Development of a core set of pea SNP markers based on KASP technology and its application

Abstract

This invention provides a set of core molecular markers for peas developed based on KASP technology and its applications, belonging to the field of molecular detection technology. This invention utilizes KASP-based SNP markers, including 25 core SNP markers, which are evenly distributed across the seven chromosomes of the pea genome, with 3-4 SNP markers on each chromosome. Based on this set of core molecular markers for peas, high-throughput SNP genotyping detection can be performed on pea resources or varieties, enabling the identification and purity testing of commercial pea varieties; it can be used to construct pea fingerprint libraries; and it can be applied to the genetic diversity analysis of pea germplasm resources; it is low-cost and highly efficient.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and molecular-assisted breeding technology, specifically to a set of pea core SNP molecular markers developed based on KASP technology and its applications. Background Technology

[0002] Peas, a diploid legume, are the world's fourth largest edible legume crop. With the shortage of rural labor resources, a key trend in pea breeding is to develop high-quality, high-yield varieties that do not require trellising and are suitable for mechanized harvesting. The whole-genome sequencing of peas was completed in 2019. Currently, my country has a large number of identified or registered pea varieties, and the planting area is continuously increasing. Existing methods for identifying and evaluating pea germplasm resources and verifying variety authenticity mainly rely on human phenotypic judgment, which is time-consuming, susceptible to human factors, and seriously affects the utilization efficiency and accuracy of identification and evaluation. Therefore, establishing methods for detecting and evaluating pea varieties, seed purity, and germplasm resources using molecular biology techniques is of great significance for protecting breeders' variety rights and safeguarding farmers' interests.

[0003] KASP stands for Kompetitive Allele Specific PCR, which can perform precise bicelestem typing of target SNPs and InDels on a wide range of genomic DNA samples, including complex ones. KASP technology's advantages, such as extremely high accuracy and transformation rate, unprecedented low cost, and simple and rapid result analysis, have led to its application in variety identification and seed purity testing of vegetable crops like peppers and watermelons. Therefore, there is an urgent need to develop low-density, highly accurate, and easy-to-operate SNP loci and their detection methods.

[0004] Therefore, providing pea core SNP molecular markers based on KASP technology and their applications is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] To meet the needs of the aforementioned fields, this invention provides a set of core SNP markers for peas developed based on KASP technology. Based on these core SNP markers, high-throughput SNP typing detection of pea materials can be achieved, thereby establishing a simpler, more efficient, and lower-cost method for identifying and evaluating the authenticity, seed purity, and germplasm resources of pea varieties, protecting breeders' variety rights and safeguarding farmers' interests.

[0006] Peas are a common species, which leads to problems such as confusion in the genotype of germplasm materials and difficulty in distinguishing them in production and when registering new varieties. At the same time, the pea genome is particularly large (about 4.5G), which makes resequencing expensive. If genotype identification is performed through resequencing, the cost is very high, resulting in a significant economic burden.

[0007] The pea genome is large and contains a great many repetitive sequences, which makes the development of a core SNP marker set for peas very difficult.

[0008] The selection of SNP sites in the pea core molecular marker set is crucial. Even with various primer changes or specially designed primers, accurate genotyping of certain SNP sites remains impossible. Therefore, it is essential to select the most suitable SNP sites to form the pea core molecular marker set for accurate identification of purebred pea materials. Through extensive screening and research experiments, this invention has finally found a pea core SNP marker set optimally suited for high-throughput SNP genotyping detection in pea materials, exhibiting high sensitivity, good specificity, and a 100% genotyping success rate.

[0009] On the one hand, this invention has developed a set of molecular markers for peas based on KASP technology, including 25 SNP markers, which are numbered SNP1 to SNP25;

[0010] The SNP1 is labeled with the bases A / G and is located on pea chromosome 1 at position 3172953.

[0011] The SNP2 is labeled with the base C / T and is located on pea chromosome 1 at position 277489395.

[0012] The SNP3 is labeled with the base T / A and is located on pea chromosome 1 at position 311989395.

[0013] The SNP4 is labeled with the base T / A and is located on pea chromosome 2 at position 1767704.

[0014] The SNP5 is labeled with the base G / C and is located on pea chromosome 2 at position 65837220.

[0015] The SNP6 is labeled with the bases A / C and is located on pea chromosome 2 at position 268353837.

[0016] The SNP7 is labeled with the bases A / G and is located on pea chromosome 2 at position 421393393.

[0017] The SNP8 is labeled with the base A / T and is located on pea chromosome 3 at position 118983751.

[0018] The SNP9 is labeled with the base G / C and is located on pea chromosome 3 at position 251295825.

[0019] The SNP10 is labeled with the base A / T and is located on pea chromosome 3 at position 295457439.

[0020] The SNP11 is labeled with the base T / C and is located on pea chromosome 4 at position 21190452.

[0021] The SNP12 is labeled with the base T / C and is located on pea chromosome 4 at position 113911433.

[0022] The SNP13 is labeled with the base C / T and is located on pea chromosome 4 at position 297836064.

[0023] The SNP14 is labeled with the base C / T and is located on pea chromosome 4 at position 410691643.

[0024] The SNP15 is labeled with the base C / T and is located on pea chromosome 5 at position 44491766.

[0025] The SNP16 is labeled with the base C / T and is located on pea chromosome 5 at position 104804482.

[0026] The SNP17 is labeled with the base C / G and is located on pea chromosome 5 at position 176342951.

[0027] The SNP18 is labeled with the base C / G and is located on pea chromosome 5 at position 527844249.

[0028] The SNP19 is labeled with the base C / A and is located on pea chromosome 6 at position 150897667.

[0029] The SNP20 is labeled with the base T / C and is located on pea chromosome 6 at position 355057090.

[0030] The SNP21 is labeled with the base G / A and is located on pea chromosome 6 at position 445826212.

[0031] The SNP22 is labeled with the base C / G and is located on pea chromosome 7 at position 115628325.

[0032] The SNP23 is labeled with the bases A / G and is located on pea chromosome 7 at position 163788370.

[0033] The SNP24 is labeled with the base G / C and is located on pea chromosome 7 at position 312150050.

[0034] The SNP25 is labeled with the base T / C and is located on pea chromosome 7 at position 460676933.

[0035] The physical location of the SNP was determined based on the pea genome sequence, which is based on the pea genome published at (https: / / urgi.versailles.inra.fr / jbrowse / gmod_jbrowse / ?data=myData / Pea / Psat_v1a / data).

[0036] Furthermore, the sequences of the first and last 100 bp of SNP1 to SNP25 are shown in the table below:

[0037] serial number First 100bp The last 100bp SNP1 Seq ID NO.76 Seq ID NO.77 SNP2 Seq ID NO.78 Seq ID NO.79 SNP3 Seq ID NO.80 Seq ID NO.81 SNP4 Seq ID NO.82 Seq ID NO.83 SNP5 Seq ID NO.84 Seq ID NO.85 SNP6 Seq ID NO.86 Seq ID NO.87 SNP7 Seq ID NO.88 Seq ID NO.89 SNP8 Seq ID NO.90 Seq ID NO.91 SNP9 Seq ID NO.92 Seq ID NO.93 SNP10 Seq ID NO.94 Seq ID NO.95 SNP11 Seq ID NO.96 Seq ID NO.97 SNP12 Seq ID NO.98 Seq ID NO.99 SNP13 Seq ID NO.100 Seq ID NO.101 SNP14 Seq ID NO.102 Seq ID NO.103 SNP15 Seq ID NO.104 Seq ID NO.105 SNP16 Seq ID NO.106 Seq ID NO.107 SNP17 Seq ID NO.108 Seq ID NO.109 SNP18 Seq ID NO.110 Seq ID NO.111 SNP19 Seq ID NO.112 Seq ID NO.113 SNP20 Seq ID NO.114 Seq ID NO.115 SNP21 Seq ID NO.116 Seq ID NO.117 SNP22 Seq ID NO.118 Seq ID NO.119 SNP23 Seq ID NO.120 Seq ID NO.121 SNP24 Seq ID NO.122 Seq ID NO.123 SNP25 Seq ID NO.124 Seq ID NO.125

[0038] Furthermore, this invention provides a KASP primer combination for detecting the pea core molecular marker set described above, comprising any one or more of 25 primer pairs corresponding to SNPs 1 to 25, and all of them. Each primer pair includes forward primer 1, forward primer 2, and reverse primer, as detailed in the table below:

[0039]

[0040]

[0041] Furthermore, the 5' end of forward primer 1 is tagged with a FAM fluorescent signal, and the 5' end of forward primer 2 is tagged with a HEX fluorescent signal.

[0042] In another aspect, the present invention provides reagents, chips or kits containing any one or more combinations of primer combinations as described above.

[0043] Furthermore, this invention provides a method for detecting pea genotypes using the primer combinations described above, comprising the following steps:

[0044] (1) Extract DNA from the pea variety to be tested;

[0045] (2) Using the DNA from step (1) as a template, add the primer combination Primer mix and KASP Master mix (LGC, Biosearch Technologies) to perform PCR amplification; the Primer mix is ​​the primer combination described in claim 3.

[0046] (3) Detect and analyze the amplification results to determine the genotype of the pea variety to be tested at the SNP molecular marker site corresponding to each set of primers.

[0047] Further, the PCR reaction system described in step (2) is as follows: 0.8 μl of 5–10 ng / μl pea genomic DNA, 0.8 μl of KASPMaster Mix, and 0.05 μl of Primer mix.

[0048] Further, the PCR reaction conditions described in step (2) are as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s; 61℃ annealing / extension for 60 s, with the annealing temperature decreasing by 0.6℃ for each cycle, for a total of 10 cycles; then 94℃ denaturation for 20 s, 55℃ annealing / extension for 60 s, for a total of 26 cycles.

[0049] Furthermore, the PCR in step (2) is performed using LGC's fully automated PCR platform.

[0050] Further, step 3) involves analysis using LGC's data analysis software Kraken or KlusterCaller.

[0051] In another aspect, the present invention provides the use of the molecular marker set, primer combination, reagent, chip, or kit described above in pea genotyping, pea variety authenticity identification, pea variety purity identification, pea SNP fingerprint library construction, pea genetic diversity analysis, or pea molecular marker-assisted breeding.

[0052] The beneficial effects of this invention are as follows: This invention provides a set of core SNP molecular markers for peas developed based on KASP technology and its applications. These SNP molecular markers are evenly distributed across the seven chromosomes of the pea genome, with 3-4 SNP markers per chromosome, totaling 25 SNP markers. Based on this set of core SNP molecular markers for peas, high-throughput SNP genotyping detection can be performed on pea germplasm resources or varieties, enabling the identification of authenticity and purity of commercial pea varieties; it can be used to construct pea fingerprint libraries; and it can be applied to the genetic diversity analysis of pea germplasm resources. Genetic analysis using the core SNP molecular markers provided by this invention can significantly reduce costs and is highly suitable for production applications. Attached Figure Description

[0053] Figure 1 This is a diagram showing the saturated SNP site information of the present invention;

[0054] Figure 2 This is a cluster analysis diagram of 135 different pea resources detected using 25 pea core SNP markers in Embodiment 1 of the present invention.

[0055] Figure 3 This is a genotyping diagram of the SNP primers used in this invention;

[0056] Figure 4 This is a cluster analysis diagram of 142 pea resources detected using 25 pea core SNP markers in Embodiment 2 of the present invention.

[0057] Figure 5 This is a genotyping result diagram corresponding to locus 234765722 on chromosome 1 in Embodiment 3 of the present invention;

[0058] Figure 6 This is a genotyping result diagram corresponding to locus 14940515 on chromosome 2 in Example 3 of the present invention;

[0059] Figure 7 This is a genotyping result diagram corresponding to the 436915076 locus on chromosome 4 in Example 3 of the present invention. Detailed Implementation

[0060] The present invention will be further described in detail below with reference to the embodiments. It should be noted that the embodiments described below are intended to facilitate understanding of the present invention and are not intended to limit it in any way. Unless otherwise specified, the embodiments are carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products that can be purchased commercially.

[0061] The KASP Master Mix used in the following examples was purchased from LGC Ltd. in the UK, with product number KBS-1016-001.

[0062] Example 1: Development of SNP molecular markers for pea cores based on KASP technology

[0063] Based on the pea genome published in 2019 (https: / / urgi.versailles.inra.fr / jbrowse / gmod_jbrowse / ?data=myData / Pea / Psat_v1a / data), the resequencing results of 21 pea germplasm accessions with significant differences in geographical origin and phenotypic characteristics were analyzed. After screening, 236 loci were obtained. 68 SNP loci with good polymorphism and even distribution on chromosomes were selected, and primers were designed to convert these 68 SNP loci into KASP markers.

[0064] The main steps include:

[0065] 1. Select a preliminary data file based on MAF (Minimum Allocative Frequency) > 5% and missing data < 10%;

[0066] 2. Based on MAF>0.4, miss rate<0.2, heterozygosity<0.2, and no other variant information within 100bp upstream and downstream of the variant site, the corresponding result files are selected.

[0067] 3. For the loci selected in step 2, calculate the PIC (polymorphism information content), He (expected heterozygosity), and Ho (observed heterozygosity) for each locus;

[0068] 4. Filter according to LD (Chain Imbalance), with an initial filtering threshold of 0.2;

[0069] 5. The filtered sites are further filtered according to non-synonymous mutations in the coding region, ultimately obtaining 236 site information.

[0070] 6. The 236 sites were manually screened to remove heterozygous sites and to ensure even distribution on the chromosome, resulting in 68 SNP sites. KASP primers were designed for PCR detection.

[0071] 7. Genotyping of 135 pea germplasm materials was performed using the 68 SNP markers selected above. To further narrow down the marker range by using the fewest SNPs to distinguish the largest number of samples, 5, 10, 15, 20, 25, 30, and 34 loci were randomly selected from the 68 KASP detection loci. The number of materials that could be distinguished by each locus was counted (pairwise comparisons were performed; any difference at any locus was considered distinguishable). 50 bootstrap tests were performed, and the average value (rounded to the nearest integer) was plotted. It was found that at least 25 SNPs could distinguish all samples. Figure 1-2Finally, a set of pea core SNP molecular markers containing 25 SNP markers with good stability and high information content was constructed (Table 1).

[0072] Table 1. Set of SNP molecular markers in pea core

[0073]

[0074]

[0075] For the pea core SNP molecular marker set shown in Table 1, KASP primer combinations for the pea core molecular marker set, as shown in Table 2, were designed. These combinations include any one or more of the 25 primer pairs corresponding to SNPs 1 to 25, or all of them. Each primer pair includes forward primer 1, forward primer 2, and a reverse primer. Forward primer 1 has a FAM fluorescent signal tag at its 5' end; forward primer 2 has a HEX fluorescent signal tag at its 5' end.

[0076] Table 2. KASP primer combinations for the pea core molecular marker set

[0077]

[0078]

[0079] Example 2: Method for identifying 142 pea resources using 25 pea core SNP markers

[0080] 1) DNA extraction

[0081] Genomic DNA was extracted from 142 pea leaves using the CTAB method.

[0082] 2) SNP genotyping was performed using LGC's fully automated PCR platform.

[0083] The specific KASP Primer mix and the universal KASP Master mix, composed of SNP markers numbered SNP1 to SNP25 from Example 1, were added to a 96-well plate containing DNA samples. After sealing the plate, water bath PCR was performed. The PCR reaction mixture consisted of: 0.8 μl of 5–10 ng / μl pea genomic DNA, 0.8 μl of KASP Master Mix, and 0.05 μl of Primer mix. The standardized PCR amplification procedure used was as follows:

[0084] Pre-denaturation at 94℃ for 15 min; denaturation at 94℃ for 20 s; annealing / extension at 61℃ for 60 s, with the annealing temperature decreasing by 0.6℃ per cycle, for a total of 10 cycles; followed by denaturation at 94℃ for 20 s, annealing / extension at 55℃ for 60 s, for a total of 26 cycles. Experimental results were analyzed using LGC's data analysis software Kraken or KlusterCaller, and the detection data were read using SNPviewer. Clicking on each primer name displays the genotyping result for each sample at that locus. Figure 3 ).

[0085] 3) Results Analysis

[0086] 142 pea germplasm materials were analyzed using 25 SNPs, and 23 groups (66 materials) were found to be completely clustered together in cluster analysis. Figure 4 The genotypes were completely identical, indicating that these were synonyms (or germplasm resources). The results show that the 25 core SNP markers are suitable for constructing fingerprint profiles of pea varieties and for resource-specific identification.

[0087] Example 3: Comparison of different pea core SNP marker sets

[0088] Following the method provided in Example 1, the inventors screened another set of 25 core SNP markers during the screening process. However, when used for large-scale genotyping of pea materials, they found that some pea materials were difficult to distinguish.

[0089] Another set of pea core SNP markers selected is designated as core marker set 2. The difference between core marker set 2 and the pea core SNP marker set (core marker set 1) provided in Example 1 is that SNP2, SNP5 and SNP14 in core marker set 2 are different. Specifically, SNP2 uses the 234765722 locus on chromosome 1 with allelic variation A / C; SNP5 uses the 14940515 locus on chromosome 2 with allelic variation A / G; and SNP14 uses the 436915076 locus on chromosome 4 with allelic variation A / G.

[0090] The primers used for locus 234765722 on chromosome 1 are:

[0091] Forward primer 1: GAAGGTGACCAAGTTCATGCTCCTTAAGATTACCCATTCCAG

[0092] Forward primer 2: GAAGGTCGGAGTCAACGGATTCCTTAAGATTACCCATTCCAT

[0093] Reverse primer: TACTTAACACTTGTAGACAAAG

[0094] The primers used for locus 14940515 on chromosome 2 are:

[0095] Forward primer 1: GAAGGTGACCAAGTTCATGCTGGTTCCTTTGCTAGAGAATGATA

[0096] Forward primer 2: GAAGGTCGGAGTCAACGGATTGGTTCCTTTGCTAGAGAATGATG

[0097] Reverse primer: GCTCTTTCTGTTCATTACCATGTGA

[0098] The primers used for locus 436915076 on chromosome 4 are:

[0099] Forward primer 1: GAAGGTGACCAAGTTCATGCTGCCATTACAAGATCCTCTTCCAACA Forward primer 2: GAAGGTCGGAGTCAACGGATTGCCATTACAAGATCCTCTTCCAACG

[0100] Reverse primer: TATGAAAGCAATAAAAGAAGGG

[0101] When using the method provided in Example 2 to perform genotyping on 135 pea germplasm resources with core marker set 1, core marker set 1 was able to distinguish the genotypes of the 135 pea germplasm resources with 100% accuracy. Figure 2 This indicates that the 25 core SNP markers are suitable for constructing fingerprint profiles of pea varieties and for resource-specific identification. However, using the second core marker set resulted in some materials being indistinguishable. Further investigation revealed issues at loci 234765722 on chromosome 1, 14940515 on chromosome 2, and 436915076 on chromosome 4, affecting genotyping. The genotyping results for locus 234765722 on chromosome 1 are as follows: Figure 5 As shown, the genotyping results corresponding to locus 14940515 on chromosome 2 are as follows: Figure 6 As shown, the phenotypic structure corresponding to locus 436915076 on chromosome 4 is as follows: Figure 7 As shown, there are samples that are difficult to genotype at all three loci. Between the red (homozygous), blue (homozygous), or purple (heterozygous) regions, there are also gray regions (not detected), indicating that none of these three loci were successfully genotyped.

[0102] After repeatedly changing the primers for these three SNP loci, it was still impossible to resolve some difficult-to-type samples. The reason may be that the genes at loci 234765722 on chromosome 1, 14940515 on chromosome 2, and 436915076 on chromosome 4 contain genes with very high homology to surrounding genes, highly homologous sequences, heterozygous gene loci, and are rich in GC. This leads to problems such as the loci not showing a peak and being undetected, amplifying homologous sequences and causing errors, or producing two genotypes when detecting these loci at once. Therefore, it is difficult to achieve simultaneous detection of all loci.

[0103] When using the core marker set 1, which involves modifying the SNP2 site to position 277489395 on chromosome 1, the SNP5 site to position 65837220 on chromosome 2, and the SNP14 site to position 410691643 on chromosome 4, it is possible to cleverly avoid GC-rich sequences and prevent amplification of homologous sequences or heterozygous sites, thereby solving the problem of difficult genotyping and greatly improving the accuracy of genotyping.

[0104] It is evident that the selection of SNP sites in the pea core molecular marker set is crucial. Even with multiple primer changes or specially designed primers, some SNP sites still cannot achieve accurate typing. The most suitable SNP sites must be selected to form the pea core molecular marker set for accurate identification of pea germplasm materials.

[0105] Example 4 uses a pea core SNP marker set to detect whether the pea variety to be tested is 'Zhewan No. 1'.

[0106] Based on a set of 25 core SNP markers for peas, the SNP fingerprint of the pea variety 'Zhewan 1' was constructed. Since SNP markers are diallelic, the fingerprint composed of 25 SNP markers consists of 50 bases. The core SNP fingerprint of the pea variety 'Zhewan 1' is: GGGGTTTTGGCCAATTGGTTTTTTCCGGAAGGGGGGCCGGCCCCTTCCAA. The SNP fingerprint was converted into a QR code using an online barcode generator to obtain the QR code for the SNP fingerprint of the pea variety 'Zhewan 1', as shown in Table 2.

[0107] Pea varieties were randomly selected for testing. After DNA preparation, genotyping was performed using a set of 25 core SNP markers to obtain their SNP fingerprints.

[0108] Results Analysis: The SNP fingerprint profile of the tested pea variety was compared with that of the authentic pea variety 'Zhewan No. 1'. If the two were completely identical, the tested pea variety was identified as 'Zhewan No. 1'; otherwise, it was not.

[0109] Table 2 SNP fingerprint information of pea variety 'Zhewan No. 1'

[0110]

[0111] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

[0112] sequence list

[0113] Seq ID NO.1

[0114] SNP1 forward primer 1: GAAGGTGACCAAGTTCATGCTAAGCCTGGAAGCTCCAAGACTTTCA

[0115] Seq ID NO.2

[0116] SNP1 forward primer 2: GAAGGTCGGAGTCAACGGATTAAGCCTGGAAGCTCCAAGACTTTCG

[0117] Seq ID NO.3

[0118] SNP1 reverse primer: TCACATGATGGTGATCAATAAG

[0119] Seq ID NO.4

[0120] SNP2 forward primer 1: GAAGGTGACCAAGTTCATGCTGGTCCAAAGGACTCTACATGTTGA

[0121] Seq ID NO.5

[0122] SNP2 forward primer 2: GAAGGTCGGAGTCAACGGATTGGTCCAAAGGACTCTACATGTTGG

[0123] Seq ID NO.6

[0124] SNP2 reverse primer: TGAGCATTACCTGCAGGTTTCCC

[0125] Seq ID NO.7

[0126] SNP3 forward primer 1: GAAGGTGACCAAGTTCATGCTCGAATATGCTTGATAGTGAGAT

[0127] Seq ID NO.8

[0128] SNP3 forward primer 2: GAAGGTCGGAGTCAACGGATTCGAATATGCTTGATAGTGAGAA

[0129] Seq ID NO.9

[0130] SNP3 reverse primer: AGGCCCAGAGCTCGATCAATAA

[0131] Seq ID NO.10

[0132] SNP4 forward primer 1: GAAGGTGACCAAGTTCATGCTTGTACTCTTATCGGGATATAT

[0133] Seq ID NO.11

[0134] SNP4 forward primer 2: GAAGGTCGGAGTCAACGGATTTGTACTCTTATCGGGATATAA

[0135] Seq ID NO.12

[0136] SNP4 reverse primer: ATGTAGTGTATGTTTAGAGGGTGAGA

[0137] Seq ID NO.13

[0138] SNP5 forward primer 1: GAGGTGACCAAGTTCATGCTCACATCACCAAGAACTTCATGAG

[0139] Seq ID NO.14

[0140] SNP5 forward primer 2: GAAGGTCGGAGTCAACGGATTCACATCACCAAGAACTTCATGAC

[0141] Seq ID NO.15

[0142] SNP5 reverse primer: GACCGCAATTTAAAACCACGAT

[0143] Seq ID NO.16

[0144] SNP6 forward primer 1: GAAGGTGACCAAGTTCATGCTGGTTTCTTTGGTTTTGCTTTCTA

[0145] Seq ID NO.17

[0146] SNP6 forward primer 2: GAAGGTCGGAGTCAACGGATTGGTTTCTTTGGTTTTGCTTTCTC

[0147] Seq ID NO.18

[0148] SNP6 reverse primer: CCATACCAAAGCAATGCCACCCG

[0149] Seq ID NO.19

[0150] SNP7 forward primer 1: GAAGGTGACCAAGTTCATGCTGGTCGTGGAGGTTCGATAATAAAAA

[0151] Seq ID NO.20

[0152] SNP7 forward primer 2: GAAGGTCGGAGTCAACGGATTGGTCGTGGAGGTTCGATAATAAAAG

[0153] Seq ID NO.21

[0154] SNP7 reverse primer: CATAGATTCCTTTACCATCCTC

[0155] Seq ID NO.22

[0156] SNP8 forward primer 1: GAAGGTGACCAAGTTCATGCTACACTTTTCCATCTGCAGGCTGACA

[0157] Seq ID NO.23

[0158] SNP8 forward primer 2: GAAGGTCGGAGTCAACGGATTACACTTTTCCATCTGCAGGCTGACT

[0159] Seq ID NO.24

[0160] SNP8 reverse primer: CAACACAGAAACAAAAAACCTAAGATTAC

[0161] Seq ID NO.25

[0162] SNP9 forward primer 1: GAAGGTGACCAAGTTCATGCTGCCAATTGAAATCTCCTTCTCCTGG

[0163] Seq ID NO.26

[0164] SNP9 forward primer 2: GAAGGTCGGAGTCAACGGATTGCCAATTGAAATCTCCTTCTCCTGC

[0165] Seq ID NO.27

[0166] SNP9 reverse primer: GGTTTTCGAACAGAGTAAGATCCG

[0167] Seq ID NO.28

[0168] SNP10 forward primer 1: GAAGGTGACCAAGTTCATGCTCAACCCACCATGCCATCATTA

[0169] Seq ID NO.29

[0170] SNP10 forward primer 2: GAAGGTCGGAGTCAACGGATTCAACCCACCATGCCATCATTT

[0171] Seq ID NO.30

[0172] SNP10 reverse primer: CCAGCAAAAGCAGTATCTAAGAAG

[0173] Seq ID NO.31

[0174] SNP11 forward primer 1: GAAGGTGACCAAGTTCATGCTGGAGGAACATGATGTCTTTGTCT

[0175] Seq ID NO.32

[0176] SNP11 forward primer 2: GAAGGTCGGAGTCAACGGATTGGAGGAACATGATGTCTTTGTCC

[0177] Seq ID NO.33

[0178] SNP11 reverse primer: TAGAGCAATTCCTCCCTCAATA

[0179] Seq ID NO.34

[0180] SNP12 forward primer 1: GAAGGTGACCAAGTTCATGCTATGTACAGGAGATGGGAAAAT

[0181] Seq ID NO.35

[0182] SNP12 forward primer 2: GAAGGTCGGAGTCAACGGATTATGTACAGGAGATGGGAAAAC

[0183] Seq ID NO.36

[0184] SNP12 reverse primer: GACCTCACTGCTTTCCTGGACT

[0185] Seq ID NO.37

[0186] SNP13 forward primer 1: GAAGGTGACCAAGTTCATGCTGACTCATTTATCCAATCTTCAGCC

[0187] Seq ID NO.38

[0188] SNP13 forward primer 2: GAAGGTCGGAGTCAACGGATTGACTCATTTATCCAATCTTCAGCT

[0189] Seq ID NO.39

[0190] SNP13 reverse primer: GGGTGATGAGGGGTTTGAGTTG

[0191] Seq ID NO.40

[0192] SNP14 forward primer 1: GAAGGTGACCAAGTTCATGCTGGTCCACATTTTGAAACATGGAATA

[0193] Seq ID NO.41

[0194] SNP14 forward primer 2: GAAGGTCGGAGTCAACGGATTGGTCCACATTTTGAAACATGGAATG

[0195] Seq ID NO.42

[0196] SNP14 reverse primer: TTAAGTCTCTTCTCCCCTCGTT

[0197] Seq ID NO.43

[0198] SNP15 forward primer 1: GAAGGTGACCAAGTTCATGCTACAGATTCTCCTCCTTGAGCA

[0199] Seq ID NO.44

[0200] SNP15 forward primer 2: GAAGGTCGGAGTCAACGGATTACAGATTCTCCTCCTTGAGCG

[0201] Seq ID NO.45

[0202] SNP15 reverse primer: TCAAACTACTAACAATTTTATAACAGGAG

[0203] Seq ID NO.46

[0204] SNP16 forward primer 1: GAAGGTGACCAAGTTCATGCTGGCTTAGAAGGTTGAACATCCA

[0205] Seq ID NO.47

[0206] SNP16 forward primer 2: GAAGGTCGGAGTCAACGGATTGGCTTAGAAGGTTGAACATCCG

[0207] Seq ID NO.48

[0208] SNP16 reverse primer: TCAGTGTCGTGTGAGTGTCTTG

[0209] Seq ID NO.49

[0210] SNP17 forward primer 1: GAAGGTGACCAAGTTCATGCTAGAACACTCGAATCGGCCAAC

[0211] Seq ID NO.50

[0212] SNP17 forward primer 2: GAAGGTCGGAGTCAACGGATTAGAACACTCGAATCGGCCAAG

[0213] Seq ID NO.51

[0214] SNP17 reverse primer: ATGAAGGAAAAGGCATGCGGTTAGCA

[0215] Seq ID NO.52

[0216] SNP18 forward primer 1: GAAGGTGACCAAGTTCATGCTTCAGTGACTCCTCTGCCTC

[0217] Seq ID NO.53

[0218] SNP18 forward primer 2: GAAGGTCGGAGTCAACGGATTTCAGTGACTCCTCTGCCTG

[0219] Seq ID NO.54

[0220] SNP18 reverse primer: TCTTCTTCACATGTTGCTTGCAA

[0221] Seq ID NO.55

[0222] SNP19 forward primer 1: GAAGGTGACCAAGTTCATGCTCAGTGATACAACACAGAGGGTTGAC

[0223] Seq ID NO.56

[0224] SNP19 forward primer 2: GAAGGTCGGAGTCAACGGATTCAGTGATACAACACAGAGGGTTGAA

[0225] Seq ID NO.57

[0226] SNP19 reverse primer: GAGAGGAAGTATCATCCTGCAA

[0227] Seq ID NO.58

[0228] SNP20 forward primer 1: GAAGGTGACCAAGTTCATGCTACAGCCTATTGGGTGTTTGCCTG

[0229] Seq ID NO.59

[0230] SNP20 forward primer 2: GAAGGTCGGAGTCAACGGATTACAGCCTATTGGGTGTTTGCCTA

[0231] Seq ID NO.60

[0232] SNP20 reverse primer: GCAACTCGTTGTGCTTCGTGAC

[0233] Seq ID NO.61

[0234] SNP21 forward primer 1: GAAGGTGACCAAGTTCATGCTCAATAATCCTTCTTTGGATTTCT

[0235] Seq ID NO.62

[0236] SNP21 forward primer 2: GAAGGTCGGAGTCAACGGATTCAATAATCCTTCTTTGGATTTCC

[0237] Seq ID NO.63

[0238] SNP21 reverse primer: ATTTGTGAACATTGTTCTCAAACATG

[0239] Seq ID NO.64

[0240] SNP22 forward primer 1: GAAGGTGACCAAGTTCATGCTGCAATTCACTCATCACTTC

[0241] Seq ID NO.65

[0242] SNP22 forward primer 2: GAAGGTCGGAGTCAACGGATTGCAATTCACTCATCACTTG

[0243] Seq ID NO.66

[0244] SNP22 reverse primer: GGAGGGTAAAGGTATTGCCATG

[0245] Seq ID NO.67

[0246] SNP23 forward primer 1: GAAGGTGACCAAGTTCATGCTGTGGGAAAGAAATTTTTGCTGAAC

[0247] Seq ID NO.68

[0248] SNP23 forward primer 2: GAAGGTCGGAGTCAACGGATTGTGGGAAAGAAATTTTTGCTGAAT

[0249] Seq ID NO.69

[0250] SNP23 reverse primer: TTCTACTCGGATTCGGTATGAG

[0251] Seq ID NO.70

[0252] SNP24 forward primer 1: GAAGGTGACCAAGTTCATGCTCTTCAATGCCATGCTTGTAAG

[0253] Seq ID NO.71

[0254] SNP24 forward primer 2: GAAGGTCGGAGTCAACGGATTCTTCAATGCCATGCTTGTAAC

[0255] Seq ID NO.72

[0256] SNP24 reverse primer: TGCAGCAAGACTCAGAAAAATT

[0257] Seq ID NO.73

[0258] SNP25 forward primer 1: GAAGGTGACCAAGTTCATGCTCGGAATAATAATTCACAACTCTAG

[0259] Seq ID NO.74

[0260] SNP25 forward primer 2: GAAGGTCGGAGTCAACGGATTCGGAATAATAATTCACAACTCTAA

[0261] Seq ID NO.75

[0262] SNP25 reverse primer: ATATTTTTGCCACGGCCGGCCGA

[0263] Seq ID NO.76

[0264] SNP1 first 100bp:

[0265] TTTAATTCTCATTAGCCTCATTGCCGCATTCCTTATCACTACATAGTTATCCATGAATTCCTCATCCACGACATCGAAGCCTGGAAGCTCCAAGACTTTC

[0266] Seq ID NO.77

[0267] 100bp after SNP1:

[0268] AAACCAAGTCACCATATGGTATCCATGTTGCTCCCTTCTTATTGATCACCATCATGTGATGGATCACAATACTGGCCCAATTTGTTTCAATTTTGTTGGC

[0269] Seq ID NO.78

[0270] 100 bp before SNP2:

[0271] AATTTGGAAGAGATACTGTGAAAATAAGTAGAGATTTTTGAAAAGTTTCTCTTATGAGCATTACCTGCAGGTTTCCCATTTAGCAGGCTTATGATCAGGA

[0272] Seq ID NO.79

[0273] 100 bp after SNP2:

[0274] CAACATGTAGAGTCCTTTGGACCTCGGGTAGATTGAAGTATATAGTGCTGTGCTTCTCGGTGCAAGGATCATACGCACTTCTGAGTCTTCGGATACCCTG

[0275] Seq ID NO.80

[0276] 100 bp before SNP3:

[0277] TCTCAAAATAGTTTGCCTACAACAATTAATGCCAAAACTGATGTCTCAGATGATTTCTCTGCCAAAGATTCAAGTACAACGAATATGCTTGATAGTGAGA

[0278] Seq ID NO.81

[0279] 100 bp after SNP3:

[0280] ATTATCCAACATTGGTGGAAATGGCTTAGCATCTGTCTCCCCTCAACTTGAAGAACTTATTGATCGAGCTCTGGGCCTTGGATCTGTTGCCAAATCAAAT

[0281] Seq ID NO.82

[0282] 100 bp before SNP4:

[0283] GTGGAGGTTTGTTGAAATCATCTTCATTTAAGAATATGTTATCGTAACTCGACGGAGGCGACGATGGATCTTCAGAATTTTGTACTCTTATCGGGATATA

[0284] Seq ID NO.83

[0285] 100 bp after SNP4:

[0286] TCCTGGAATAGTAGAAGTTACAATTAGTCTCACCCTCTAAACATACACTACATGTACATGACCGTCTTTTTATTACCGCATATGGCCCGAATATGTGGTT

[0287] Seq ID NO.84

[0288] 100 bp before SNP5:

[0289] GTGACCGCAATTTAAAAGCAACCGTGACCGCAATTTAAAACCACGATATATGTAAATAATATACATATATATCTAATTACATGCCTTGATGTTAGGAAGA

[0290] Seq ID NO.85

[0291] 100 bp after SNP5:

[0292] TCATGAAGTTCTTGGTGATGTGAACAACTAGCTTGTCCATAAAAGCAGGAGCAATGTAAAATCCATCCATCATATTGTCAAGGTTGTACCTGAAATAACC

[0293] Seq ID NO.86

[0294] 100 bp before SNP6:

[0295] GATAAGAGACCAGATATCTTTATTGTAATGTAAACTTGGTCTGACCCTAACAATTTAATTCGTACTTTTTCTAGATTAGGTTTCTTTGGTTTTGCTTTCT

[0296] Seq ID NO.87

[0297] 100 bp after SNP6:

[0298] TGAATCTCGAGGCTTTGCGGGTGGCATTGCTTTGGTATGGGAATCTAATTGAATGTAAATGACCATTTTAAAGCAACACTTTTAATTCCTTCACTCTAAA

[0299] Seq ID NO.88

[0300] 100 bp before SNP7:

[0301] TTTCCGATTAGTACTCCGCATAATGAAGAGGTTGCAAATGCGGTTAAGATCATTGAAGAACACTTGAAGGTTCATCGGTCGTGGAGGTTCGATAATAAAA

[0302] Seq ID NO.89

[0303] 100 bp after SNP7:

[0304] CAATGCAAGTTGTGAGGATGGTAAAGGAATCTATGTGTATGATTTACCATCTAAGTTTAACAAGGATTTGGTTGGTCAGTGCAGTGACATGCTTCCATGG

[0305] Seq ID NO.90

[0306] 100 bp before SNP8:

[0307] TTATGAAGAAACAAATTGTATGGAAATTTCCAACACAGAAACAAAAAACCTAAGATTACCTTTCTCCATGGACCAGCTCTTGATGAGGAATGAGTCTGGC

[0308] Seq ID NO.91

[0309] 100 bp after SNP8:

[0310] GTCAGCCTGCAGATGGAAAAGTGTAAAGGGAAGATAACTTAGCCAAGCAGAATGACCTTGGATGAATAGCAATACGAAAAAGAAAATAAAAAAAATCTGA

[0311] Seq ID NO.92

[0312] 100 bp before SNP9:

[0313] GCTAAAATCATGTTAGTAGATGCTTGTTTTTTGTTAGAGCTTCTCATATCAAAAGAGTTAGATCATGAACTACCATGCCAATTGAAATCTCCTTCTCCTG

[0314] Seq ID NO.93

[0315] 100 bp after SNP9:

[0316] ACCTCAACTCCTGAGAGACGAAGATGTCTTGTCGGATCTTACTCTGTTCGAAAACCAGATCCCGGTTTTCGTCCTCCACGAGCTTTCTCGAAAGCTTTTC

[0317] Seq ID NO.94

[0318] 100 bp before SNP10:

[0319] TGGTGTAGGAAACAGTCCAATCAAAGTAAACAAAAGGGTCACCAGCAAAAGCAGTATCTAAGAAGAAACATAGAAGTGTTGTGAAGAACCAACAAGAGGA

[0320] Seq ID NO.95

[0321] 100 bp after SNP10:

[0322] AATGATGGCATGGTGGGTTGAAAGTACTATCGCTTATCTTCTCAAGATGTATAGAGAGAGGGAGGAAGAAGGTGTTGAGGTGAATGAAAGTTGGCAGAGA

[0323] Seq ID NO.96

[0324] 100 bp before SNP11:

[0325] AAGTAAACAAAGTGTTAACCTTCATTAGGAGGTGGGTGTGGCAGTGTCTTGGAAGAAGAACCAGGTGGAGATACTCTAGGAGGAACATGATGTCTTTGTC

[0326] Seq ID NO.97

[0327] 100 bp after SNP11:

[0328] ACCAGTTGTTGGTGATATTGAGGGAGGAATTGCTCTATCTAGATCAAAAATTGTAAAATTAAAACTACTTATATATTCAGTTGCAGCCAAGAACTTTATT

[0329] Seq ID NO.98

[0330] 100 bp before SNP12:

[0331] TTTCGTTAAATTAATAAAACATGATTATTTGAAAACATTGATCACAAAATTTGCAAAAACAAAGAGTGTATTGAGGTGTTATGTACAGGAGATGGGAAAA

[0332] Seq ID NO.99

[0333] 100 bp after SNP12:

[0334] TGTGCGTCCAGTTGACGAGTCCAGGAAAGCAGTGAGGTCAACCATGTCTTCTACATTTAAATTGATGCCCGTCTCTTCTTCAACCTGTTAGAATCACAAC

[0335] Seq ID NO.100

[0336] 100 bp before SNP13:

[0337] ACCACCTATTTCTCCTATCCATTTCCTTGATATAAGCACCTTTATTAACATGCACAATATCATAGTTATTTTCAAATGACTCATTTATCCAATCTTCAGC

[0338] Seq ID NO.101

[0339] 100 bp after SNP13:

[0340] ATTGTCAATATCTCCAACTCAAACCCCTCATCACCCCTCTTTTGGTTTCTATTACCGTTTAGTTCTTCTTTGAAAAATTCCAACAAAACTATACCTAAAT

[0341] Seq ID NO.102

[0342] 100 bp before SNP14:

[0343] GTGGTGAATGTGTGAAGAACCTTGTATGACCATTTCTGCCATGTTAAGTCTCTTCTCCCCTCGTTGAGACCATTCAACGAAATTGGACCAAGAACACCAG

[0344] Seq ID NO.103

[0345] 100 bp after SNP14:

[0346] ATTCCATGTTTCAAAATGTGGACCAACATTCTGCATATAAATGCGCCAAATGATTAGCATGGACTAAAGTTCATATTTTTAGACACGGGGATGCTTAGAG

[0347] Seq ID NO.104

[0348] 100 bp before SNP15:

[0349] ATCTTCTTCATCTGGTTATTCAAATTCAAACTACTAACAATTTTATAACAGGAGATTATGAAATCTTGTCCAAAGAAAGACATTGAGAATTTCAAAGATT

[0350] Seq ID NO.105

[0351] 100 bp after SNP15:

[0352] GCTCAAGGAGGAGAATCTGTATATAAATATAGAGGTAAGTATAATTAACGAAATAATTCGATTTTTCTGAAATTTGAATTTTGAATCGATATTTTTGTTA

[0353] Seq ID NO.106

[0354] 100 bp before SNP16:

[0355] AATGAATACATTGTACGTATTCACAGGTGTTAGTAATCACAAGTGTCAGTGTCGTGTGAGTGTCTTGAGGATTTAACATTAGTGATCATCACCACGAATA

[0356] Seq ID NO.107

[0357] 100 bp after SNP16:

[0358] GGATGTTCAACCTTCTAAGCCAAATAGAATGAATAGTCATATGGAAAATATCGAACCTCTTTGGTGGCTCATTAGGAACAAAATGCCTTTGAACTTGTTT

[0359] Seq ID NO.108

[0360] 100 bp before SNP17:

[0361] GCTGGAGTTGTTCGCGTGTTGCGGATCAGTGTGCCAGTATTCCGATGAAGGAAAAGGCATGCGGTTAGCAGTTGCATGTGCTGTGGAATGAGTTGCCCCG

[0362] Seq ID NO.109

[0363] 100 bp after SNP17:

[0364] TTGGCCGATTCGAGTGTTCTAAAGAGAGGGACTTGGTATCGTATGCACCCCATGAATTGGTTGACAGAAGAGAGAGAGCACGGTTAACATCTTGCGTAGC

[0365] Seq ID NO.110

[0366] 100 bp before SNP18:

[0367] ACAACACCTTAAAAACCGTGTTAGATCGTATTCTCAAGCAAAGAATCTCTCTTCTTCTTCACATGTTGCTTGCAAATTGAAGGATGAGAAGAAGGCAAAG

[0368] Seq ID NO.111

[0369] 100 bp after SNP18:

[0370] AGGCAGAGGAGTCACTGAGGACTGTCATGTACTTGAGCTGCTGGGGTCCCAATTGATTCATTGCTACATATATAGTGATACAAGATTAATTATGTCTAAT

[0371] Seq ID NO.112

[0372] 100 bp before SNP19:

[0373] TCTCAGTTAATTAATTTAGTAAAATATGCATTGAACTATTTCAGGAACTGCATGAGAAAAGTGGTTCGCCAAAGGACAGTGATACAACACAGAGGGTTGA

[0374] Seq ID NO.113

[0375] 100 bp after SNP19:

[0376] AGAAAAACCATTCAAACTTCAACAGGTTCAGCAACTTTTAGTATTGCAGGATGATACTTCCTACTCTACTATTATGCATCAATAAATTGTCTATTAGATG

[0377] Seq ID NO.114

[0378] 100 bp before SNP20:

[0379] TTCAGATCTTGCACAGTTTTGAGAAGCAACTCGTTGTGCTTCGTGACGAGCATGTTTAACGTTTCATTGCACTTAGCAAAAGAAGATGGTGTAGTTATTA

[0380] Seq ID NO.115

[0381] 100 bp after SNP20:

[0382] AGGCAAACACCCAATAGGCTGTAATAATCCAACTGTTATTTTCTTTATTCCTACGCTATGAATGCGTTTAAGATTTAATGACAATTGGTTTATAAGTGAA

[0383] Seq ID NO.116

[0384] 100 bp before SNP21:

[0385] TAGAAATTGTCTTGGATTTGACAATTGTGAACATTGTTCTCAAACATGTTTGCAACATTTTTTGAGGTTTCTGAAGTTTCTAGTGGCTGTGAAAATTGGA

[0386] Seq ID NO.117

[0387] 100 bp after SNP21:

[0388] GAAATCCAAAGAAGGATTATTGAAGCTGGTTGACACATGATTTCCACTTGAAGCATCTATGAATCCTCTTTGTGAAGGAACATTGAAGCTTGTTGAGATT

[0389] Seq ID NO.118

[0390] 100 bp before SNP22:

[0391] ATTAGATAGCTTAGAGTTTGACATATCCATATAAAATATAACATTCAAGCTAGCTTCTCCTCAAGTAGAAGAAAAAAAAGATGCAATTCACTCATCACTT

[0392] Seq ID NO.119

[0393] 100 bp after SNP22:

[0394] ATAAGATCATTGGCAATAGGTAGTGCTTTACACAGAGCCTTAAATGGGATCTTTTTAGGCATGGCAATACCTTTACCCTCCATCATATCAAGTTTCTCCT

[0395] Seq ID NO.120

[0396] 100 bp before SNP23:

[0397] GGCTTGTTCTAACACATTTGGACTTGTTACTGGTGCATTTCTACTCGGATTCGGTATGAGTGAAATTCCAAAGGGTATTTGGTTGAATGCAAATTGGACC

[0398] Seq ID NO.121

[0399] 100 bp after SNP23:

[0400] TTCAGCAAAAATTTCTTTCCACAAGTTGCAAAAATGGCTGTCAAATTAGACGACGCTCATCAAGATTTTTCAAATGCTATCGGTTGTAAGTTATTCATA

[0401] Seq ID NO.122

[0402] SNP24 first 100bp:

[0403] GTTGTATTAAAAGACCAGTAGCTAAACAGAAGGTAAATAACCATAATCTAGGCAACACATAATTGTTTGATGTACTATTTCTTCAATGCCATGCTTGTAA

[0404] Seq ID NO.123

[0405] SNP24 followed by 100bp:

[0406] TAAAACCCAAATCTTTTAGCTTCTTTGATGAAATTTCAGAAGGAACCTTATCATAATTTTTCTGAGTCTTGCTGCATGTATATGTAGGAATAACATCAGA

[0407] Seq ID NO.124

[0408] SNP25 first 100bp:

[0409] GATAGAAGTTTTGATGAAAACGGTTCTTGAATATTTTTGCCACGGCCGGCCGAGGTACGACTTAACCGAGCTAGGGTTTAGGTCCGACGCGGCCGTGACC

[0410] Seq ID NO.125

[0411] SNP25 followed by 100bp:

[0412] TAGAGTTGTGAATTATTCCGGTGTTTTGGTTTGGATCGGTTCGGCCTTGAGCCGTAACAGTGATTGTTTTTTGAGGAGGGTTTCTCGCGATAATGTT sequence list <110> Zhejiang Academy of Agricultural Sciences <120> A Pea Core SNP Molecular Marker Set Developed Based on KASP Technology and Its Application <160> 125 <170> SIPOSequenceListing 1.0 <210> 1 <211> 46 <212> DNA <213> Artificial Sequence <400> 1 gaaggtgacc aagttcatgc taagcctgga agctccaaga ctttca 46 <210> 2 <211> 46 <212> DNA <213> Artificial Sequence <400> 2 gaaggtcgga gtcaacggat taagcctgga agctccaaga ctttcg 46 <210> 3 <211> twenty two <212> DNA <213> Artificial Sequence <400> 3 tcacatgatg gtgatcaata ag 22 <210> 4 <211> 45 <212> DNA <213> Artificial Sequence <400> 4 gaaggtgacc aagttcatgc tggtccaaag gactctacat gttga 45 <210> 5 <211> 45 <212> DNA <213> Artificial Sequence <400> 5 gaaggtcgga gtcaacggat tggtccaaag gactctacat gttgg 45 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <400> 6 tgagcattac ctgcaggttt ccc 23 <210> 7 <211> 43 <212> DNA <213> Artificial Sequence <400> 7 gaaggtgacc aagttcatgc tcgaatatgc ttgatagtga gat 43 <210> 8 <211> 43 <212> DNA <213> Artificial Sequence <400> 8 gaaggtcgga gtcaacggat tcgaatatgc ttgatagtga gaa 43 <210> 9 <211> twenty two <212> DNA <213> Artificial Sequence <400> 9 aggcccagag ctcgatcaat aa 22 <210> 10 <211> 42 <212> DNA <213> Artificial Sequence <400> 10 gaaggtgacc aagttcatgc ttgtactctt atcggggatat at 42 <210> 11 <211> 42 <212> DNA <213> Artificial Sequence <400> 11 gaaggtcgga gtcaacggat ttgtactctt atcggggatat aa 42 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <400> 12 atgtagtgta tgtttagagg gtgaga 26 <210> 13 <211> 44 <212> DNA <213> Artificial Sequence <400> 13 gaaggtgacc aagttcatgc tcacatcacc aagaacttca tgag 44 <210> 14 <211> 44 <212> DNA <213> Artificial Sequence <400> 14 gaaggtcgga gtcaacggat tcacatcacc aagaacttca tgac 44 <210> 15 <211> twenty two <212> DNA <213> Artificial Sequence <400> 15 gaccgcaatt taaaaccacg at 22 <210> 16 <211> 44 <212> DNA <213> Artificial Sequence <400> 16 gaaggtgacc aagttcatgc tggtttcttt ggttttgctt tcta <210> 17 <211> 44 <212> DNA <213> Artificial Sequence <400> 17 gaaggtcgga gtcaacggat tggtttcttt ggttttgctt tctc <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <400> 18 ccataccaaa gcaatgccac ccg <210> 19 <211> 46 <212> DNA <213> Artificial Sequence <400> 19 gaaggtgacc aagttcatgc tggtcgtgga ggttcgataa taaaaa <210> 20 <211> 46 <212> DNA <213> Artificial Sequence <400> 20 gaaggtcgga gtcaacggat tggtcgtgga ggttcgataa taaaag <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <400> 21 catagattcc tttaccatcc tc <210> 22 <211> 46 <212> DNA <213> Artificial Sequence <400> twenty two gaaggtgacc aagttcatgc tacacttttc catctgcagg ctgaca 46 <210> twenty three <211> 46 <212> DNA <213> Artificial Sequence <400> twenty three gaaggtcgga gtcaacggat tacacttttc catctgcagg ctgact 46 <210> twenty four <211> 29 <212> DNA <213> Artificial Sequence <400> twenty four caacacagaa acaaaaaacc taagattac 29 <210> 25 <211> 46 <212> DNA <213> Artificial Sequence <400> 25 gaaggtgacc aagttcatgc tgccaattga aatctccttc tcctgg 46 <210> 26 <211> 46 <212> DNA <213> Artificial Sequence <400> 26 gaaggtcgga gtcaacggat tgccaattga aatctccttc tcctgc 46 <210> 27 <211> twenty four <212> DNA <213> Artificial Sequence <400> 27 ggttttcgaa cagagtaaga tccg 24 <210> 28 <211> 42 <212> DNA <213> Artificial Sequence <400> 28 gaaggtgacc aagttcatgc tcaacccacc atgccatcat ta 42 <210> 29 <211> 42 <212> DNA <213> Artificial Sequence <400> 29 gaaggtcgga gtcaacggat tcaacccacc atgccatcat tt 42 <210> 30 <211> twenty four <212> DNA <213> Artificial Sequence <400> 30 ccagcaaaag cagtatctaa gaag 24 <210> 31 <211> 44 <212> DNA <213> Artificial Sequence <400> 31 gaaggtgacc aagttcatgc tggaggaaca tgatgtcttt gtct 44 <210> 32 <211> 44 <212> DNA <213> Artificial Sequence <400> 32 gaaggtcgga gtcaacggat tggaggaaca tgatgtcttt gtcc 44 <210> 33 <211> twenty two <212> DNA <213> Artificial Sequence <400> 33 tagagcaatt cctccctcaa ta 22 <210> 34 <211> 42 <212> DNA <213> Artificial Sequence <400> 34 gaaggtgacc aagttcatgc tatgtacagg agatgggaaa at 42 <210> 35 <211> 42 <212> DNA <213> Artificial Sequence <400> 35 gaaggtcgga gtcaacggat tatgtacagg agatgggaaa ac 42 <210> 36 <211> twenty two <212> DNA <213> Artificial Sequence <400> 36 gacctcactg ctttcctgga ct 22 <210> 37 <211> 45 <212> DNA <213> Artificial Sequence <400> 37 gaaggtgacc aagttcatgc tgactcattt atccaatctt cagcc 45 <210> 38 <211> 45 <212> DNA <213> Artificial Sequence <400> 38 gaaggtcgga gtcaacggat tgactcattt atccaatctt cagct 45 <210> 39 <211> twenty two <212> DNA <213> Artificial Sequence <400> 39 gggtgatgag gggtttgagt tg 22 <210> 40 <211> 46 <212> DNA <213> Artificial Sequence <400> 40 gaaggtgacc aagttcatgc tggtccacat tttgaaacat ggaata 46 <210> 41 <211> 46 <212> DNA <213> Artificial Sequence <400> 41 gaaggtcgga gtcaacggat tggtccacat tttgaaacat ggaatg 46 <210> 42 <211> twenty two <212> DNA <213> Artificial Sequence <400> 42 ttaagtctct tctcccctcg tt 22 <210> 43 <211> 42 <212> DNA <213> Artificial Sequence <400> 43 gaaggtgacc aagttcatgc tacagattct cctccttgag ca 42 <210> 44 <211> 42 <212> DNA <213> Artificial Sequence <400> 44 gaaggtcgga gtcaacggat tacagattct cctccttgag cg 42 <210> 45 <211> 29 <212> DNA <213> Artificial Sequence <400> 45 tcaaactact aacaatttta taacaggag 29 <210> 46 <211> 43 <212> DNA <213> Artificial Sequence <400> 46 gaaggtgacc aagttcatgc tggcttagaa ggttgaacat cca 43 <210> 47 <211> 43 <212> DNA <213> Artificial Sequence <400> 47 gaaggtcgga gtcaacggat tggcttagaa ggttgaacat ccg 43 <210> 48 <211> twenty two <212> DNA <213> Artificial Sequence <400> 48 tcagtgtcgt gtgagtgtct tg 22 <210> 49 <211> 42 <212> DNA <213> Artificial Sequence <400> 49 gaaggtgacc aagttcatgc tagaacactc gaatcggcca ac 42 <210> 50 <211> 42 <212> DNA <213> Artificial Sequence <400> 50 gaaggtcgga gtcaacggat tagaacactc gaatcggcca ag 42 <210> 51 <211> 26 <212> DNA <213> Artificial Sequence <400> 51 atgaaggaaa aggcatgcgg ttagca 26 <210> 52 <211> 40 <212> DNA <213> Artificial Sequence <400> 52 gaaggtgacc aagttcatgc ttcagtgact cctctgcctc 40 <210> 53 <211> 40 <212> DNA <213> Artificial Sequence <400> 53 gaaggtcgga gtcaacggat ttcagtgact cctctgcctg 40 <210> 54 <211> twenty three <212> DNA <213> Artificial Sequence <400> 54 tcttcttcac atgttgcttg caa 23 <210> 55 <211> 46 <212> DNA <213> Artificial Sequence <400> 55 gaaggtgacc aagttcatgc tcagtgatac aacacagagg gttgac 46 <210> 56 <211> 46 <212> DNA <213> Artificial Sequence <400> 56 gaaggtcgga gtcaacggat tcagtgatac aacacagagg gttgaa 46 <210> 57 <211> twenty three <212> DNA <213> Artificial Sequence <400> 57 gagtaggaag tatcatcctg caa 23 <210> 58 <211> 44 <212> DNA <213> Artificial Sequence <400> 58 gaaggtgacc aagttcatgc tacagcctat tgggtgtttg cctg 44 <210> 59 <211> 44 <212> DNA <213> Artificial Sequence <400> 59 gaaggtcgga gtcaacggat tacagcctat tgggtgtttg ccta 44 <210> 60 <211> twenty two <212> DNA <213> Artificial Sequence <400> 60 gcaactcgtt gtgcttcgtg ac 22 <210> 61 <211> 44 <212> DNA <213> Artificial Sequence <400> 61 gaaggtgacc aagttcatgc tcaataatcc ttctttggat ttct 44 <210> 62 <211> 44 <212> DNA <213> Artificial Sequence <400> 62 gaaggtcgga gtcaacggat tcaataatcc ttctttggat ttcc 44 <210> 63 <211> 25 <212> DNA <213> Artificial Sequence <400> 63 attgtgaaca ttgttctcaa acatg 25 <210> 64 <211> 40 <212> DNA <213> Artificial Sequence <400> 64 gaaggtgacc aagttcatgc tgcaattcac tcatcacttc 40 <210> 65 <211> 40 <212> DNA <213> Artificial Sequence <400> 65 gaaggtcgga gtcaacggat tgcaattcac tcatcacttg 40 <210> 66 <211> twenty two <212> DNA <213> Artificial Sequence <400> 66 ggagggtaaa ggtattgcca tg 22 <210> 67 <211> 45 <212> DNA <213> Artificial Sequence <400> 67 gaaggtgacc aagttcatgc tgtgggaaag aaatttttgc tgaac 45 <210> 68 <211> 45 <212> DNA <213> Artificial Sequence <400> 68 gaaggtcgga gtcaacggat tgtgggaaag aaatttttgc tgaat 45 <210> 69 <211> twenty two <212> DNA <213> Artificial Sequence <400> 69 ttctactcgg attcggtatg ag 22 <210> 70 <211> 42 <212> DNA <213> Artificial Sequence <400> 70 gaaggtgacc aagttcatgc tcttcaatgc catgcttgta ag 42 <210> 71 <211> 42 <212> DNA <213> Artificial Sequence <400> 71 gaaggtcgga gtcaacggat tcttcaatgc catgcttgta ac 42 <210> 72 <211> twenty two <212> DNA <213> Artificial Sequence <400> 72 tgcagcaaga ctcagaaaaa tt 22 <210> 73 <211> 45 <212> DNA <213> Artificial Sequence <400> 73 gaaggtgacc aagttcatgc tcggaataat aattcacaac tctag 45 <210> 74 <211> 45 <212> DNA <213> Artificial Sequence <400> 74 gaaggtcgga gtcaacggat tcggaataat aattcacaac tctaa 45 <210> 75 <211> twenty three <212> DNA <213> Artificial Sequence <400> 75 atatttttgc cacggccggc cga 23 <210> 76 <211> 100 <212> DNA <213> Artificial Sequence <400> 76 tttaattctc attagcctca ttgccgcatt ccttatcact acatagttat ccatgaattc 60 ctcatccacg acatcgaagc ctggaagctc caagactttc 100 <210> 77 <211> 100 <212> DNA <213> Artificial Sequence <400> 77 aaaccaagtc accatatggt atccatgttg ctcccttctt attgatcacc atcatgtgat 60 ggatcacaat actggcccaa tttgtttcaa ttttgttggc 100 <210> 78 <211> 100 <212> DNA <213> Artificial Sequence <400> 78 aatttggaag agatactgtg aaaataagta gagatttttg aaaagtttct cttatgagca 60 ttacctgcag gtttcccatt tagcaggctt atgatcagga 100 <210> 79 <211> 100 <212> DNA <213> Artificial Sequence <400> 79 caacatgtag agtcctttgg acctcgggta gattgaagta tatagtgctg tgcttctcgg 60 tgcaaggatc atacgcactt ctgagtcttc ggataccctg 100 <210> 80 <211> 100 <212> DNA <213> Artificial Sequence <400> 80 tctcaaaata gtttgcctac aacaattaat gccaaaactg atgtctcaga tgatttctct 60 gccaaagatt caagtacaac gaatatgctt gatagtgaga 100 <210> 81 <211> 100 <212> DNA <213> Artificial Sequence <400> 81 attatccaac attggtggaa atggcttagc atctgtctcc cctcaacttg aagaacttat 60 tgatcgagct ctgggccttg gatctgttgc caaatcaaat 100 <210> 82 <211> 100 <212> DNA <213> Artificial Sequence <400> 82 gtggaggttt gttgaaatca tcttcattta agaatatgtt atcgtaactc gacggaggcg 60 acgatggatc ttcagaattt tgtactctta tcgggatata 100 <210> 83 <211> 100 <212> DNA <213> Artificial Sequence <400> 83 tcctggaata gtagaagtta caattagtct caccctctaa acatacacta catgtacatg 60 accgtctttt tattaccgca tatggcccga atatgtggtt 100 <210> 84 <211> 100 <212> DNA <213> Artificial Sequence <400> 84 gtgaccgcaa tttaaaagca accgtgaccg caatttaaaa ccacgatata tgtaaataat 60 atacatatat atctaattac atgccttgat gttaggaaga 100 <210> 85 <211> 100 <212> DNA <213> Artificial Sequence <400> 85 tcatgaagtt cttggtgatg tgaacaacta gcttgtccat aaaagcagga gcaatgtaaa 60 atccatccat catattgtca aggttgtacc tgaaataacc 100 <210> 86 <211> 100 <212> DNA <213> Artificial Sequence <400> 86 gataagagac cagatatctt tattgtaatg taaacttggt ctgaccctaa caatttaatt 60 cgtacttttt ctagattagg tttctttggt tttgctttct 100 <210> 87 <211> 100 <212> DNA <213> Artificial Sequence <400> 87 tgaatctcga ggctttgcgg gtggcattgc tttggtatgg gaatctaatt gaatgtaaat 60 gaccatttta aagcaacact tttaattcct tcactctaaa 100 <210> 88 <211> 100 <212> DNA <213> Artificial Sequence <400> 88 tttccgatta gtactccgca taatgaagag gttgcaaatg cggttaagat cattgaagaa 60 cacttgaagg ttcatcggtc gtggaggttc gataataaaa 100 <210> 89 <211> 100 <212> DNA <213> Artificial Sequence <400> 89 caatgcaagt tgtgaggatg gtaaaggaat ctatgtgtat gatttaccat ctaagtttaa 60 caaggatttg gttggtcagt gcagtgacat gcttccatgg 100 <210> 90 <211> 100 <212> DNA <213> Artificial Sequence <400> 90 ttatgaagaa acaaattgta tggaaatttc caacacagaa acaaaaaacc taagattacc 60 tttctccatg gaccagctct tgatgaggaa tgagtctggc 100 <210> 91 <211> 100 <212> DNA <213> Artificial Sequence <400> 91 gtcagcctgc agatggaaaa gtgtaaaggg aagataactt agccaagcag aatgaccttg 60 gatgaatagc aatacgaaaa agaaaataaa aaaaatctga 100 <210> 92 <211> 100 <212> DNA <213> Artificial Sequence <400> 92 gctaaaatca tgttagtaga tgcttgtttt ttgttagagc ttctcatatc aaaagagtta 60 gatcatgaac taccatgcca attgaaatct ccttctcctg 100 <210> 93 <211> 100 <212> DNA <213> Artificial Sequence <400> 93 acctcaactc ctgagagacg aagatgtctt gtcggatctt actctgttcg aaaaccagat 60 cccggttttc gtcctccacg agctttctcg aaagcttttc 100 <210> 94 <211> 100 <212> DNA <213> Artificial Sequence <400> 94 tggtgtagga aacagtccaa tcaaagtaaa caaaagggtc accagcaaaa gcagtatcta 60 agaagaaaca tagaagtgtt gtgaagaacc aacaagagga 100 <210> 95 <211> 100 <212> DNA <213> Artificial Sequence <400> 95 aatgatggca tggtgggttg aaagtactat cgcttatctt ctcaagatgt atagagagag 60 ggaggaagaa ggtgttgagg tgaatgaaag ttggcagaga 100 <210> 96 <211> 100 <212> DNA <213> Artificial Sequence <400> 96 aagtaaacaa agtgttaacc ttcattagga ggtgggtgtg gcagtgtctt ggaagaagaa 60 ccaggtggag atactctagg aggaacatga tgtctttgtc 100 <210> 97 <211> 100 <212> DNA <213> Artificial Sequence <400> 97 accagttgtt ggtgatattg agggaggaat tgctctatct agatcaaaaa ttgtaaaatt 60 aaaactactt atatattcag ttgcagccaa gaactttatt 100 <210> 98 <211> 100 <212> DNA <213> Artificial Sequence <400> 98 tttcgttaaa ttaataaaac atgattattt gaaaacattg atcacaaaat ttgcaaaaac 60 aaagagtgta ttgaggtgtt atgtacagga gatgggaaaa 100 <210> 99 <211> 100 <212> DNA <213> Artificial Sequence <400> 99 tgtgcgtcca gttgacgagt ccaggaaagc agtgaggtca accatgtctt ctacatttaa 60 attgatgccc gtctcttctt caacctgtta gaatcacaac 100 <210> 100 <211> 100 <212> DNA <213> Artificial Sequence <400> 100 accacctatt tctcctatcc atttccttga tataagcacc tttattaaca tgcacaatat 60 catagttatt ttcaaatgac tcatttatcc aatcttcagc 100 <210> 101 <211> 100 <212> DNA <213> Artificial Sequence <400> 101 attgtcaata tctccaactc aaacccctca tcacccctct tttggtttct attaccgttt 60 agttcttctt tgaaaaattc caacaaaact atacctaaat 100 <210> 102 <211> 100 <212> DNA <213> Artificial Sequence <400> 102 gtggtgaatg tgtgaagaac cttgtatgac catttctgcc atgttaagtc tcttctcccc 60 tcgttgagac cattcaacga aattggacca agaacaccag 100 <210> 103 <211> 100 <212> DNA <213> Artificial Sequence <400> 103 attccatgtt tcaaaatgtg gaccaacatt ctgcatataa atgcgccaaa tgattagcat 60 ggactaaagt tcatattttt agacacgggg atgcttagag 100 <210> 104 <211> 100 <212> DNA <213> Artificial Sequence <400> 104 atcttcttca tctggttatt caaattcaaa ctactaacaa ttttataaca ggagattatg 60 aaatcttgtc caaagaaaga cattgagaat ttcaaagatt 100 <210> 105 <211> 100 <212> DNA <213> Artificial Sequence <400> 105 gctcaaggag gagaatctgt atataaatat agaggtaagt ataattaacg aaataattcg 60 atttttctga aatttgaatt ttgaatcgat atttttgtta 100 <210> 106 <211> 100 <212> DNA <213> Artificial Sequence <400> 106 aatgaataca ttgtacgtat tcacaggtgt tagtaatcac aagtgtcagt gtcgtgtgag 60 tgtcttgagg atttaacatt agtgatcatc accacgaata 100 <210> 107 <211> 100 <212> DNA <213> Artificial Sequence <400> 107 ggatgttcaa ccttctaagc caaatagaat gaatagtcat atggaaaata tcgaacctct 60 ttggtggctc attaggaaca aaatgccttt gaacttgttt 100 <210> 108 <211> 100 <212> DNA <213> Artificial Sequence <400> 108 gctggagttg ttcgcgtgtt gcggatcagt gtgccagtat tccgatgaag gaaaaggcat 60 gcggttagca gttgcatgtg ctgtggaatg agttgccccg 100 <210> 109 <211> 100 <212> DNA <213> Artificial Sequence <400> 109 ttggccgatt cgagtgttct aaagagaggg acttggtatc gtatgcaccc catgaattgg 60 ttgacagaag agagagagca cggttaacat cttgcgtagc 100 <210> 110 <211> 100 <212> DNA <213> Artificial Sequence <400> 110 acaacacctt aaaaaccgtg ttagatcgta ttctcaagca aagaatctct cttcttcttc 60 acatgttgct tgcaaattga aggatgagaa gaaggcaaag 100 <210> 111 <211> 100 <212> DNA <213> Artificial Sequence <400> 111 aggcagagga gtcactgagg actgtcatgt acttgagctg ctggggtccc aattgattca 60 ttgctacata tatagtgata caagattaat tatgtctaat 100 <210> 112 <211> 100 <212> DNA <213> Artificial Sequence <400> 112 tctcagttaa ttaatttagt aaaatatgca ttgaactatt tcaggaactg catgagaaaa 60 gtggttcgcc aaaggacagt gatacaacac agagggttga 100 <210> 113 <211> 100 <212> DNA <213> Artificial Sequence <400> 113 agaaaaacca ttcaaacttc aacaggttca gcaactttta gtattgcagg atgatacttc 60 ctactctact attatgcatc aataaattgt ctattagatg 100 <210> 114 <211> 100 <212> DNA <213> Artificial Sequence <400> 114 ttcagatctt gcacagtttt gagaagcaac tcgttgtgct tcgtgacgag catgtttaac 60 gtttcattgc acttagcaaa agaagatggt gtagttatta 100 <210> 115 <211> 100 <212> DNA <213> Artificial Sequence <400> 115 aggcaaacac ccaataggct gtaataatcc aactgttatt ttctttattc ctacgctatg 60 aatgcgttta agatttaatg acaattggtt tataagtgaa 100 <210> 116 <211> 100 <212> DNA <213> Artificial Sequence <400> 116 tagaaattgt cttggatttg acaattgtga acattgttct caaacatgtt tgcaacattt 60 tttgaggttt ctgaagtttc tagtggctgt gaaaattgga 100 <210> 117 <211> 100 <212> DNA <213> Artificial Sequence <400> 117 gaaatccaaa gaaggattat tgaagctggt tgacacatga tttccacttg aagcatctat 60 gaatcctctt tgtgaaggaa cattgaagct tgttgagatt 100 <210> 118 <211> 100 <212> DNA <213> Artificial Sequence <400> 118 attagatagc ttagagtttg acatatccat ataaaatata acattcaagc tagcttctcc 60 tcaagtagaa gaaaaaaaag atgcaattca ctcatcactt 100 <210> 119 <211> 100 <212> DNA <213> Artificial Sequence <400> 119 ataagatcat tggcaatagg tagtgcttta cacagagcct taaatgggat ctttttaggc 60 atggcaatac ctttaccctc catcatatca agtttctcct 100 <210> 120 <211> 100 <212> DNA <213> Artificial Sequence <400> 120 ggcttgttct aacacatttg gacttgttac tggtgcattt ctactcggat tcggtatgag 60 tgaaattcca aagggtattt ggttgaatgc aaattggacc 100 <210> 121 <211> 100 <212> DNA <213> Artificial Sequence <400> 121 ttcagcaaaa atttctttcc cacaaagttg caaaaatggc tgtcaaatta gacgacgctc 60 atcaagattt ttcaaatgct atcgttgtaa gttattcata 100 <210> 122 <211> 100 <212> DNA <213> Artificial Sequence <400> 122 gttgtattaa aagaccagta gctaaacaga aggtaaataa ccataatcta ggcaacacat 60 aattgtttga tgtactattt cttcaatgcc atgcttgtaa 100 <210> 123 <211> 100 <212> DNA <213> Artificial Sequence <400> 123 taaaacccaa atcttttagc ttctttgatg aaatttcaga aggaacctta tcataatttt 60 tctgagtctt gctgcatgta tatgtaggaa taacatcaga 100 <210> 124 <211> 100 <212> DNA <213> Artificial Sequence <400> 124 gatagaagtt ttgatgaaaa cggttcttga atatttttgc cacggccggc cgaggtacga 60 cttaaccgag ctagggttta ggtccgacgc ggccgtgacc 100 <210> 125 <211> 100 <212> DNA <213> Artificial Sequence <400> 125 tagagttgtg aattattatt ccggtgtttt ggtttggatc ggttcggcct tgagccgtaa 60 cagtgattgt tttttgagga gggtttctcg cgataatgtt 100

Claims

1. A KASP primer combinatorial for detecting SNP molecular markers in pea cores, characterized in that, The primer combination includes all of the 25 primer pairs corresponding to SNPs 1 to 25. Each primer pair includes forward primer 1, forward primer 2, and reverse primer, as detailed below: 。 2. The primer combination as described in claim 1, characterized in that, The 5' end of forward primer 1 is tagged with FAM fluorescent signal; the 5' end of forward primer 2 is tagged with HEX fluorescent signal.

3. A reagent, chip, or kit containing the primer combination as described in claim 1 or 2.

4. A method for detecting pea genotypes using primer combinations as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Extract DNA from the pea variety to be tested; (2) Using the DNA from step (1) as a template, add the primer combination Primer mix and KASP Master mix to perform PCR amplification; the Primer mix is ​​the primer combination described in claim 1 or 2; (3) Detect and analyze the amplification results to determine the genotype of the pea variety to be tested at the SNP molecular marker site corresponding to each set of primers.

5. The method as described in claim 4, characterized in that, The PCR amplification system described in step (2) is as follows: 0.8 μl of 5~10 ng / μl pea genomic DNA, 0.8 μl of KASP Master Mix, and 0.05 μl of Primer mix.

6. The method as described in claim 5, characterized in that, The PCR amplification program in step (2) is as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s; 61℃ annealing / extension for 60 s, with the annealing temperature decreasing by 0.6℃ for each cycle, for a total of 10 cycles; then 94℃ denaturation for 20 s, 55℃ annealing / extension for 60 s, for a total of 26 cycles.

7. The method as described in claim 6, characterized in that, The PCR described in step (2) is performed using the high-throughput IntelliQube genotyping platform.

8. The method as described in claim 7, characterized in that, Step 3) Perform analysis using LGC's data analysis software Kraken or KlusterCaller.

9. The use of a KASP-based pea core SNP molecular marker set, or the primer combination of claim 1 or 2, or the reagent, chip, or kit of claim 3, in identifying pea genotypes, pea variety authenticity, pea variety purity, constructing a pea SNP fingerprint library, and analyzing pea genetic diversity. The molecular marker set includes 25 core SNP markers, numbered SNP1 to SNP25; the pea genome corresponding to each SNP is Pisum sativum v1a; SNP1 to SNP25 are shown below: 。

Images