Molecular marker closely linked to the major QTL for resistance to fusarium wilt in mungbean and its application

Abstract

The application discloses a molecular marker closely linked to a main-effect QTL site fom1 of mung bean resistance to fusarium wilt and application thereof, and belongs to the technical field of mung bean molecular breeding. The application takes a separation population and a recombination inbred line population formed by "Wei green 9002-341" and "V1128" as test materials, uses an SSR marker, obtains a disease resistance correlation site fom1, and the site is located on a 5th chromosome, a genetic distance is 2.94 cM, a LOD is 9.28, a phenotype contribution rate is 20.90%, and an additive effect is -8.33, and the site is closely linked to a SSR marker C5-228. The molecular marker closely linked to the main-effect QTL site fom1 of mung bean resistance to fusarium wilt can be used for early selection of mung bean resistance to fusarium wilt, and has important significance for improving the breeding efficiency of mung bean resistance to fusarium wilt.

Description

Technical Field

[0001] This invention relates to a molecular marker tightly linked to a major QTL site for resistance to Fusarium wilt in mung beans and its application. This invention belongs to the field of mung bean molecular breeding technology. Background Technology

[0002] Mung bean wilt is a soil-borne fungal disease widely distributed in mung bean producing areas, causing significant yield reductions or even complete crop failure. Because the pathogen's chlamydospores can survive in the soil for extended periods, traditional methods are ineffective, making the development of resistant varieties the most effective control method. However, current research on mung bean wilt is limited both domestically and internationally, and no resistance genes have been identified. This study constructed an F11 resistant variety using "Weilv 9002-341" and "V1128". 2:3 By using BSA resequencing and genetic linkage analysis, the major resistance gene in segregating populations and F8 recombinant inbred lines was located on chromosome 5 of mung bean and closely linked to the SSR marker C5-228. This provides a theoretical basis for the breeding of disease-resistant varieties and the study of resistance mechanisms in mung bean wilt. Summary of the Invention

[0003] The purpose of this invention is to provide a major QTL site for mung bean resistance to Fusarium wilt and a closely linked SSR molecular marker C5-228, which can be applied to molecular marker-assisted breeding technology for mung bean resistance to Fusarium wilt.

[0004] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0005] This invention proposes an SSR molecular marker tightly linked to a major QTL site for resistance to Fusarium wilt in mung beans. The major QTL site, named fom1, is located on chromosome 5 (2.15-2.32 Mb) and tightly linked to the SSR molecular marker C5-228. The SSR molecular marker C5-228 is obtained by PCR amplification of mung bean genomic DNA using primers C5-228F and C5-228R. The primer sequences are as follows:

[0006] C5-228F:CCCCCCACCTTCCTTTTCAT;

[0007] C5-228R:AAGAGCTAGGGTGATGCCACA.

[0008] Furthermore, this invention also proposes a primer pair for obtaining the SSR molecular marker tightly linked to the major QTL site fom1 for resistance to Fusarium wilt in mung beans. The primer pair is C5-228F / R, and the primer sequence is as follows:

[0009] C5-228F:CCCCCCACCTTCCTTTTCAT;

[0010] C5-228R:AAGAGCTAGGGTGATGCCACA.

[0011] Furthermore, this invention also proposes the application of the SSR molecular markers closely linked to the major QTL sites for resistance to Fusarium wilt in mung beans and the primer pairs described therein in molecular marker-assisted selection breeding for resistance to Fusarium wilt in mung beans.

[0012] Furthermore, this invention also proposes a method for obtaining a new mung bean variety resistant to Fusarium wilt, comprising the following steps:

[0013] (1) Using the highly resistant mung bean wilt variety Weilv 9002-341 as the male parent and the highly susceptible variety V1128 as the female parent, a recombinant inbred line F8 population was obtained;

[0014] (2) Genomic DNA was extracted from individual plants of the paternal parent, maternal parent, and F8 population as templates, and PCR amplification was performed using primer pair C5-228F / R; the primer sequences are shown below:

[0015] C5-228F:CCCCCCACCTTCCTTTTCAT;

[0016] C5-228R: AAGAGCTAGGGTGATGCCACA;

[0017] (3) Detect the amplification products to determine the genotype of each sample. If the amplification result is the same as the paternal genotype, the sample is highly resistant to Fusarium wilt; if the amplification result is different from the paternal genotype, the sample is weakly resistant to Fusarium wilt.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] This invention used an F8 recombinant inbred line population of “Weilv 9002-341” and “V1128” as experimental materials. Using SSR markers, the major-effect QTL locus fom1 was detected on chromosome 5, located at 2.15-2.32 Mb, and tightly linked to the SSR marker C5-228. The genetic distance between the markers was 2.94 cM, the LOD was 9.28, the phenotypic contribution rate was 20.90%, and the additive effect was -8.33. The molecular markers obtained in this invention, tightly linked to the mung bean wilt resistance locus fom1, can be used for screening mung bean wilt-resistant materials, which is of great significance for improving the breeding efficiency of wilt-resistant mung beans. Attached Figure Description

[0020] Figure 1 These are the resequencing localization results;

[0021] Figure 2 The QTL localization results for mung bean resistance to Fusarium wilt within the associated interval;

[0022] Figure 3 Genotyping results for molecular marker C5-228. Detailed Implementation

[0023] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become clearer as a result. However, these examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0024] Example 1: Development of QTL sites for resistance to Fusarium wilt in mung beans and their closely linked SSR markers

[0025] 1. Construction of mung bean populations resistant to wilt

[0026] Using the wilt-susceptible mung bean resource V1128 as the female parent and the wilt-resistant variety Weilv 9002-341 as the male parent, the F1 generation was obtained through hybridization. The F1 generation was then self-crossed to form the F2 generation. 2:3 Segregating populations and F8 recombinant inbred line populations were used for QTL mapping studies on mung bean resistance to Fusarium wilt.

[0027] 2. BSA resequencing

[0028] Phenotypic identification: F1 seeds were sown in a greenhouse. 2:3 Twenty-eight individual plants from the segregating population were inoculated with the pathogen using the root-cutting spore-dipping method after the two true leaves of the seedlings had fully unfolded. Phenotypic results were assessed 14 days after inoculation. The male and female parents served as resistant and susceptible controls, respectively. The disease index of the population was calculated and classified into five disease grades (highly resistant, HR; resistant, R; moderately resistant, MR; susceptible, S; highly susceptible, HS).

[0029] Resequencing analysis and localization: Thirty highly resistant and 30 highly susceptible individuals were selected from the population to construct pools (RF and SF), and 10 individuals from each parent were used to construct pools (RP and SP). Sequencing was performed on all four pools. The raw data were filtered and aligned to the reference genome Vigna_radiata.VrJL7.genome using bwa software. GATK was then used for variant detection. High-quality SNPs and Indels were screened from the detected variants, and analyzed using SNP-index and Indel-index algorithms respectively. Both algorithms detected associated intervals when the confidence interval was 90%. Finally, the intersection of two associated intervals was taken as the candidate associated interval, located in the 0.36-2.32 Mb range on chromosome 5. Figure 1 ).

[0030] 3. QTL positioning

[0031] Phenotypic identification: The plant material used was the recombinant inbred line F8 population, containing 239 lines. Resistance identification was conducted three times, in June 2022, November 2022, and May 2023.

[0032] Molecular marker screening: Using MISA software, polymorphic sites were searched within and flanking the candidate association region on chromosome 5, and 188 SSR molecular markers were designed. After parental screening, 14 markers were finally obtained. These markers exhibited polymorphism between the parents and had clear bands, making them suitable for subsequent linkage analysis.

[0033] Linkage analysis and mapping: 239 individual plants from the F8 population were sown in germination boxes, and young trifoliate leaves were collected for genomic DNA extraction using the CTAB method. Using the genomic DNA as a template, 14 selected markers and markers at both ends of candidate association intervals were used as primers, and the genotypes of each individual plant in the F8 population were detected by 0.8% agarose gel electrophoresis. Combined with the tertiary phenotypic data, genetic linkage analysis was performed using QTL IciMapping 4.0 software, ultimately locating a major locus associated with resistance to Fusarium wilt (Table 1). Figure 2 The gene was located between molecular markers C5-228 and C5-273. The genetic distance between the two markers was 2.94 cM, the LOD ranged from 2.96 to 10.90, the additive effect ranged from -10.75 to -6.86, and the phenotypic contribution rate ranged from 7.31% to 25.26%.

[0034] The intersection of this locus and the candidate associated region was taken as the disease resistance locus fom1, located in the range of 2.15-2.32 Mb on chromosome 5, containing 24 annotated genes. The molecular marker C5-228 is located at 2.15 Mb on chromosome 5, and the primer sequences are as follows:

[0035] C5-228F:CCCCCCACCTTCCTTTTCAT;

[0036] C5-228R:AAGAGCTAGGGTGATGCCACA.

[0037] Table 1. QTL localization within candidate association intervals

[0038]

[0039] Example 2: Application of the SSR marker C5-228 linked to the fom1 site in mung bean wilt resistance breeding.

[0040] method:

[0041] (1) Using the highly resistant mung bean wilt variety Weilv 9002-341 as the male parent and the highly susceptible variety V1128 as the female parent, a recombinant inbred line F8 population was obtained;

[0042] (2) Genomic DNA was extracted from individual plants of the paternal parent, maternal parent, and F8 population as templates, and PCR amplification was performed using primer pair C5-228F / R; the primer sequences are shown below:

[0043] C5-228F:CCCCCCACCTTCCTTTTCAT;

[0044] C5-228R: AAGAGCTAGGGTGATGCCACA;

[0045] (3) The amplification products were detected by polyacrylamide gel electrophoresis to determine the disease resistance of each sample. If the amplification result is the same as the paternal genotype, it indicates that the sample has strong resistance to Fusarium wilt; if the amplification result is different from the paternal genotype, it indicates that the sample has weak resistance to Fusarium wilt.

[0046] result:

[0047] The results of genotyping and three resistance tests for the molecular marker C5-228 are shown in Table 2. Figure 3 Of the 116 RILs, the genotypes were identical to the paternal parent, with average disease severity grades of 3.43±1.67, 6.73±2.38, and 3.73±1.57; 113 RILs had genotypes different from the paternal parent, with average disease severity grades of 5.64±2.25, 7.74±2.09, and 4.98±2.10. The T-test results from three resistance identification tests all showed that, at the 0.01 level, the disease severity grades of RILs with the same genotype as the paternal parent were significantly lower than those of RILs with different genotypes.

[0048] Table 2 shows the selection of the RIL population using SSR markers C5-228 and C5-273.

[0049]

[0050] Therefore, the SSR marker C5-228 can be used to screen mung bean materials resistant to Fusarium wilt, which is of great significance for improving the breeding efficiency of Fusarium wilt-resistant mung beans.

Claims

1. A primer pair, characterized in that, This involves obtaining primer pairs for SSR molecular markers tightly linked to the major QTL site for mung bean resistance to Fusarium wilt. The major QTL site for mung bean resistance to Fusarium wilt is named... fom1 Located on chromosome 5 at 2.15-2.32 Mb, it is tightly linked to the SSR molecular marker C5-228. The primer pair is C5-228F / R, and the primer sequences are shown below: C5-228F:CCCCCCACCTTCCTTTTCAT; C5-228R:AAGAGCTAGGGTGATGCCACA.

2. The application of the primer pair described in claim 1 in molecular marker-assisted selection breeding of mung beans resistant to Fusarium wilt.

3. A method for obtaining a new mung bean variety resistant to Fusarium wilt, characterized in that, Includes the following steps: (1) Using the highly resistant mung bean wilt variety Weilv 9002-341 as the male parent and the highly susceptible variety V1128 as the female parent, a recombinant inbred line F8 population was obtained; (2) Genomic DNA was extracted from individual plants of the paternal parent, maternal parent, and F8 population as samples and used as templates for PCR amplification using primer pair C5-228F / R; the primer sequences are shown below: C5-228F:CCCCCCACCTTCCTTTTCAT; C5-228R: AAGAGCTAGGGTGATGCCACA; (3) The amplification products were detected by polyacrylamide gel electrophoresis to determine the disease resistance of each sample. If the amplification result is the same as the paternal genotype, the resistance to Fusarium wilt is strong; if the amplification result is different from the paternal genotype, the resistance to Fusarium wilt is weak.

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