SSR marker primer combinations and their applications for identification of Leymus chinensis germplasm resources

Abstract

The present invention discloses an SSR marker primer combination for identifying Leymus chinensis germplasm resources and its application. The present invention provides an SSR marker primer combination for identifying Leymus chinensis germplasm resources, which consists of 20 pairs of SSR primers. The present invention also provides an application of the SSR marker primer combination for identifying Leymus chinensis germplasm resources in constructing a molecular identity card of Leymus chinensis germplasm resources and identifying Leymus chinensis germplasm resources, and identifies 105 Leymus chinensis germplasm resources through the constructed molecular identity card of grass germplasm resources. The present invention provides a technical basis for the protection of Leymus chinensis germplasm resources, the authenticity identification of varieties, the identification of the kinship of germplasm resources and biological breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of plant germplasm resource identification, and specifically relates to an SSR marker primer combination for identifying Leymus chinensis germplasm resources and an application thereof. Background Art

[0002] Leymus chinensis, a member of the genus Leymus in the Poaceae family, is a dominant species in my country's meadow steppes and typical grasslands. The total area of ​​​​Leymus chinensis in the world is 420,000 km 2 , of which about 50% are found in my country, and it is an important native grass species in my country. Leymus chinensis is cold-resistant (-47.5℃), drought-resistant, salt-alkali-resistant, trample-resistant, has high nutritional value and good palatability. It is not only an important wild fine forage grass in grassland areas, but also an ideal grass species for artificial grassland construction, grassland ecological restoration and salinized grassland management, and has important ecological functions.

[0003] The habitat in the distribution area of ​​Leymus chinensis is complex, the intraspecific variation diversity is high, and there are obvious differences in leaf color and yield traits. The traditional identification of Leymus chinensis germplasm resources mainly relies on the morphological differences of nutrition and reproductive growth, which is easily affected by environmental factors, and it is difficult to accurately identify germplasm resources by leaves and plant type before flowering. With the continuous deepening of Leymus chinensis germplasm resources and breeding work, the number of germplasm resources and the number of varieties have gradually increased, and the problems of resource purity and variety authenticity have become increasingly serious. However, since there is no unified and standardized resource and variety identification standard, the phenomenon of mixed or fake varieties in the market has brought great troubles to scientific research, production and seed conservation. With the continuous development of molecular biology, DNA molecular marker technology has been widely used in genetic evaluation, biodiversity variation, genetic map construction, molecular marker-assisted breeding, and whole genome selection breeding due to its characteristics of rapidity, accuracy, and large amount of information. In recent years, with the rapid development of high-throughput sequencing technology and the development of various bioinformatics software, the development of molecular markers has become more convenient.

[0004] SSR (Simple Sequence Repeats) is a simple sequence repeat. It refers to a tandem repeat sequence of dozens of nucleotides consisting of 1-6 nucleotides as repeating units in the genome. It is widely distributed in the genome of eukaryotic organisms. Due to the different number of repetitions of the repeat sequence, polymorphism between alleles is generated. The two ends of SSR are mostly relatively conservative single-copy sequences, which can be amplified by PCR by designing primers. SSR markers are an ideal molecular marker with co-dominance, high polymorphism, abundant quantity, uniform distribution, and easy detection. It overcomes the shortcomings of RFLP and RAPD markers, and fully meets the four basic criteria for germplasm resource identification: environmental stability, identifiability of inter-germplasm variation, minimum intra-variety variation, and stable reliability of experimental results. It can also detect genetic variation in organisms efficiently and accurately. Therefore, the development of SSR molecular marker primer combinations using the genome sequence of Leymus chinensis and the construction of a molecular ID card for Leymus chinensis play an important role in the identification of Leymus chinensis germplasm resources, variety identification, hybrid identification, kinship analysis, QTL positioning, and molecular marker-assisted breeding.

[0005] At present, there are reports on the use of SSR markers to construct molecular ID cards of germplasm resources in the identification of rice, corn, and wheat germplasm resources, but there are no reports on the SSR molecular marker primer combination of Leymus chinensis and the construction of a molecular ID card of Leymus chinensis by the SSR molecular marker primer combination in the prior art. There is an urgent need in the prior art for a Leymus chinensis SSR marker primer combination and the application of the primer combination to construct a molecular ID card of Leymus chinensis, so as to quickly, accurately, and efficiently identify Leymus chinensis germplasm resources. Summary of the invention

[0006] The first purpose of the present invention is to overcome the shortcomings of the prior art and provide an SSR marker primer combination for identifying Leymus chinensis germplasm resources.

[0007] The first object of the present invention is achieved by the following technical scheme: an SSR marker primer combination for identifying Leymus chinensis germplasm resources, the SSR marker primer combination consists of 20 pairs of SSR primers, and the nucleotide sequences of the SSR primers are shown in the following table:

[0008] Primer Name Forward Primer Reverse Primer GW01-3 CAGGAGTGCACTTGCATGG (SEQ ID NO:1) ATTTCAGAACAACCGTTGGC (SEQ ID NO:2) GW01-5 ATCACGTCTTGCATGCTCTG (SEQ ID NO:3) AAGCAGTTCTCTCGTCCAGC (SEQ ID NO:4) GW01-6 CTTGATCCAAGGCCAGGTTA (SEQ ID NO:5) GCTATGTTATGGCGCCTTGT (SEQ ID NO:6) GW02-3 CCTTTCACCTTCGAATCCAG (SEQ ID NO:7) TCGTGAAGTTGTCACAAGTGG (SEQ ID NO:8) GW02-6 CGAGTTCTTGCAACTGGACA (SEQ ID NO:9) GAAGGACCGGAATATCACCA (SEQ ID NO:10) GW03-1 AATCGACGACCTTCCATACG (SEQ ID NO:11) ATGGCCACTCGTATGCTTTC (SEQ ID NO:12) GW03-7 CAATTAGGCACTTCCGTTGG (SEQ ID NO:13) GCACTGCTGGCATAAAGGTT (SEQ ID NO:14) GW13-3 TCCGTGTAAGGAGACCATCC (SEQ ID NO:15) GCTCCGTAAGAGCAGTGAGG (SEQ ID NO:16) GW05-2 GGCTAGTTGCTACTCTGCGG (SEQ ID NO:17) CACGAAAGCAACACACATCC (SEQ ID NO:18) GW13-5 CTTGCTATCCATGTGCCAGA (SEQ ID NO:19) AACGGAGTGCTCATCAGCTT (SEQ ID NO:20) GW06-2 GGCGGACTAGTAAGTGCTGG (SEQ ID NO:21) GGATGAGGACGATGCTGATT (SEQ ID NO:22) GW06-3 GCAACTCTCTCTGACGGTCC (SEQ ID NO:23) GGTGCCTGGTTCAATAGGAA (SEQ ID NO:24) GW06-6 AACGCCGAACTAGAGAATGC (SEQ ID NO:25) CTTCGGTATTACGCTCCGAC (SEQ ID NO:26) GW06-7 TGAACAAGGAATGTGCAAGG (SEQ ID NO:27) CCATCATATCAGCGTTGTGG (SEQ ID NO:28) GW07-1 TAGCTCGTCGACCATTAGGC (SEQ ID NO:29) GAAGCAGAAGACTGCATCGC (SEQ ID NO:30) GW07-4 TCTGTAGCTCAATCATGGCG (SEQ ID NO:31) TAAGATGCTCCGGTGTTTCC (SEQ ID NO:32) GW13-7 TTTGATCTCGTGATGATGGC (SEQ ID NO:33) CCGAGCTCTGAAAACCTGTTC (SEQ ID NO:34) GW09-6 CAGAAACAAGACACGAGGCA (SEQ ID NO:35) TGATCTGCTGCAATGTCTCC (SEQ ID NO:36) GW11-7 GGTGAAGAAGCGACTGAAGG (SEQ ID NO:37) TCCATGTGCCAGAACCATTA (SEQ ID NO:38) GW12-4 GGCCTGTTCTGGTACTCTGC (SEQ ID NO:39) TACTTGCAGGTTGCAGTTGG (SEQ ID NO:40)

[0009] The 5' ends of the forward primer labeled GW01-3, the reverse primer labeled GW01-3, the forward primer labeled GW01-5, the reverse primer labeled GW01-5, the forward primer labeled GW06-6, the reverse primer labeled GW06-6, the forward primer labeled GW07-4, the reverse primer labeled GW07-4, the forward primer labeled GW12-4, and the reverse primer labeled GW12-4 are connected with a fluorescent label NED;

[0010] The 5' ends of the forward primer labeled GW01-6, the reverse primer labeled GW01-6, the forward primer labeled GW03-7, the reverse primer labeled GW03-7, the forward primer labeled GW13-5, the reverse primer labeled GW13-5, the forward primer labeled GW06-3, the reverse primer labeled GW06-3, the forward primer labeled GW09-6, and the reverse primer labeled GW09-6 are connected with a fluorescent label FAM;

[0011] The 5' ends of the forward primer labeled GW02-3, the reverse primer labeled GW02-3, the forward primer labeled GW03-1, the reverse primer labeled GW03-1, the forward primer labeled GW13-3, the reverse primer labeled GW13-3, the forward primer labeled GW06-7, the reverse primer labeled GW06-7, the forward primer labeled GW11-7, and the reverse primer labeled GW11-7 are connected with a fluorescent label ROX;

[0012] The 5' ends of the forward primer labeled GW02-6, the reverse primer labeled GW02-6, the forward primer labeled GW05-2, the reverse primer labeled GW05-2, the forward primer labeled GW06-2, the reverse primer labeled GW06-2, the forward primer labeled GW07-1, the reverse primer labeled GW07-1, the forward primer labeled GW13-7, and the reverse primer labeled GW13-7 are connected to a fluorescent label HEX.

[0013] The second purpose of the present invention is to overcome the shortcomings of the prior art and provide an SSR marker primer combination for the identification of Leymus chinensis germplasm resources for use in the construction of a molecular identity card for Leymus chinensis germplasm resources and the identification of Leymus chinensis germplasm resources.

[0014] The beneficial effects of the present invention are as follows: the present invention provides an SSR marker primer combination consisting of 20 pairs of SSR primer markers, and constructs a molecular identity card for grass germplasm resources, thereby identifying 105 Leymus chinensis germplasm resources. The present invention provides a technical basis for the protection of Leymus chinensis germplasm resources, the authenticity identification of varieties, the identification of germplasm resource kinship, and biological breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the capillary electrophoresis of the PCR amplification product of A01 using fluorescent GW02-3 primers;

[0016] Figure 2 is the capillary electrophoresis of the PCR amplification product of A01 using the fluorescent GW06-6 primer pair;

[0017] Figure 3is the capillary electrophoresis of the PCR amplification product of A01 using fluorescent GW03-7 primers;

[0018] Figure 4 is the capillary electrophoresis of the PCR amplification product of A01 using the fluorescent GW07-4 primer pair;

[0019] Figure 5 This is the capillary electrophoresis diagram of the PCR amplification product of A01 using fluorescent GW13-5 primer pair. DETAILED DESCRIPTION

[0020] The present invention is described in detail below with reference to the accompanying drawings.

[0021] Example Development of 20 pairs of SSR primer markers and construction of a molecular ID card for Leymus chinensis germplasm resources

[0022] 1. Collection of Leymus chinensis germplasm resources

[0023] The 105 Leymus chinensis germplasm resources and new varieties collected were from Mongolia and Inner Mongolia, Heilongjiang, Jilin, Liaoning, Hebei, Shanxi and other places in my country. These 105 Leymus chinensis germplasm materials were planted and preserved in the National Perennial Forage Germplasm Resource Garden (Hohhot). The germplasm resource numbers and specific sources of the 105 Leymus chinensis are shown in Table 1.

[0024] Table 1 Number and source of Leymus chinensis germplasm resources

[0025] serial number source serial number source A01 Shuozhou City, Shanxi Province A54 Zhangjiakou City, Hebei Province A02 National Northern Forage Germplasm Resource Bank (Hohhot) A55 National Northern Forage Germplasm Resource Bank (Hohhot) A03 National Northern Forage Germplasm Resource Bank (Hohhot) A56 Tongliao City, Inner Mongolia Autonomous Region A04 Lvliang City, Shanxi Province A57 Bulgan Province, Mongolia A05 National Northern Forage Germplasm Resource Bank (Hohhot) A58 Haixi Mongolian and Tibetan Autonomous Prefecture, Qinghai Province A06 Kent Province, Mongolia A59 Xilin Gol League, Inner Mongolia Autonomous Region A07 Linfen City, Shanxi Province A60 Hulunbuir City, Inner Mongolia Autonomous Region A08 Kent Province, Mongolia A61 National Northern Forage Germplasm Resource Bank (Hohhot) A09 National Northern Forage Germplasm Resource Bank (Hohhot) A62 Jilin City, Jilin Province A10 National Northern Forage Germplasm Resource Bank (Hohhot) A63 Xilin Gol League, Inner Mongolia Autonomous Region A11 Xilin Gol League, Inner Mongolia Autonomous Region A64 Xilin Gol League, Inner Mongolia Autonomous Region A12 National Northern Forage Germplasm Resource Bank (Hohhot) A65 Hulunbuir City, Inner Mongolia Autonomous Region A13 Xilin Gol League, Inner Mongolia Autonomous Region A66 Eastern Province of Mongolia A14 Sukhbaatar Province, Mongolia A67 Central Province of Mongolia A15 National Northern Forage Germplasm Resource Bank (Hohhot) A68 National Northern Forage Germplasm Resource Bank (Hohhot) A16 Eastern Province of Mongolia A69 Kent Province, Mongolia A17 Jinzhou City, Liaoning Province A70 Sukhbaatar Province, Mongolia A18 Hulunbuir City, Inner Mongolia Autonomous Region A71 Tongliao City, Inner Mongolia Autonomous Region A19 Linfen City, Shanxi Province A72 National Northern Forage Germplasm Resource Bank (Hohhot) A20 Hulunbuir City, Inner Mongolia Autonomous Region A73 Hulunbuir City, Inner Mongolia Autonomous Region A21 Jinzhou City, Liaoning Province A74 Xilin Gol League, Inner Mongolia Autonomous Region A22 Eastern Province of Mongolia A75 Xilin Gol League, Inner Mongolia Autonomous Region A23 National Northern Forage Germplasm Resource Bank (Hohhot) A76 Eastern Province of Mongolia A24 Songyuan City, Jilin Province A77 Siping City, Jilin Province A25 Hulunbuir City, Inner Mongolia Autonomous Region A78 Arkhangai Province, Mongolia A26 Eastern Province of Mongolia A79 Zhangjiakou City, Hebei Province A27 Shihezi City, Xinjiang Uygur Autonomous Region A80 Xilin Gol League, Inner Mongolia Autonomous Region A28 Baicheng City, Jilin Province A81 Xilin Gol League, Inner Mongolia Autonomous Region A29 National Northern Forage Germplasm Resource Bank (Hohhot) A82 Linfen City, Shanxi Province A30 National Northern Forage Germplasm Resource Bank (Hohhot) A83 National Northern Forage Germplasm Resource Bank (Hohhot) A31 National Northern Forage Germplasm Resource Bank (Hohhot) A84 Jilin City, Jilin Province A32 Chifeng City, Inner Mongolia Autonomous Region A85 Hulunbuir City, Inner Mongolia Autonomous Region A33 Linfen City, Shanxi Province A86 Kent Province, Mongolia A34 Linfen City, Shanxi Province A87 Xing'an League, Inner Mongolia Autonomous Region A35 Eastern Province of Mongolia A88 National Northern Forage Germplasm Resource Bank (Hohhot) A36 National Northern Forage Germplasm Resource Bank (Hohhot) A89 Mongolian Province A37 Hulunbuir City, Inner Mongolia Autonomous Region A90 Tacheng Prefecture, Xinjiang Uygur Autonomous Region A38 National Northern Forage Germplasm Resource Bank (Hohhot) A91 National Northern Forage Germplasm Resource Bank (Hohhot) A39 Mongolian Province A92 National Northern Forage Germplasm Resource Bank (Hohhot) A40 Xilin Gol League, Inner Mongolia Autonomous Region A93 Yan'an City, Shaanxi Province A41 Hulunbuir City, Inner Mongolia Autonomous Region A94 National Northern Forage Germplasm Resource Bank (Hohhot) A42 Xilin Gol League, Inner Mongolia Autonomous Region A95 National Northern Forage Germplasm Resource Bank (Hohhot) A43 National Northern Forage Germplasm Resource Bank (Hohhot) A96 National Northern Forage Germplasm Resource Bank (Hohhot) A44 Hulunbuir City, Inner Mongolia Autonomous Region A97 National Northern Forage Germplasm Resource Bank (Hohhot) A45 Changchun City, Jilin Province A98 National Northern Forage Germplasm Resource Bank (Hohhot) A46 Linfen City, Shanxi Province A99 National Northern Forage Germplasm Resource Bank (Hohhot) A47 Xilin Gol League, Inner Mongolia Autonomous Region A100 National Northern Forage Germplasm Resource Bank (Hohhot) A48 Chifeng City, Inner Mongolia Autonomous Region A101 New varieties of Leymus chinensis from West Ujimqin A49 Xilin Gol League, Inner Mongolia Autonomous Region A102 New strains A50 National Northern Forage Germplasm Resource Bank (Hohhot) A103 New strains A51 National Northern Forage Germplasm Resource Bank (Hohhot) A104 New strains A52 Hulunbuir City, Inner Mongolia Autonomous Region A105 New strains A53 National Northern Forage Germplasm Resource Bank (Hohhot)

[0026] 2. DNA extraction of Leymus chinensis germplasm resources

[0027] Young leaves of Leymus chinensis were collected, and each sample was divided into 3 parts and stored in 2 mL centrifuge tubes. After quick freezing in liquid nitrogen, they were frozen in a -80 °C refrigerator for later use. After being taken out and ground, 105 parts of Leymus chinensis genomic DNA were extracted using a DNA kit (Tiangen kit), detected by electrophoresis on 1% agarose gel, and the DNA concentration was determined by a spectrophotometer.

[0028] 3. Development and screening of SSR primer markers for Leymus chinensis

[0029] According to the reference sequence of the whole genome of Leymus chinensis (https: / / bigd.big.ac.cn / , PRJCA010499), SSR molecular markers were developed using MISA software, and the upstream and downstream primers and annealing temperatures of SSR were designed using Primer 3 v2.4.0 software. 100 pairs of primers with repeating motifs of 2-6 bases and evenly distributed on the 14 chromosomes of the Leymus chinensis genome were selected. After polymorphism screening, 20 pairs of SSR primers with stable amplification, clear bands and polymorphism were obtained. The information of the 20 pairs of SSR markers is shown in Table 2, and the nucleotide sequences of the 20 pairs of SSR primers are shown in Table 3.

[0030] Table 2 Information of 20 pairs of SSR markers

[0031] Primer name Repeat Type Base composition Repetitions chromosome Starting position End position GW01-3 P3 GAA 17 Chr01 17059658 17059708 GW01-5 P2 AT 12 Chr01 7771565 7771588 GW01-6 P2 TC 16 Chr01 30124074 30124105 GW02-3 P3 TTC 11 Chr08 77335097 77335129 GW02-6 P3 AAC 13 Chr08 23510156 23510194 GW03-1 P2 TC 18 Chr02 14013400 14013435 GW03-7 P2 TG 10 Chr02 39978400 39978419 GW13-3 P3 TCT 10 Chr07 163736033 163736062 GW05-2 P2 AG 20 Chr03 43523818 43523857 GW13-5 P3 TTG 15 Chr07 309806282 309806326 GW06-2 P2 CT 15 Chr10 62296208 62296237 GW06-3 P2 TC 10 Chr10 87240747 87240766 GW06-6 P2 AG 20 Chr10 325612827 325612866 GW06-7 P2 AG 12 Chr10 468080799 468080822 GW07-1 P2 GA 14 Chr04 4842877 4842904 GW07-4 P2 AG 10 Chr04 47377265 47377284 GW13-7 P2 CT 10 Chr07 629126115 629126134 GW09-6 P3 AAC 12 Chr05 408051302 408051337 GW11-7 P3 AAC 11 Chr06 474379137 474379169 GW12-4 P2 TC 13 Chr13 269057862 269057887

[0032] Table 3 Nucleotide sequences of 20 pairs of SSR primers

[0033] Primer name Forward primer sequence Reverse primer sequence Annealing Temperature (°C) Product Length (bp) Fluorescent Label GW01-3 CAGGAGTGCACTTGCATGG (SEQ ID NO :1) ATTTCAGAACAACCGTTGGC (SEQ ID NO :2) 60 222 NED GW01-5 ATCACGTCTTGCATGCTCTG (SEQ ID NO :3) AAGCAGTTCTCTCGTCCAGC (SEQ ID NO :4) 60 221 NED GW01-6 CTTGATCCAAGGCCAGGTTA (SEQ ID NO :5) GCTATGTTATGGCGCCTTGT (SEQ ID NO :6) 60 173 FAM GW02-3 CCTTTCACCTTCGAATCCAG (SEQ ID NO :7) TCGTGAAGTTGTCACAAGTGG (SEQ ID NO :8) 59 246 ROX GW02-6 CGAGTTCTTGCAACTGGACA (SEQ ID NO :9) GAAGGACCGGAATATCACCA (SEQ ID NO :10) 60 212 HEX GW03-1 AATCGACGACCTTCCATACG (SEQ ID NO :11) ATGGCCACTCGTATGCTTTC (SEQ ID NO :12) 60 232 ROX GW03-7 CAATTAGGCACTTCCGTTGG (SEQ ID NO :13) GCACTGCTGGCATAAAGGTT (SEQ ID NO :14) 60 152 FAM GW13-3 TCCGTGTAAGGAGACCATCC (SEQ ID NO :15) GCTCCGTAAGAGCAGTGAGG (SEQ ID NO :16) 60 262 ROX GW05-2 GGCTAGTTGCTACTCTGCGG (SEQ ID NO :17) CACGAAAGCAACACACATCC (SEQ ID NO :18) 60 206 HEX GW13-5 CTTGCTATCCATGTGCCAGA (SEQ ID NO :19) AACGGAGTGCTCATCAGCTT (SEQ ID NO :20) 60 162 FAM GW06-2 GGCGGACTAGTAAGTGCTGG (SEQ ID NO :21) GGATGAGGACGATGCTGATT (SEQ ID NO :22) 60 211 HEX GW06-3 GCAACTCTCTCTGACGGTCC (SEQ ID NO :23) GGTGCCTGGTTCAATAGGAA (SEQ ID NO :24) 60 166 FAM GW06-6 AACGCCGAACTAGAGAATGC (SEQ ID NO :25) CTTCGGTATTACGCTCCGAC (SEQ ID NO: 26) 59 224 NED GW06-7 TGAACAAGGAATGTGCAAGG (SEQ ID NO: 27) CCATCATATCAGCGTTGTGG (SEQ ID NO: 28) 59 258 ROX GW07-1 TAGCTCGTCGACCATTAGGC (SEQ ID NO: 29) GAAGCAGAAGACTGCATCGC (SEQ ID NO: 30) 60 196 HEX GW07-4 TCTGTAGCTCAATCATGGCG (SEQ ID NO: 31) TAAGATGCTCCGGTGTTTCC (SEQ ID NO: 32) 60 228 NED GW13-7 TTTGATCTCGTGATGATGGC (SEQ ID NO: 33) CCGAGCTCTGAAACCTGTTC (SEQ ID NO: 34) 59 205 HEX GW09-6 CAGAAACAAGACACGAGGCA (SEQ ID NO: 35) TGATCTGCTGCAATGTCTCC (SEQ ID NO: 36) 60 172 FAM GW11-7 GGTGAAGAAGCGACTGAAGG (SEQ ID NO: 37) TCCATGTGCCAGAACCATTA (SEQ ID NO: 38) 60 251 ROX GW12-4 GGCCTGTTCTGGTACTCTGC (SEQ ID NO: 39) TACTTGCAGGTTGCAGTTGG (SEQ ID NO: 40) 60 218 NED

[0034] 4. PCR reaction system and reaction procedure

[0035] 4.1 PCR reaction system

[0036] The M13 universal linker sequence (TGTAAAACGACGGCCAGT) was added to the 5' end of each primer pair to synthesize simple sequence repeat (SSR) primer combinations with different fluorescent groups (FAM, HEX, NED, ROX, see Table 2) (all synthesized at Sangon Biotechnology (Shanghai) Co., Ltd.). The PCR system volume was 25 μL and contained the following components: DNA template (concentration of 20-50 ng·μL -1 )) 1 μL, forward primer and reverse primer (10 μmol·L -1 ) 0.5 μL each, dNTP (mix) 0.5 μL, TaqBuffer (containing MgCl2, 10×) 2.5 μL, Taq enzyme 0.2μL, double distilled water 19.8μL.

[0037] 4.2 PCR reaction procedure

[0038] First, perform initial denaturation at 95°C for 5 min; then perform 30 cycles, each cycle including denaturation at 94°C for 0.5 min, annealing time for 0.5 min (annealing temperature is 55--60°C, see Table 2 for details), and extension at 72°C for 0.5 min; finally, perform final extension at 72°C for 10 min and store at 4°C.

[0039] 5. Capillary electrophoresis detection

[0040] 990 μL HIDI and 10 uL LIZ500 were aspirated, mixed, and added to a 96-well reaction plate, 10 μL per well. Centrifuge at 1200 rpm for 15 seconds, add 1 μL of PCR amplification product, centrifuge at 1200 rpm for 15 seconds, seal the 96-well plate with a sealing film, shake, and centrifuge at 1200 rpm for 30 seconds. Denature at 98°C for 5 minutes in a PCR instrument, and immediately place the 96-well plate on an ice-water mixture to rapidly cool (cool to the same temperature as the ice-water mixture), centrifuge at 1200 rpm for 15 seconds, and use an ABI 3730xI automated DNA analyzer based on capillary electrophoresis and fluorescent labeling technology for detection, inject at 1 kV for 10 seconds, and electrophoresis at 15 kV for 30 minutes.

[0041] 6. Primer amplification and data analysis

[0042] The 20 pairs of polymorphic SSR primers screened were used to perform SSR typing on 105 samples of Leymus chinensis germplasm resources. The peak graph of the amplification results of primer pairs GW02-3, GW06-6, GW03-7, GW07-4, and GW13-5 on A01 germplasm resources is shown as an example (see Figures 1 - 5 ). GeneMapper 6.0 software was used to compare the peak positions in each figure with the molecular weight internal standards in the lanes to calculate the exact sizes of the DNA fragments. The capillary electrophoresis test was repeated three times and the average value was taken to obtain the number of bands amplified by the corresponding 20 primer pairs for 105 germplasm resources and the sizes of the corresponding bands.

[0043] 7. Construction of molecular ID card of Leymus chinensis germplasm resources

[0044] The SSR molecular ID card of Leymus chinensis germplasm resources was formed according to the peak combination of each pair of SSR primers. To facilitate the construction of the molecular ID card, we grouped the amplified fragments of each pair of primers in increments of 10bp, and assigned values ​​from 1 to 9 to different groups, with missing parts represented by 0. Each pair of primers contains a numerical range of 4 sites; if the fragment range exceeds the range of 9 groups, it is recorded in the 9th group. The order of SSR molecular markers in the molecular ID card is GW07-1, GW03-1, GW13-7, GW03-7, GW07-4, GW02-3, GW06-6, GW13-5, GW01-3, GW05-2, GW06-3, GW11-7, GW06-2, GW06-7, GW09-6, GW12-4, GW01-5, GW01-6, GW02-6 and GW13-3. The molecular ID cards of 105 Leymus chinensis are shown in the following table.

[0045] Table 4 Molecular IDs of 105 samples of Leymus chinensis

[0046] Resource number Molecular identity card information A01 00003400334012342400233423302240380010002233120026001100000022330000100022001400 A02 00003334247025602200233322341370238020001122200026002000133512345000100011503300 A03 44002336300023003000233423341246558000001233230026602234123822345000100040002000 A04 44003366233423002235344533451112255825552333200012443400135023445700100034002300 A05 56006667333022002233233323441112223813371233250033442300112323356000111011202200 A06 00003344333022002000233322442350380016002223100023552200120030001000100030006700 A07 00002333234633001400233023401223280037701233220055552340880022330000100025002400 A08 66773334247025602200233324001370238022701122200026002600150012345000100011503300 A09 00003000330012002300233323443344238012272233120017003000240022237000110016002400 A10 00003300335622402200233422451333800013301223220015002200100022339000100070001200 A11 00002334334734003000233424551220158023002222200023002300120012300000110078901400 A12 30003344300012231300230024661245338018001223200066663440120022347000100056001500 A13 40002334335055601200233522351330280033001123200020002300100022337000100078002400 A14 00003300333023342000333423551222280033001122200020003300124022347000111155002400 A15 13003400300033002000333423551233235810001220220020002230127023505000100048002200 A16 50003770360022332333334423402330248012701125120044402400115023000000100037002200 A17 30002300340033341000233422441300380033441233230066703444120022345900110060003400 A18 13003400333023001122334523552370558012702223200020002400100033449000100030002300 A19 30003400300020001400334424441330238011272223230023303444300022335000111110001000 A20 44442334360020002000233524451230128030001122200033662300120022301000120023002200 A21 00003400300020002300233324003340238012702233134055673000134022307000110036602400 A22 00001334340033002300234422551300480014601223130034453300120022340000110044002300 A23 40003366340022333300335623401240480055801233300022701000115033440000100030003000 A24 00002300233023352000233322401230680018001223200013001000160022349000100014002000 A25 46003340333620003600333523401125280033301234000010004566158022301600110056001300 A26 00003400330030002000333422501246348014001233470033302300114023001000100012001550 A27 00003340300034002350335024401200258015772333220020002200120023450000000034004000 A28 00003330340012001200334523401200228015552222200012002244134022330000111133002000 A29 00003400333423001000334522451100380022601222100044661770240022336000111120003300 A30 00001333335544000000334522342347380011001334123020005777100023456700100022001300 A31 77883466335023002000233024451230244800001233220033001000200023500000111120002300 A32 35003440330033001260233424662355380010001222000034663340230022347000100035004500 A33 35002330337033002600233422361345480011701223200034552400330023340000100035002400 A34 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[0047] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A SSR marker primer combination for identifying Leymus chinensis germplasm resources, characterized in that: The SSR marker primer combination consists of 20 pairs of SSR primers, and the nucleotide sequences of the SSR primers are shown in the following table: 。 2. The SSR marker primer combination for identifying Leymus chinensis germplasm resources according to claim 1, characterized in that: The 5' ends of the forward primer labeled GW01-3, the reverse primer labeled GW01-3, the forward primer labeled GW01-5, the reverse primer labeled GW01-5, the forward primer labeled GW06-6, the reverse primer labeled GW06-6, the forward primer labeled GW07-4, the reverse primer labeled GW07-4, the forward primer labeled GW12-4, and the reverse primer labeled GW12-4 are connected with a fluorescent label NED; The 5' ends of the forward primer labeled GW01-6, the reverse primer labeled GW01-6, the forward primer labeled GW03-7, the reverse primer labeled GW03-7, the forward primer labeled GW13-5, the reverse primer labeled GW13-5, the forward primer labeled GW06-3, the reverse primer labeled GW06-3, the forward primer labeled GW09-6, and the reverse primer labeled GW09-6 are connected with a fluorescent label FAM; The 5' ends of the forward primer labeled GW02-3, the reverse primer labeled GW02-3, the forward primer labeled GW03-1, the reverse primer labeled GW03-1, the forward primer labeled GW13-3, the reverse primer labeled GW13-3, the forward primer labeled GW06-7, the reverse primer labeled GW06-7, the forward primer labeled GW11-7, and the reverse primer labeled GW11-7 are connected with a fluorescent label ROX; The 5' ends of the forward primer labeled GW02-6, the reverse primer labeled GW02-6, the forward primer labeled GW05-2, the reverse primer labeled GW05-2, the forward primer labeled GW06-2, the reverse primer labeled GW06-2, the forward primer labeled GW07-1, the reverse primer labeled GW07-1, the forward primer labeled GW13-7, and the reverse primer labeled GW13-7 are connected to a fluorescent label HEX.

3. Application of the SSR marker primer combination for identification of Leymus chinensis germplasm resources according to claim 2 in constructing a molecular identity card of Leymus chinensis germplasm resources and identifying Leymus chinensis germplasm resources.

Images