCTT 2 GBJT31 F TGGATCCGGAGGAGAACT 3 (GCC) ₅ , (CCG) ₄ 267-279 58 3 R TCATATCGATGCTTGGGG 4 GBJT32 F GATGGATCGAGACAAGCG 5 (TGGCTGC) ₄ 263-270 58 3 R TGGAGGTGTGTGCCTACC 6 GBJT41 F CGCTTGAGGAAGCATCAC 7 (CTT) ₃ TTC (CTT) ₃ TTC (CTT) ₂ 238-244 50 3 R CCTACGTCATCTACGGCG 8 GBJT68 F GAGGCGCTTTGACACTTG 9 (CTCCTG) ₂ (CTC) ₄ 172-253 50 4 R TCACGGGATGATCCAAGA 10 GBJT130 F CCACCGTGTCTCTTTCCA 11 (GGC) ₆ 215-230 56 3 R CGCCATGAAACAGCTCTC 12 GBJT136 F GTGTGTGCCAGTGATCCA 13 (CATG) ₃ , (CGA) ₄ , (GAG) ₄ 205-289 56 4 R ATTCCCGGAACGACCTT 14 GBJT149 F AAGCAGAAGAACTCCGCC 15 (TTCAT) ₄ 230-279 58 5 R GGAATCGATGCAACCAAG 16 GBJT157 F CTGGGGCCAGGAAACTAC 17 (GCT) ₄ 194 58 One R CTCAGGAGCGATCAGACG 18 GBJT161 F ATTCGTTCGGGGATCAAG 19 (CAT) ₆ 188-194 58 3 R GCTGCATGCACATCACAC 20 GBJT164 F TTGTTTGCCTTCACCAGG 21 (CCTCCG) ₂ 195-203 58 2 R ACAGTGGAACGGTGGTTG 22 GBJT170 F CGGACGCCTGATGTGA 23 (CTG) ₁ T (CTG) ₂ CTC (CTG) ₁ T (CTG) ₄ 199-205 60 2 R CGTCTACGGTATCGGCTG 24 GBJT174 F CAGCGACAGCAGATCACA 25 (GAGGA) ₃ 212-297 58 2 R TAGCAGCAGCAGCTCAGG 26 GBJT181 F TCCACACAGCAACAACCA 27 (AG) ₁₃ 152-156 56 3 R CGTGCCAAGATCCAGAAG 28 GBJT183 F ATAGACAGGCAGCGGACA 29 (CGC) ₄ 207-210 56 2 R GTGGGTGAAGTTCCAGCA 30 GBJT185 F TCAAGCCAGCCAAAAGAA 31 (GGC) ₅ 248-277 58 3 R AGCCCTAACCCTAGGACG 32 GBJT198 F ATCTGTCGTCGTTGCTGC 33 (GAT) ₄ GAGGA (GGAC) ₃ 232-248 60 3 R CACGCACCTCCGACTC 34

^a F: forward primer ^b R: reverse primer

SSR primer pairs provided in the present invention can be usefully used for detecting DNA polymorphism and analyzing genetic diversity in the silkworm. The SSR primer according to the present invention can be used to efficiently evaluate and preserve the genetic resources of yulmu. Accordingly, the present invention provides a method for detecting DNA polymorphism of Yulmu, including performing PCR using the SSR primer pair.

Specifically, Yulmu's DNA polymorphism detection method

(a) extracting genomic DNA from yulmu;

(b) performing PCR using the extracted genomic DNA as a template and the SSR primer pair provided by the present invention; And

(c) separating the PCR products by size.

Extraction of genomic DNA in step (a) is performed by phenol / chloroform extraction method, SDS extraction method (Tai et al ., Plant Mol . Biol . Reporter , 8: 297-303, 1990), CTAB separation method ( Cetyl Trimethyl Ammonium Bromide; Murray et al. , Nuc . Res. , 4321-4325, 1980) or commercially available DNA extraction kits.

In addition, the PCR in the step (b) may be performed using a PCR reaction mixture containing a variety of components known in the art required for the PCR reaction. The PCR reaction mixture includes a suitable amount of DNA polymerase, dNTP, PCR buffer solution and water (dH ₂ O) in addition to genomic DNA extracted from Yulmu to be analyzed and the SSR primer pair provided in the present invention. The PCR buffer solution includes Tris-HCl, MgCl ₂ , KCl, and the like. At this time, the MgCl ₂ concentration greatly affects the specificity and yield of amplification, and may be preferably used in the range of 1.5-2.5 mM. In general, when Mg ^{2 +} is insufficient if the excess is increased nonspecific PCR amplification products and, Mg ^{2 +} PCR The yield of the product is reduced. The PCR buffer may further include an appropriate amount of Triton X-100. PCR also denatured the template DNA at 94-95 ° C., followed by denaturation; Annealing; And a cycle of extension, followed by general PCR reaction conditions which are finally elongated at 72 ° C. The denaturation and amplification may be performed at 94-95 ° C. and 72 ° C., respectively, and the temperature at the time of binding may vary depending on the type of primer. Preferably it is 52-57 degreeC, More preferably, it is 55 degreeC. The time and number of cycles in each step can be determined according to the conditions generally made in the art. The optimal reaction conditions when performing PCR using the SSR primer pair according to the present invention are as follows: After denature the template DNA for 3 minutes at 95 ℃, 30 seconds at 95 ℃; 30 seconds at 55 ° C .; And repeating 36 cycles of 1 minute 30 seconds at 72 ° C., and finally reacting at 72 ° C. for 5 minutes.

In step (c), DNAs of PCR products may be separated according to sizes according to methods well known in the art. Preferably it can be confirmed by an agarose gel (agarose gel) or polyacrylamide gel electrophoresis or fluorescence analysis (ABI prism 3100 genetic analyzer-electropherogram). At this time, in order to use the fluorescence analyzer PCR is performed in the step (b) using a primer pair attached to a fluorescent die known in the art. PCR amplification results can be preferably confirmed by polyacrylamide gel electrophoresis, more preferably modified polyacrylamide gel electrophoresis. Electrophoresis results can be analyzed by silver staining after electrophoresis. General PCR performance and resulting analysis methods are well known in the art.

In addition, the present invention provides a method for identifying varieties of yulmu, including performing PCR using the SSR primer pair.

Specifically, how to identify varieties of Yulmu

(a) extracting genomic DNA from the silkworm and control jelly varieties;

(b) performing PCR using each of the extracted genomic DNA as a template and the SSR primer pair provided by the present invention;

(c) separating each PCR product by size; And

(d) comparing the separation results for each size of step (c) with each other.

Genomic DNA extraction, PCR execution and separation of PCR products in steps (a) to (c) are as described above, and in step (d) the comparison of the yeast and control yeast varieties was carried out in the same combination of SSR primers. This is done by comparing the size of each PCR product of the yulmu, which is a sample for the pair, with the control yulmu variety. By comparing the size comparison results for each pair of SSR primers, if the results are consistent with the results of the yulmu and the control yulmu varieties, the yulmu as the sample can be identified as the same varieties as the yulmu control varieties. The identification method of the variety can be useful when the identification of the breed, such as judging whether there is a violation of the labeling of origin through judging whether the overlapping when introducing a new genetic resource and confirm the origin.

Genomic DNA extraction, PCR, and size separation of PCR products for the control yulmu variety may be performed simultaneously with the yulmu as a sample, but may be performed before the yulmu as a sample and prepared as a reference for identification. Using such a reference table can be useful because it can be compared with the reference table by performing genomic DNA extraction, PCR, and size separation of PCR products for the yulmu that becomes a sample.

The present invention also provides a kit for detecting DNA polymorphism of Yulmu containing the SSR primer pair according to the present invention. In addition, DNA polymerase and a PCR reaction buffer of the above-described composition may be additionally included in order to easily perform a PCR reaction, and components necessary for performing electrophoresis to confirm amplification of a PCR product are present. It may be further included in the kit of the invention.

The present invention also provides a kit for identifying varieties of yulmu containing SSR primer pairs according to the present invention. The breed identification method is as described above. In addition to the DNA polymerase and the PCR reaction buffer of the above-described composition in order to be able to easily perform the PCR reaction may be further included, the components or known to perform electrophoresis to confirm whether the PCR product amplification or not Identification criteria for selected varieties may be further included in the kits of the present invention.

The rule of thumb which can be applied to the present invention is Cokes Guatea J. Coix gigantea J. Konig ex Roxb., Coix Lacrima-Jobie var. GIANTTEA (J. Conic X Roxb .) Staff lachryma - jobi var. gigantea (J. Konig ex Roxb.) Stapf), Cokes Lacrima-Jobie var. Meiyuan staff ( Coix lachryma -jobi var. mayuen Stapf) and variants thereof, but is not limited thereto. Preferably Cokes Lacrima-Jobie var. May be Coix lachryma-jobi var. Mayuen Stapf.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Example  1> Genomic DNA Extraction

Genomic DNA was extracted from the cultivar Yulmu (Genetic Resources Research Institute of Agricultural Biotechnology, Conservation No .: K040438) using SDS (Tai et al ., Plant Mol . Biol . Reporter , 8: 297-303, 1990). This will be described in detail as follows. 0.5 g of leaf tissues were taken from young juvenile radish plants at 1 month of growth and placed in a 1.5 ml tube and quenched with liquid nitrogen. The leaf was then ground finely with a plastic rod immediately. Before the ground leaves were dissolved, 750 μl of extraction buffer (200 mM Tris-HCl, pH 8.0), 200 mM NaCl, 25 mM EDTA, 0.5% SDS) was added and the mixture was stored at 65 ° C. for 30 minutes. An equivalent amount of a phenol (chloro): chloroform: isoamylalchol (25: 24: 1) solution was further added thereto, followed by stirring for about 15 minutes. It was centrifuged for 15 minutes at 13,000 rpm. The supernatant was taken and transferred to a new tube. Equal amount of isopropanol (-20 ° C.) was added to the tube and stored at −20 ° C. for 1 hour. Thereafter, the mixture was centrifuged at 13,000 rpm for 15 minutes. After discarding the supernatant, the precipitated DNA was washed with 70% ethanol. After the DNA was dried, it was sufficiently dissolved by adding TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 7.4) containing about 300 µl of RNase (50 µg / ml), followed by 1 hour at 37 ° C. Stored.

< Example  2> With adapter Ligation (ligation)

500 ng of genomic DNA obtained in Example 1 was digested with Eco RV, Dra I, Sma I, Pvu I, Alu I, Hae III and Rsa I according to the instructions of each restriction enzyme manufacturer. After cleaved DNA was electrophoresed in a 1.2% agarose gel, only DNA fragments of about 300-1500 bp size were obtained by elution from the gel. The DNA fragments obtained were then ligated overnight at 12 ° C. using two adapters (AP-11 and AP-12, 50 ng each) and a T4 DNA ligase (Promega, USA). At this time, the AP-12 adapter phosphorylated the 5 'end with alkaline phosphatase (manufacturer and manufacturer), and the composition of the reaction solution during ligation is as follows: 1X ligation buffer (Promega, USA), 50 ng AP11 adapter, 50 ng AP12 adapter, 150 ng DNA fragment, 3 Unit T4 ligase (Promega, USA).

Adapter used in the present invention Adapter name Sequence SEQ ID NO: AP-11 5'-CTCTTGCTTAGATCTGGACTA-3 ' 35 AP-12 5'-TAGTCCAGATCTAAGCAAGAGCACA-3 ' 36

< Example  3> Supersatellite  Selection and Separation of Containing DNA Fragments

<3-1> Magnetic Bead Preparation

The solution was removed after collecting 0.6 ml of magnetic beads (SA-PMPs, Promega, USA; 1 mg / ml) with a magnetic stand (Promega, USA). 0.6 ml of 0.5x Saline-Sodium Citrate (SSC) buffer was added thereto, washed, and collected again using a magnetic separator to remove the solution. Washing with 0.5 × SSC buffer was performed three times. 100 μl of 0.5 × SSC buffer was added to the washed magnetic beads and used within 30 minutes.

<3-2> hybridization

Distilled water was added to the ligation product prepared in Example 2 to 500 μl, and then denatured at 65 ° C. for 10 minutes. Biotin-labeled SSR probe 1.5 μl (20ng) and 20 μl SSC buffer 13 μl were added thereto and left for 10 minutes at room temperature to induce binding. The eight SSR probes used for hybridization are listed in Table 3 below, and the SSR probes were labeled with biotin according to conventional methods known in the art.

Sequence of probe for the separation of Yulmu supersatellite order SEQ ID NO: Biotin- (GA) ₂₀ 37 Biotin- (AT) ₂₀ 38 Biotin- (CA) ₂₀ 39 Biotin- (AGC) ₁₅ 40 Biotin- (GGC) ₁₅ 41 Biotin- (AAG) ₁₅ 42 Biotin- (AAC) ₁₅ 43 Biotin- (AGG) ₁₅ 44

Through hybridization reaction, DNA fragments having the repeat base sequence of genomic DNA fragments will complementarily bind to biotin-labeled SSR probes, which are called SSR probe-template hybrids.

<3-3> Separation of Template

The magnetic bead solution prepared in <3-1> was placed in the SSR probe-template hybrid solution in <3-2> and allowed to stand at room temperature for 10 minutes. Magnetic beads in which the SSR probe-template hybrid was bound were collected by using a magnetic separator in the reaction solution, and the supernatant was removed. The magnetic beads combined with the SSR probe-template hybrid were washed four times with 300 μl of 0.1 × SSC, and then 50 μl of distilled water was added and heated at 90 ° C. for 5 minutes to separate the template.

<3-4> PCR reaction

The PCR reaction was performed using the AP-11 adapter as a primer on the template isolated in <3-3>. The composition of the PCR reaction mixture (20 μl) is as follows: 250 ng adapter-ligated DNA, 10 pmol AP11 primer, 2 mM dNTP, 2 units Taq DNA polymerase, 5 μl 10 × buffer. PCR reactions were heated at 72 ° C. for 2 minutes, 94 ° C. for 3 minutes, and then at 94 ° C. for 30 seconds; 30 seconds at 56 ° C .; And 1 minute at 72 ° C. for 24 cycles, and finally at 72 ° C. for 10 minutes.

In this manner, in the present invention, 500 DNA fragments containing supersatellites were isolated.

< Example  4> Sequencing of isolated DNA fragments and primer  design

DNA fragments containing the supersatellite isolated in Example 3 were cloned into pGEM T-easy vector vector (Promega, USA) to analyze their sequence. Sequence analysis was performed by requesting from Genotech. Only fragments containing 5 or more repeat units were selected based on the analyzed sequences. Based on the part containing the repeating unit, a primer in both directions was designed to amplify the supersatellite. A total of 200 pairs of primer pairs were designed (results not shown).

< Example  5> SSR Of marker  Selection

Among the primer pairs designed in Example 4, in order to select SSR primer combinations showing a lot of polymorphic variation in various yeast strains, DNA profiling through PCR was carried out for the 30-point cultivated yeast or yeast varieties of Table 4 below. profiling) analysis was performed.

Rate used in SSR analysis Deposit number Depositary Institution Collection country (country of origin) Type K040438 Agriculture Promotion Agency Republic of Korea Coix lachryma - jobi var. mayuen Stapf K040439 Agriculture Promotion Agency Republic of Korea Coix lachryma - jobi var. mayuen Stapf K040442 Agriculture Promotion Agency Republic of Korea Coix lachryma - jobi var. mayuen Stapf K040450 Agriculture Promotion Agency Republic of Korea Coix lachryma - jobi var. mayuen Stapf K040458 Agriculture Promotion Agency Republic of Korea Coix lachryma - jobi var. mayuen Stapf K040466 Agriculture Promotion Agency Republic of Korea Coix lachryma - jobi var. mayuen Stapf K040468 Agriculture Promotion Agency Republic of Korea Coix lachryma - jobi var. mayuen Stapf K040473 Agriculture Promotion Agency Republic of Korea Coix lachryma - jobi var. mayuen Stapf K040477 Agriculture Promotion Agency Republic of Korea Coix lachryma - jobi var. mayuen Stapf K040480 Agriculture Promotion Agency Republic of Korea Coix lachryma - jobi var. mayuen Stapf GXY-01 Agriculture Promotion Agency China Coix lachryma - jobi var. mayuen Stapf GXY-03 Agriculture Promotion Agency China Coix lachryma - jobi var. mayuen Stapf GXY-08 Agriculture Promotion Agency China Coix lachryma - jobi var. mayuen Stapf GXY-09 Agriculture Promotion Agency China Coix lachryma - jobi var. mayuen Stapf GXY-39 Agriculture Promotion Agency China Coix lachryma - jobi var. mayuen Stapf GXY-40 Agriculture Promotion Agency China Coix lachryma - jobi var. mayuen Stapf GXY-48 Agriculture Promotion Agency China Coix lachryma - jobi var. mayuen Stapf GXY-49 Agriculture Promotion Agency China Coix lachryma - jobi var. mayuen Stapf GXY-58 Agriculture Promotion Agency China Coix lachryma - jobi var. mayuen Stapf GXY-59 Agriculture Promotion Agency China Coix lachryma - jobi var. mayuen Stapf GXY-63 Agriculture Promotion Agency China Coix lachryma - jobi var. mayuen Stapf GXY-67 Agriculture Promotion Agency China Coix lachryma - jobi var. mayuen Stapf GXY-80 Agriculture Promotion Agency China Coix lachryma - jobi var. mayuen Stapf JP197 Agriculture Promotion Agency Japan Coix lachryma - jobi var. mayuen Stapf JP199 Agriculture Promotion Agency Japan Coix lachryma - jobi var. mayuen Stapf JP202 Agriculture Promotion Agency Japan Coix lachryma - jobi var. mayuen Stapf JP203 Agriculture Promotion Agency Japan Coix lachryma - jobi var. mayuen Stapf JP83417 Agriculture Promotion Agency Japan Coix lachryma - jobi var. mayuen Stapf JP83418 Agriculture Promotion Agency Japan Coix lachryma - jobi var. mayuen Stapf JP83428 Agriculture Promotion Agency Japan Coix lachryma - jobi var. mayuen Stapf

The composition of each PCR reaction mixture (20 [mu] l) was as follows: Yulmu template DNA isolated by the method of Example 1 40 ng, forward primer 0.2 [mu] M, reverse primer 0.6 [mu] M, fluorescently labeled M13 primer (TGTAAAACGACGGCCAGT) 0.6 μM, 1 × PCR reaction solution (10 mM Tris-HCl, pH 8.8), 1.5 mM MgCl ₂ , 50 mM KCl, 0.1% Triton X-100), 1 unit DNA polymerase. PCR reaction was performed after denaturing the template DNA for 3 minutes at 94 ℃, 30 seconds at 94 ℃; 30 seconds at the temperature corresponding to the Tm of each primer (see Table 1); And 35 repetitions under conditions of 45 seconds at 72 ° C., and then 30 seconds at 94 ° C .; 45 sec at 53 ° C .; And 15 repetitions at 45 ° C. for 45 seconds, and finally at 72 ° C. for 15 minutes.

An automated sequencing device (ABI 3100 Genetic Analyzer, Applied Biosystems, USA) was used to analyze the size of PCR product fragments amplified with different fluorescent materials. For the analysis, 1.5 μl of PCR product, 9.2 μl of Hi-di formamide, and 0.3 μl of internal size standard (Genscan-500 ROX) were mixed and denatured at 94 ° C. for 3 minutes. It was.

As a result, we identified 17 pairs of SSR primer pairs showing polymorphism in the yeast cultivars and yeast varieties collected domestically and internationally, and then GBJT25 (primary pairs represented by SEQ ID NOs: 1 and 2) and GBJT31 (SEQ ID NOs: 3 and 4). Primer pairs shown), GBJT32 (primer pairs shown by SEQ ID NOs: 5 and 6), GBJT41 (primer pairs shown by SEQ ID NOs: 7 and 8), GBJT68 (primer pairs shown by SEQ ID NOs: 9 and 10), and GBJT130 ( Primer pairs represented by SEQ ID NOs: 11 and 12), GBJT136 (primer pairs represented by SEQ ID NOs: 13 and 14), GBJT149 (primer pairs represented by SEQ ID NOs: 15 and 16), GBJT157 (SEQ ID NOs: 17 and 18) Primer pairs), GBJT161 (primary pairs represented by SEQ ID NOs: 19 and 20), GBJT164 (primary pairs represented by SEQ ID NOs: 21 and 22), GBJT170 (primary pairs represented by SEQ ID NOs: 23 and 24), GBJT174 (SEQ ID NO: Primer pairs represented by 25 and 26), GBJT181 ( Primer pairs represented by SEQ ID NOs: 27 and 28, GBJT183 (primer pairs represented by SEQ ID NOs: 29 and 30), GBJT185 (primer pairs represented by SEQ ID NOs: 31 and 32), and GBJT198 (SEQ ID NOs: 33 and 34) Primer pairs) were selected (see Table 1). The size of each PCR fragment amplified by the 17 pairs of primers is shown in Table 5 below.

Kinds and Sizes of Amplified Supersatellites According to Primers K040438 K040439 K040442 K040450 K040458 K040466 K040468 K040473 K040477 K040480 GBJT25 278 278 278 278 278 278 278 278 278 278 GBJT31 279 279 279 279 279 279 279 279 279 279 GBJT32 267 267 267 267 267 267 267 267 267 267 GBJT41 238 238 238 238 238 238 238 238 238 238 GBJT68 253 253 253 253 253 172 253 253 253 253 GBJT130 215 221 221 215 230 215 221 215 215 215 GBJT136 282 282 282 282 282 278 282 282 282 282 GBJT149 241 230 194 241 241 241 241 241 241 241 GBJT157 194 194 194 194 194 194 194 194 194 194 GBJT161 191 191 191 188 191 191 191 188 191 191 GBJT164 195 195 195 195 195 195 195 195 195 195 GBJT170 205 205 205 205 205 205 205 205 205 205 GBJT174 297 212 212 212 212 212 212 297 297 212 GBJT181 154 154 154 154 154 154 154 154 152 154 GBJT183 210 210 210 210 210 210 210 210 210 210 GBJT185 248 248 248 248 248 248 248 248 248 248 GBJT198 232 232 248 232 232 232 232 232 232 232

GXY-01 GXY-03 GXY-08 GXY-09 GXY-39 GXY-40 GXY-48 GXY-49 GXY-58 GXY-59 GBJT25 275 275 275 278 278 275 275 275 275 275 GBJT31 267 267 267 279 267 267 279 279 270 279 GBJT32 270 270 270 263 263 270 270 270 263 270 GBJT41 238 238 238 238 238 238 238 238 238 238 GBJT68 244 244 244 244 253 244 241 172 172 172 GBJT130 221 215 215 221 215 215 215 215 215 215 GBJT136 282 282 282 282 282 282 289 282 282 282 GBJT149 274 274 194 274 274 274 274 274 274 274 GBJT157 194 194 194 194 194 194 194 194 194 194 GBJT161 188 188 188 188 188 188 194 194 188 194 GBJT164 195 195 195 195 195 195 195 195 195 195 GBJT170 205 205 205 205 205 205 199 205 199 205 GBJT174 212 212 212 212 212 212 212 212 212 212 GBJT181 156 156 154 154 154 154 156 156 152 156 GBJT183 207 207 207 207 207 207 207 210 207 210 GBJT185 277 277 248 248 248 277 277 277 277 277 GBJT198 232 232 232 232 235 232 235 232 235 232

GXY-63 GXY-67 GXY-80 JP197 JP199 JP202 JP203 JP83417 JP83418 JP83428 GBJT25 275 275 275 262 278 278 278 278 278 278 GBJT31 270 270 279 279 279 279 279 279 279 279 GBJT32 270 270 270 267 267 267 267 267 267 267 GBJT41 241 238 238 241 238 238 238 238 238 244 GBJT68 172 244 172 253 253 253 253 253 253 253 GBJT130 221 215 215 230 221 215 221 215 215 221 GBJT136 282 282 282 205 282 282 282 205 282 282 GBJT149 274 274 274 241 241 279 241 194 194 241 GBJT157 194 194 194 194 194 194 194 194 194 194 GBJT161 188 188 188 188 188 188 194 194 188 194 GBJT164 203 195 195 195 195 195 195 195 195 195 GBJT170 205 205 205 205 205 205 205 205 205 205 GBJT174 212 212 212 297 212 212 212 297 212 212 GBJT181 156 156 154 154 154 154 156 156 152 156 GBJT183 207 207 207 207 207 207 207 210 207 210 GBJT185 248 277 277 248 248 248 251 248 248 248 GBJT198 232 232 232 232 232 232 232 235 232 232

As described above, in the present invention, the first SSR marker was developed in Yulmu. The SSR primer pair provided in the present invention can be very useful for effectively detecting the DNA polymorphism of yulmu and preparing the DNA profile of yulmu. Through efficient evaluation of the genetic resources of Yulmu, it is possible to effectively carry out analysis of redundancy with existing resources and establish novelty when introducing new genetic resources, and to identify varieties of yulmu.

Attach sequence list electronic file

Claims (7)

Primer pairs represented by SEQ ID NOs: 1 and 2; Primer pairs represented by SEQ ID NOs: 3 and 4; Primer pairs represented by SEQ ID NOs: 5 and 6; Primer pairs represented by SEQ ID NOs: 7 and 8; Primer pairs represented by SEQ ID NOs: 9 and 10; Primer pairs represented by SEQ ID NOs: 11 and 12; Primer pairs represented by SEQ ID NOs: 13 and 14; Primer pairs represented by SEQ ID NOs: 15 and 16; Primer pairs represented by SEQ ID NOs: 17 and 18; Primer pairs represented by SEQ ID NOs: 19 and 20; Primer pairs represented by SEQ ID NOs: 21 and 22; Primer pairs represented by SEQ ID NOs: 23 and 24; Primer pairs represented by SEQ ID NOs: 25 and 26; Primer pairs represented by SEQ ID NOs: 27 and 28; Primer pairs represented by SEQ ID NOs: 29 and 30; Primer pairs represented by SEQ ID NOs: 31 and 32; And an SSR primer pair selected from the group consisting of primer pairs represented by SEQ ID NOs: 33 and 34. delete The SSR primer pair according to claim 1, which is derived from Coix lachryma-jobi . (a) extracting genomic DNA from yulmu; (b) using the extracted genomic DNA as a template and performing PCR using the SSR primer pair of claim 1; And (C) Yulemu DNA polymorphism detection method comprising the step of separating the PCR product by size. (a) extracting genomic DNA from the silkworm and control jelly varieties; (b) using each extracted genomic DNA as a template and performing PCR using the SSR primer pair of claim 1; (c) separating each PCR product by size; And (d) a method of identifying varieties of yulmu comprising the step of comparing the results of the separation by size of the yulmu and control yulmu varieties. Yulmu's DNA polymorphism detection kit comprising the SSR primer pair of claim 1. A kit for identifying varieties of yulmu comprising the SSR primer pair of claim 1.