Patent Publication Number: US-2022228227-A1

Title: Two PARMS-SNP Molecular Markers for Identifying Resistant Gene VrTAF5 of Vigna radiata (Linn.) Wilczek Cercospora Leaf Spot Disease

Description:
TECHNICAL FIELD 
     The invention relates to the field of molecular genetic breeding, in particular to two PARMS-SNP (Penta-primer amplification refractory mutation system-single nucleotide polymorphism) molecular markers for identifying a resistant gene VrTAF5 of  Vigna radiata  (Linn.) Wilczek  Cercospora  leaf spot disease (CLS). 
     BACKGROUND 
       Vigna radiata  (Linn.) Wilczek leaf spot disease is the main fungal disease in the growth of  Vigna radiata  (Linn.) Wilczek, which occurs in China and other  Vigna radiata  (Linn.) Wilczek producing area in Asia. The disease is caused by several species of  Cercospora , among which  Cercospora canescens  Ellis and Martin is the main pathogen, which mainly infects the leaves of plants, and suffers the most in flowering and pod bearing period. Selecting resistant varieties can effectively reduce the occurrence of diseases in  Vigna radiata  (Linn.) Wilczek planting. Although many germplasm resources of  Vigna radiata  (Linn.) Wilczek resistant to leaf spot disease have been found, however, only one study has reported QTL-qCLS, which can explain 65.5-80.5% of phenotypic variation of resistance. 
     Phenotypic identification of  Vigna radiata  (Linn.) Wilczek leaf spot disease costs lots of time and laborious, and identification in field is easily affected by environment. Therefore, compared with the phenotypic identification, molecular markers relate to disease resistance traits have the advantages of accuracy, economy and rapidity. Functional molecular markers are molecular markers developed according to polymorphic sequences within functional genes closely relate to phenotypic traits with advantages that they can accurately and reliably lock target genes, accurately respond to genetic variations of functional alleles, and have more reliable genetic effects. Single nucleotide polymorphism (SNP) is a polymorphism produced by single nucleotide variation, so it is difficult to distinguish its polymorphism by using the difference in length between conventional Polymerase chain reaction (PCR) and gel electrophoresis. Penta-primer amplification refractory mutation system (PARMS) is a new SNP typing technology, which has lower cost than imported detection reagent Kompetitive Allele Specific PCR (KASP), and when DNA is alkaline cracked, the PARMS is more densely clustered than the KASP. 
     SUMMARY 
     In order to solve the problems existing in the prior art, an objective of the invention is to provide PARMS-SNP molecular markers for identifying resistant gene VrTAF5 of  Vigna radiata  (Linn.) Wilczek  Cercospora  leaf spot disease and a primer thereof, which can quickly identify  Vigna radiata  (Linn.) Wilczek resistant varieties, and meet needs of assisting  Vigna radiata  (Linn.) Wilczek molecular breeding and shortening a breeding time. 
     To realize the above objective, in an aspect, the invention provides two PARMS-SNP molecular markers for identifying the resistant gene VrTAF5 of  Vigna radiata  (Linn.) Wilczek  Cercospora  leaf spot disease, which include SNP sites PARMS-1517 and PARMS-10010; a 240 bp sequence from before to after the SNP site PARMS-1517 is shown in SEQ ID NO.1, a 131st position is the SNP site PARMS-1517, and a polymorphism is A/C; a 240 bp sequence from before to after the SNP site PARMS-10010 is shown in SEQ ID NO.2, a 117th position is the SNP site PARMS-10010, and a polymorphism is A/G. 
     In another aspect, the invention further provides a primer set for identifying the two PARMS-SNP molecular markers, and the primer set includes a PARMS-1517 primer set and a PARMS-10010 primer set. The PARMS-1517 primer set includes primers PARMS- 1517F1, PARMS-1517F2 and PARMS-1517R: nucleotide sequences of PARMS-1517F1 are shown in SEQ ID NO.3, that of PARMS-1517F2 are shown in SEQ ID NO.4, and that of PARMS-1517R are shown in SEQ ID NO.5. The PARMS-10010 primer set includes primers PARMS-10010F1, PARMS-10010F2 and PARMS-10010R: nucleotide sequences of PARMS-10010F1 are as shown in SEQ ID NO.6, that of PARMS-10010F2 are as shown in SEQ ID NO.7, and that of PARMS-10010R are as shown in SEQ ID NO.8. 
     In still another aspect, the invention further provides an application of the two PARMS-SNP molecular markers or the primer set in assisting  Vigna radiata  (Linn.) Wilczek breeding. 
     In even another aspect, the invention further provides an application of the two PARMS-SNP molecular markers in identifying the resistant gene VrTAF5 of  Vigna radiata  (Linn.) Wilczek  Cercospora  leaf spot disease. 
     In further another aspect, the invention further provides an application method of the primer set, the primer set is applied to identify the resistant gene VrTAF5 of  Vigna radiata  (Linn.) Wilczek  Cercospora  leaf spot disease. 
     In an embodiment of the invention, specific steps for identifying the resistant gene VrTAF5 of  Vigna radiata  (Linn.) Wilczek  Cercospora  leaf spot disease are as follows: performing a PCR amplification on  Vigna radiata  (Linn.) Wilczek genome DNA by using the primer set; reading fluorescent signals after the PCR amplification, analyzing and converting the fluorescent signals, and identifying the analyzed and converted fluorescent signals to obtain genotypes belonging to susceptible homozygous AA type, resistant homozygous CC or GG type, and heterozygous CA or AG type. 
     In an embodiment of the invention, a PCR amplification reaction system of the PARMS-1517 primer set contains 1 μL of template DNA, 5 μL of 2×PARMS master mix, 0.15 μL of primer PARMS-1517F1 in 10 μM, 0.15 μL of primer PARMS-1517F2 in 10 μM, 0.4 μL of primer PARMS-1517R in 10 μM and 3.3 μL of ddH 2 O. 
     A PCR amplification reaction system of PARMS-10010 primer set contains 1 μL of template DNA, 5 μL of 2×PARMS master mix, 0.15 μL of primer PARMS-10010F1 in 10 μM, 0.15 μL of primer PARMS-10010F2 in 10 μM, 0.4 μL of primer PARMS-10010R in 10 μM and 3.3 μL of ddH 2 O. 
     In an embodiment of the invention, procedures of the PCR amplification are as follow: thermal activating at 94° C. for 15 min, denaturing at 94° C. for 20 s, annealing and extension at 57-65° C. for 1 min, 10 cycles; denaturing at 94° C. for 20 s, annealing and extending at 57° C. for 1 min, 30 cycles. 
     In an embodiment of the invention, the fluorescence signals are read by TECAN infinite M1000 microplate reader. On-line software SNP Decoder (http://www.snpway.com/snpdecoder/) is used to analyze and convert the fluorescence signals. 
     In even further another aspect, the invention further provides a  Vigna radiata  (Linn.) Wilczek  Cercospora  leaf spot disease resistance detection kit, which includes the primer set for identifying the two PARMS-SNP molecule makers. 
     The invention discloses the following technical effects: the invention provides two PARMS-SNP molecular markers for identifying a resistant gene VrTAF5 of  Vigna radiata  (Linn.) Wilczek  Cercospora  leaf spot disease and a primer set for identifying the two PARMS-SNP molecular markers, which have the characteristics of specificity, accuracy, reliability, simple operation so as to quickly identify  Vigna radiata  (Linn.) Wilczek resistant varieties, meet the needs of assisting  Vigna radiata  (Linn.) Wilczek molecular breeding and shorten a breeding period. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
       In order to explain the embodiments of the invention or the technical solution in the prior art more clearly, the following will briefly introduce the drawings used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the invention, and for those skilled in the art, other drawings can be obtained according to these drawings without paying creative labor. 
         FIG. 1  is a comparative picture of proteins encoded by resistant gene VrTAF5 of  Vigna radiata  (Linn.) Wilczek  Cercospora  leaf spot disease of resistant parent V4718 and susceptible parent KPS1. 
         FIG. 2A  is PARMS-1517 typing diagram of 186 samples to be tested. 
         FIG. 2B  is PARMS-10010 typing diagram of 186 samples to be tested. 
     
    
    
     DETAILED DESCRIPTION OF EMBODIMENTS 
     The following will clearly and completely describe the technical solution in the embodiments of the invention with reference to the drawings in the embodiments of the invention. Obviously, the described embodiments are only part of the embodiments of the invention, not all of them. Based on the embodiments of the invention, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the invention. 
     In order to make the above objectives, features and advantages of the invention clearer and easier to understand, the invention will be further explained in detail with reference to the drawings and illustrated embodiments. 
     Embodiment 1 
     Design PARMS-SNP Molecular Marker of Resistant Gene VrTAF5 of  Vigna radiata  (Linn.) Wilczek  Cercospora  Leaf Spot Disease 
     The major QTL-qCLS has been finely located in genomic region of −13 kilobases (Kb) on chromosome 6 by using BC 8 F 2  and F2 populations of  Vigna radiata  (Linn.) Wilczek  Cercospora  leaf spot disease resistant variety V4718 and susceptible variety KPS1. The genomic region contains only one annotated gene LOC106765332 (named “VrTAF5”) encoding TATA binding protein related factor 5(TAFS), which is a subunit of TFIID and SAGA complex. 
     Sequencing the Sequences of Resistant Gene VrTAF5 in the Susceptible Variety KPS1 and the Resistant Variety V4718 
     Comparing the sequences of the resistant gene VrTAF5 between the two parents mentioned above, it shows multiple single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels), among which 8 SNPs exist in 8 different exons, while one SNP(G4,932C) exists in exon 8, which causes amino acid mutation (S250T). The mutation results are shown in  FIG. 1 , and the mutation sites are marked by horizontal lines in  FIG. 1 . 
     Seven PARMS-SNP molecular markers: PARMS-1517, PARMS-1739, PARMS-4932, PARMS-9548, PARMS-9884, PARMS-10010 and PARMS-10228 are designed and developed. According to the seven single nucleotide differences of gene sequences between the V4718 and the KPS1, PARMS primer sets containing seven SNP differences are designed by Primer5.0, and each primer set contains two specific primers and one universal primer. 
     According to the invention, PARMS-SNP detection technology and a high-throughput SNP detection platform GeneMatrix are adopted for sample analysis and detection. 
     According to the results of resistance evaluation, the PARMS-1517 and the PARMS-10010 have a higher screening rate of molecular markers. The PARMS-1517 is based on the nucleotide difference at position 32622352 on chromosome 6 of  Vigna radiata  (Linn.) Wilczek. The sequence of 240 base pairs (bp) before and after the PARMS-1517 is shown in Sequence (SEQ) ID NO.1, and the 131st position is the PARMS-1517, with polymorphism of Adenine/Cytosine (A/C). The PARMS-10010 is based on the nucleotide difference at position 32613913 on chromosome 6 of  Vigna radiata  (Linn.) Wilczek. The sequence of 240 bp before and after the PARMS-10010 is shown in SEQ ID NO.2, and the 117th position is the PARMS-10010 with polymorphism of Adenine/Guanine (A/G), which can be used to identify the resistance of  Vigna radiata  (Linn.) Wilczek varieties to  Cercospora  leaf spot disease. 
     Primer sequences of PARMS-1517 molecular markers are shown as follows: 
     
       
         
           
               
            
               
                 Primer 1: PARMS-1517F1: 
               
               
                 5′ GAAGGTGACCAAGTTCATGCTGTTCTGCTAGATACCACGATGGA 3′ 
               
               
                   
               
               
                 Primer 2: PARMS-1517F2: 
               
               
                 5′ GAAGGTCGGAGTCAACGGATTTTCTGCTAGATACCACGATGGC 3′ 
               
               
                   
               
               
                 Primer 3: PARMS-1517R: 
               
               
                 5′ GACCTATATGCCCATGATCTCAG 3′. 
               
            
           
         
       
     
     Primer sequences of PARMS-10010 molecular markers are shown as follows: 
     
       
         
           
               
            
               
                 Primer 1: PARMS-10010F1: 
               
               
                 5′GAAGGTGACCAAGTTCATGCTTTTAATGATCTGAGTCTGTTAGCAT 
               
               
                 TT 3′ 
               
               
                   
               
               
                 Primer 2: PARMS-10010F2: 
               
               
                 5′GAAGGTCGGAGTCAACGGATTTTTAATGATCTGAGTCTGTTAGCAT 
               
               
                 TC 3′ 
               
               
                   
               
               
                 Primer 3: PARMS-10010R: 
               
               
                 5′TCTTTAGGAATTTTCTTTTCTAATGG 3′. 
               
            
           
         
       
     
     Embodiment 2 
     Application of PARMS-SNP Markers of  Vigna radiata  (Linn.) Wilczek  Cercospora  Leaf Spot Disease Resistant Gene in Screening  Vigna radiata  (Linn.) Wilczek Resistant Varieties 
     Genome DNA of 186  Vigna radiata  (Linn.) Wilczek varieties to be detected (as shown in Table 1) is extracted, and each DNA concentration is determined by spectrophotometer. Take that DNA of  Vigna radiata  (Linn.) Wilczek variety to be detected as a template, and carry out polymerase chain reaction (PCR) amplification by using the primer set, namely PARMS reaction detection. 
     PCR system (10 microliters (μL)): 5 μL of 2×PARMS master mix, 0.15 μL of primer 1 (10 micromoles per liter (μM)) ,0.15 μL of primer 2 (10 μM), 0.4 μL of primer 3 (10 μM), 1 μL of template DNA (50 ng/μL), 3.3 μL of double distilled H 2 O (ddH 2 O). 
     PCR reaction procedures: heat activating at 94° C. for 15 minutes (min); denaturing at 94° C. for 20 seconds (s), annealing at 57-65° C., extending for 1 min, 10 cycles; denaturing at 94° C. for 20 s, annealing and extending at 57° C. for 1 min, 30 cycles. 
     After PCR, the fluorescence signals are read by TECAN infinite M1000 microplate reader. The fluorescence signals are further analysed and converted by online software SNP Decoder (http://www.snpway.com/snpdecoder/), so that a clear and intuitive typing map is obtained, and the genotype results are output according to different colors. 
     If only base A is detected at SNP site PARMS-1517, it is identified that the  Vigna radiata  (Linn.) Wilczek sample to be detected has a susceptible homozygous genotype; if only base C is detected, the  Vigna radiata  (Linn.) Wilczek sample is detected to have a resistant homozygous genotype; if both base A and base C are detected, it is determined that the  Vigna radiata  (Linn.) Wilczek sample to be detected has a heterozygous genotype. 
     If only base A is detected at SNP site PARMS-10010, it is identified that the  Vigna radiata  (Linn.) Wilczek sample to be detected has a susceptible homozygous genotype; if only base G is detected, the  Vigna radiata  (Linn.) Wilczek sample to be detected is identified to have a resistant homozygous genotype; if both base A and base G are detected, it is judged that the  Vigna radiata  (Linn.) Wilczek sample to be detected has a heterozygous genotype. 
     The indoor inoculation resistance of  Vigna radiata  (Linn.) Wilczek varieties is identified, and the specific steps are as follows: 
     the resistance of  Vigna radiata  (Linn.) Wilczek is evaluated by indoor artificial inoculation, and healthy  Vigna radiata  (Linn.) Wilczek plants are inoculated at the leaf-regrowth stage, which is kept in a moisture shed for 36 hours and then grew normally at 25° C. 
     The incidence of  Cercospora  leaf spot disease is observed 15 days after inoculation. 
     Evaluation of resistance refers to  Disease Classification Standard  (Wu Quan&#39; an, 1991): 
     Grade 0: no visible infection on leaves; 
     Grade 1: there are only small disease spots on the leaves, accounting for less than 2% of the leaf area; 
     Grade 3: the spots are small, with a diameter of 1-2 millimeters (mm) and no halo of chlorosis, accounting for 3%-25% of the leaf area; 
     Grade 5: the spot has a larger diameter of 2.1-5 mm, and has chlorotic halo, accounting for 26%-50% of the leaf area; 
     Grade 7: the disease spots are large, the diameter is more than 5.1 mm accounting for 51%-75% of the leaf area, and some leaves are dead; 
     Grade 9: the disease spots are connected into a piece, accounting for more than 76% of the leaf area, with a large number of sporulation and serious deciduous leaves. 
     Evaluation criteria of resistance (classified by disease index): 
     resistance-(R): disease index of 0-20.0; middle resistance (MR): disease index of 20.0-40.0; middle susceptibility (MS): disease index of 40.1-60.0; susceptibility (S) disease index of 60.1-80.0; high susceptibility (HS): disease index of 80.1-100. 
       Disease index=(number of disease plants at all levels×disease grade value)/(total number of investigated strains×highest level value) ×100  Calculation of disease index:
 
     The results of C/A genotype and disease index are shown in Table 1, and the genotyping results are shown in  FIG. 2A . There are 12 genotypes of CC and CA, among which 9 are resistant varieties, accounting for 75%. A total of 174 AA genotypes are detected, of which 141 are susceptible, accounting for 81.03%. 
     The corresponding results of G/A genotype and disease index are shown in Table 1, and the genotyping results are shown in  FIG. 2B . There are 12 genotypes of GG and GA, among which 9 are resistant varieties, accounting for 75%. A total of 174 AA genotypes are detected, of which 141 are susceptible, accounting for 81.03%. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Corresponding results of genotype and disease index 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Resistance 
                 PARMS- 
                 PARMS- 
               
               
                 NO. 
                 Variety 
                 evaluation 
                 1517 
                 10010 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 Xinyu 
                 HS 
                 AA 
                 AA 
               
               
                   
                 L13012 
               
               
                 2 
                 Xinyu 
                 S 
                 AA 
                 AA 
               
               
                   
                 L13013 
               
               
                 3 
                 Xinyu 
                 S 
                 AA 
                 AA 
               
               
                   
                 L13014 
               
               
                 4 
                 Xinyu 
                 S 
                 AA 
                 AA 
               
               
                   
                 L13015 
               
               
                 5 
                 Xinyu 
                 MS 
                 AA 
                 AA 
               
               
                   
                 L13016 
               
               
                 6 
                 Xinyu 
                 S 
                 AA 
                 AA 
               
               
                   
                 L13017 
               
               
                 7 
                 Xinyu 
                 MS 
                 AA 
                 AA 
               
               
                   
                 L13018 
               
               
                 8 
                 Xinyu 
                 S 
                 AA 
                 AA 
               
               
                   
                 L13019 
               
               
                 9 
                 Xinyu 
                 MS 
                 AA 
                 AA 
               
               
                   
                 L13020 
               
               
                 10 
                 Xinyu 
                 S 
                 AA 
                 AA 
               
               
                   
                 L13021 
               
               
                 11 
                 Xinyu 
                 R 
                 AA 
                 AA 
               
               
                   
                 L13022 
               
               
                 12 
                 Xinyu 
                 MR 
                 AA 
                 AA 
               
               
                   
                 L13023 
               
               
                 13 
                 L67-1 
                 S 
                 AA 
                 AA 
               
               
                 14 
                 AL68 
                 S 
                 AA 
                 AA 
               
               
                 15 
                 AL81 
                 S 
                 AA 
                 AA 
               
               
                 16 
                 Sanxia 
                 MS 
                 AA 
                 AA 
               
               
                   
                 NO. 4 
               
               
                 17 
                 Sanxia 
                 HS 
                 AA 
                 AA 
               
               
                   
                 NO. 5 
               
               
                 18 
                 Mizhi  Vigna   
                 MS 
                 AA 
                 AA 
               
               
                   
                 
                   radiata 
                 
               
               
                   
                 (Linn.) 
               
               
                   
                 Wilczek 
               
               
                 19 
                 NO. 6 Mizhi 
                 R 
                 CC 
                 GG 
               
               
                   
                 
                   Vigna 
                 
               
               
                   
                 
                   radiata 
                 
               
               
                   
                 (Linn.) 
               
               
                   
                 Wilczek 
               
               
                 20 
                 Yanglinghei 
                 R 
                 AA 
                 AA 
               
               
                   
                 
                   Vigna 
                 
               
               
                   
                 
                   radiata 
                 
               
               
                   
                 (Linn.) 
               
               
                   
                 Wilczek 
               
               
                 21 
                 Yan&#39;an 
                 MS 
                 AA 
                 AA 
               
               
                   
                 NO. 4 
               
               
                 22 
                 Sanxia 
                 MS 
                 AA 
                 AA 
               
               
                   
                 NO. 2 
               
               
                 23 
                 Sulyu NO. 1 
                 MS 
                 AA 
                 AA 
               
               
                 24 
                 Sulyu NO. 4 
                 HS 
                 AA 
                 AA 
               
               
                 25 
                 Sulyu NO. 5 
                 MS 
                 AA 
                 AA 
               
               
                 26 
                 Sulyu NO. 6 
                 MS 
                 AA 
                 AA 
               
               
                 27 
                 Sulyu NO. 7 
                 MR 
                 CC 
                 GG 
               
               
                 28 
                 Yan&#39;an 
                 HS 
                 AA 
                 AA 
               
               
                   
                 
                   Vigna 
                 
               
               
                   
                 
                   radiata 
                 
               
               
                   
                 (Linn.) 
               
               
                   
                 Wilczek 
               
               
                 29 
                 Danlyu 
                 HS 
                 AA 
                 AA 
               
               
                   
                 NO. 1 
               
               
                 30 
                 Kanglyu 
                 MR 
                 AA 
                 AA 
               
               
                   
                 NO. 4 
               
               
                 31 
                 Zhonglyu 
                 HS 
                 AA 
                 AA 
               
               
                   
                 NO. 3 
               
               
                 32 
                 Jilyu NO. 2 
                 MS 
                 AA 
                 AA 
               
               
                 33 
                 Wild 
                 HS 
                 AA 
                 AA 
               
               
                   
                 variety 1 
               
               
                 34 
                 Wild 
                 MR 
                 AA 
                 AA 
               
               
                   
                 variety 2 
               
               
                 35 
                 Wild 
                 MS 
                 AA 
                 AA 
               
               
                   
                 variety 3 
               
               
                 36 
                 Wild 
                 MS 
                 AA 
                 AA 
               
               
                   
                 variety 4 
               
               
                 38 
                 Wild 
                 S 
                 AA 
                 AA 
               
               
                   
                 variety 6 
               
               
                 39 
                 Wild 
                 MR 
                 AA 
                 AA 
               
               
                   
                 variety 7 
               
               
                 40 
                 Wild 
                 S 
                 AA 
                 AA 
               
               
                   
                 variety 8 
               
               
                 41 
                 V1197 
                 HS 
                 AA 
                 AA 
               
               
                 42 
                 Wild 
                 S 
                 AA 
                 AA 
               
               
                   
                 variety 10 
               
               
                 43 
                 V4718 
                 R 
                 CC 
                 GG 
               
               
                 44 
                 RUM5 
                 S 
                 AA 
                 AA 
               
               
                 45 
                 KPS2 
                 HS 
                 AA 
                 AA 
               
               
                 46 
                 CN84-1 
                 MR 
                 AA 
                 AA 
               
               
                 47 
                 JP229096 
                 MR 
                 AA 
                 AA 
               
               
                 48 
                 VC1560A 
                 MR 
                 AA 
                 AA 
               
               
                 49 
                 JP229175 
                 MS 
                 AA 
                 AA 
               
               
                 50 
                 V2533 
                 MS 
                 AA 
                 AA 
               
               
                 51 
                 V1067AG 
                 MS 
                 AA 
                 AA 
               
               
                 52 
                 Zhenglyu 
                 S 
                 AA 
                 AA 
               
               
                   
                 NO. 9 
               
               
                 53 
                 JP229241 
                 MR 
                 CC 
                 GG 
               
               
                 54 
                 JP229121 
                 MR 
                 AA 
                 AA 
               
               
                 55 
                 V1945AG 
                 MS 
                 AA 
                 AA 
               
               
                 56 
                 V1616AG 
                 S 
                 AA 
                 AA 
               
               
                 57 
                 Tao 98502 
                 MS 
                 AA 
                 AA 
               
               
                 58 
                 JP231233 
                 MR 
                 AACC 
                 AAGG 
               
               
                 59 
                 Pinlyuyouzi 
                 S 
                 AA 
                 AA 
               
               
                 60 
                 B23 
                 MR 
                 AA 
                 AA 
               
               
                 61 
                 V2808BG 
                 S 
                 AA 
                 AA 
               
               
                 62 
                 JP240329 
                 MS 
                 AA 
                 AA 
               
               
                 63 
                 JP226698 
                 HS 
                 AA 
                 AA 
               
               
                 64 
                 JP240379 
                 S 
                 AA 
                 AA 
               
               
                 65 
                 JP78926 
                 S 
                 AA 
                 AA 
               
               
                 66 
                 Bailyu 
                 MR 
                 AA 
                 AA 
               
               
                   
                 NO. 8 
               
               
                 67 
                 RG066 
                 S 
                 AA 
                 AA 
               
               
                 68 
                 V2481AG 
                 MS 
                 AA 
                 AA 
               
               
                 69 
                 B30 
                 S 
                 AA 
                 AA 
               
               
                 70 
                 C2969 
                 S 
                 AA 
                 AA 
               
               
                 72 
                 V2191BG 
                 S 
                 AA 
                 AA 
               
               
                 74 
                 Gaoyang 
                 HS 
                 AA 
                 AA 
               
               
                   
                 
                   Vigna 
                 
               
               
                   
                 
                   radiata 
                 
               
               
                   
                 (Linn.) 
               
               
                   
                 Wilczek 
               
               
                 75 
                 B37 
                 MR 
                 AA 
                 AA 
               
               
                 76 
                 Yulyu NO. 2 
                 MR 
                 CC 
                 GG 
               
               
                 77 
                 JP78938 
                 S 
                 AA 
                 AA 
               
               
                 78 
                 Liaolyu 
                 S 
                 AA 
                 AA 
               
               
                   
                 NO. 8 
               
               
                 79 
                 V2984BG 
                 S 
                 AA 
                 AA 
               
               
                 80 
                 V1586BG 
                 S 
                 AA 
                 AA 
               
               
                 81 
                 JP229403 
                 MS 
                 AA 
                 AA 
               
               
                 82 
                 Zhonglyu 
                 MS 
                 AA 
                 AA 
               
               
                   
                 NO. 8 
               
               
                 83 
                 Lyufeng 
                 HS 
                 AA 
                 AA 
               
               
                   
                 NO. 2 
               
               
                 84 
                 JP229254 
                 S 
                 AA 
                 AA 
               
               
                 85 
                 V1394AG 
                 S 
                 AA 
                 AA 
               
               
                 86 
                 JP78938 
                 S 
                 AA 
                 AA 
               
               
                 87 
                 B49 
                 S 
                 AA 
                 AA 
               
               
                 88 
                 V1377AG 
                 MS 
                 AA 
                 AA 
               
               
                 89 
                 P22008 
                 MR 
                 AA 
                 AA 
               
               
                 90 
                 V2272AG 
                 MR 
                 CC 
                 GG 
               
               
                 91 
                 JP202271 
                 HS 
                 AA 
                 AA 
               
               
                 92 
                 V2709BG 
                 S 
                 AA 
                 AA 
               
               
                 93 
                 B55 
                 S 
                 AA 
                 AA 
               
               
                 94 
                 JP229130 
                 MS 
                 AA 
                 AA 
               
               
                 95 
                 JP240384 
                 MS 
                 AA 
                 AA 
               
               
                 96 
                 Zhonglyu 
                 MR 
                 AA 
                 AA 
               
               
                   
                 NO. 11 
               
               
                 97 
                 JP240338 
                 MR 
                 AA 
                 AA 
               
               
                 98 
                 Tonglyu 
                 S 
                 AA 
                 AA 
               
               
                   
                 918 
               
               
                 99 
                 JP229193 
                 MS 
                 AA 
                 AA 
               
               
                 100 
                 JP229216 
                 S 
                 AA 
                 AA 
               
               
                 101 
                 V1188 
                 MS 
                 AA 
                 AA 
               
               
                 102 
                 V22T8AG 
                 MR 
                 AA 
                 AA 
               
               
                 103 
                 KPS1 
                 HS 
                 AA 
                 AA 
               
               
                 104 
                 B69 
                 S 
                 AA 
                 AA 
               
               
                 105 
                 22172 
                 S 
                 AA 
                 AA 
               
               
                 106 
                 JP229190 
                 MS 
                 CC 
                 GG 
               
               
                 107 
                 V1387AG 
                 MR 
                 AA 
                 AA 
               
               
                 108 
                 V2066BG 
                 MS 
                 AA 
                 AA 
               
               
                 109 
                 V2419 
                 MR 
                 AA 
                 AA 
               
               
                 110 
                 P24029 
                 S 
                 AA 
                 AA 
               
               
                 111 
                 V1948AG 
                 S 
                 AA 
                 AA 
               
               
                 113 
                 AL009 
                 HS 
                 AA 
                 AA 
               
               
                 114 
                 AL010 
                 MR 
                 AA 
                 AA 
               
               
                 115 
                 AL028 
                 S 
                 AA 
                 AA 
               
               
                 116 
                 IS0028 
                 MR 
                 AA 
                 AA 
               
               
                 117 
                 AL153 
                 MR 
                 AA 
                 AA 
               
               
                 118 
                 AL155*1 
                 S 
                 AA 
                 AA 
               
               
                 119 
                 AL152*1 
                 S 
                 AA 
                 AA 
               
               
                 120 
                 LD368 
                 MR 
                 AA 
                 AA 
               
               
                 121 
                 L158 
                 MS 
                 AA 
                 AA 
               
               
                 122 
                 AL090*1 
                 MR 
                 AA 
                 AA 
               
               
                 123 
                 AL152*2 
                 MS 
                 AA 
                 AA 
               
               
                 124 
                 AL160 
                 S 
                 AA 
                 AA 
               
               
                 125 
                 AL156 
                 MS 
                 CC 
                 GG 
               
               
                 126 
                 AL154 
                 MR 
                 AA 
                 AA 
               
               
                 127 
                 AL158*1 
                 S 
                 AA 
                 AA 
               
               
                 128 
                 IS0018 
                 S 
                 AA 
                 AA 
               
               
                 129 
                 AL084*2 
                 S 
                 AA 
                 AA 
               
               
                 130 
                 CO1522 
                 MS 
                 AA 
                 AA 
               
               
                 131 
                 CO1595 
                 HS 
                 AA 
                 AA 
               
               
                 132 
                 COOO1718 
                 S 
                 AA 
                 AA 
               
               
                 133 
                 AL084*1 
                 S 
                 AA 
                 AA 
               
               
                 134 
                 COOO1724 
                 HS 
                 AA 
                 AA 
               
               
                 135 
                 COOO1725 
                 S 
                 AA 
                 AA 
               
               
                 136 
                 COOO1726 
                 HS 
                 AA 
                 AA 
               
               
                 137 
                 COOO2976 
                 S 
                 AA 
                 AA 
               
               
                 138 
                 COOO2979 
                 S 
                 AA 
                 AA 
               
               
                 139 
                 A17 
                 HS 
                 AA 
                 AA 
               
               
                 140 
                 COOO2982 
                 MR 
                 AA 
                 AA 
               
               
                 141 
                 COOO3403 
                 HS 
                 AA 
                 AA 
               
               
                 142 
                 COOO4252 
                 S 
                 AA 
                 AA 
               
               
                 143 
                 23403 
                 S 
                 AA 
                 AA 
               
               
                 144 
                 L151 
                 MS 
                 AA 
                 AA 
               
               
                 145 
                 AL085 
                 MS 
                 AA 
                 AA 
               
               
                 146 
                 JP240383 
                 S 
                 AA 
                 AA 
               
               
                 147 
                 V1301BG 
                 MS 
                 AA 
                 AA 
               
               
                 148 
                 JP31224 
                 S 
                 AA 
                 AA 
               
               
                 149 
                 JP107875 
                 MR 
                 AACC 
                 AAGG 
               
               
                 150 
                 V2013BG 
                 MS 
                 AA 
                 AA 
               
               
                 151 
                 A44 
                 S 
                 AA 
                 AA 
               
               
                 152 
                 A45 
                 MS 
                 AA 
                 AA 
               
               
                 153 
                 A46 
                 S 
                 AA 
                 AA 
               
               
                 154 
                 JP229185 
                 S 
                 AA 
                 AA 
               
               
                 155 
                 Tao 9806 
                 HS 
                 AA 
                 AA 
               
               
                 156 
                 Jilyu NO. 5 
                 S 
                 AA 
                 AA 
               
               
                 157 
                 Nanyang 
                 MR 
                 AA 
                 AA 
               
               
                   
                 
                   Vigna 
                 
               
               
                   
                 
                   radiata 
                 
               
               
                   
                 (Linn.) 
               
               
                   
                 Wilczek 
               
               
                 158 
                 Bailyu 
                 MR 
                 AA 
                 AA 
               
               
                   
                 NO. 9 
               
               
                 159 
                 Zhonglyu 
                 MS 
                 AA 
                 AA 
               
               
                   
                 NO. 5 
               
               
                 160 
                 Huailyu 
                 MS 
                 AA 
                 AA 
               
               
                   
                 NO. 8 
               
               
                 161 
                 Jinlyu NO. 8 
                 MS 
                 AA 
                 AA 
               
               
                 162 
                 Inner 
                 MR 
                 AA 
                 AA 
               
               
                   
                 Mongolia 
               
               
                   
                 
                   Vigna 
                 
               
               
                   
                 
                   radiata 
                 
               
               
                   
                 (Linn.) 
               
               
                   
                 Wilczek 
               
               
                 163 
                 Zhonglyu 
                 MS 
                 AA 
                 AA 
               
               
                   
                 NO. 15 
               
               
                 164 
                 A57 
                 HS 
                 AA 
                 AA 
               
               
                 165 
                 Zhonglyu 
                 MS 
                 AA 
                 AA 
               
               
                   
                 NO. 4 
               
               
                 166 
                 Yulyu NO. 1 
                 MR 
                 AA 
                 AA 
               
               
                 167 
                 A18 
                 S 
                 AA 
                 AA 
               
               
                 168 
                 V2010BG 
                 MR 
                 AA 
                 AA 
               
               
                 169 
                 Liaolyu 
                 MS 
                 AA 
                 AA 
               
               
                   
                 NO.3 
               
               
                 170 
                 V2036AG 
                 MS 
                 AA 
                 AA 
               
               
                 171 
                 JP240386 
                 MS 
                 CC 
                 GG 
               
               
                 172 
                 JP231216 
                 MS 
                 AA 
                 AA 
               
               
                 173 
                 JP229144 
                 MR 
                 AA 
                 AA 
               
               
                 174 
                 JP240327 
                 S 
                 AA 
                 AA 
               
               
                 175 
                 JP240342 
                 HS 
                 AA 
                 AA 
               
               
                 176 
                 Oil  Vigna   
                 MS 
                 AA 
                 AA 
               
               
                   
                 
                   radiata 
                 
               
               
                   
                 (Linn.) 
               
               
                   
                 Wilczek 
               
               
                 177 
                 V1944BY 
                 S 
                 AA 
                 AA 
               
               
                 178 
                 V1411AG 
                 S 
                 AA 
                 AA 
               
               
                 179 
                 Baolyu 942 
                 MS 
                 AA 
                 AA 
               
               
                 180 
                 V1953bg 
                 MS 
                 AA 
                 AA 
               
               
                 181 
                 Dayang 
                 MS 
                 AA 
                 AA 
               
               
                   
                 
                   Vigna 
                 
               
               
                   
                 
                   radiata 
                 
               
               
                   
                 (Linn.) 
               
               
                   
                 Wilczek 
               
               
                 182 
                 JP99006 
                 S 
                 AA 
                 AA 
               
               
                 183 
                 V2007BG 
                 MS 
                 AA 
                 AA 
               
               
                 184 
                 V3726BG 
                 R 
                 CC 
                 GG 
               
               
                 185 
                 P81015 
                 S 
                 AA 
                 AA 
               
               
                 186 
                 V1476AG 
                 MS 
                 AA 
                 AA 
               
               
                 187 
                 JP240343 
                 MS 
                 AA 
                 AA 
               
               
                 188 
                 JP98811 
                 MS 
                 AA 
                 AA 
               
               
                 189 
                 A82 
                 MR 
                 AA 
                 AA 
               
               
                 190 
                 V4908AB 
                 S 
                 AA 
                 AA 
               
               
                   
               
            
           
         
       
     
     It can be seen from the Table 1 that the PARMS-SNP markers for identifying the resistant gene of  Vigna radiata  (Linn.) Wilczek  Cercospora  leaf spot disease disclosed by the invention has the characteristics of specificity, accuracy, reliability, simple operation and the like, and can quickly identify resistant varieties, assist molecular breeding and shorten a breeding period. 
     The above embodiments only describe the preferred mode of the invention, but do not limit the scope of the invention. On the premise of not departing from the design spirit of the invention, various modifications and changes made by those skilled in the field to the technical solution of the invention shall fall within the protection scope determined by the claims of the invention.