A molecular marker closely linked to a major QTL VD-6.1 of cowpea resistance to fusarium wilt, primer thereof and application

Abstract

The application discloses a molecular marker closely linked with a cowpea anti-fusarium wilt major QTL VD-6.1, a primer thereof and an application. The molecular marker is a KASP type 3_00159, a SNP site of which is located at 43673168 bp of a No. 6 chromosome of a cowpea variety G98 reference genome, a polymorphism is A / T, and a nucleotide sequence is shown as SEQ ID NO. 1. The molecular marker is closely linked with the major QTL VD-6.1, a typing accuracy reaches more than 99%, a rapid and accurate identification of a cowpea anti-fusarium wilt genotype can be realized at a seedling stage, is not limited by environmental conditions, a breeding period is shortened from 2-3 years to one month, and breeding efficiency is significantly improved and breeding cost is reduced, thereby providing a reliable molecular tool for cowpea anti-fusarium wilt variety source creation and genetic improvement.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, specifically relating to a molecular marker closely linked to the major QTL VD-6.1 for cowpea resistance to Fusarium wilt, its primers and applications, as well as the application of the molecular marker, primer set and kit, and a method for breeding cowpea varieties resistant to Fusarium wilt based on the primer set. Background Technology

[0002] cowpea( Vigna unguiculata *L. Walp.*, belonging to the genus *Vigna* in the family Fabaceae, is a globally important legume crop, used as food, feed, and vegetable. It is also an important coarse grain and vegetable crop in my country, playing a vital role in ensuring vegetable supply, increasing farmers' income, and promoting rural revitalization. *Fusarium wilt* is caused by *Fusarium oxysporum* var. *hypoxanthin* (a type of *Vigna*). Fusarium oxysporum f.sp. Tracheiphilum , Fot Fusarium wilt (FUL) is one of the three major diseases of cowpea and a major cause of continuous cropping obstacles in cowpea. This disease can occur throughout the entire growth period of cowpea. The pathogen infects the vascular bundles through the roots, causing browning of the vascular bundles, yellowing and shedding of leaves, and ultimately plant death. Infection in the seedling stage can lead to devastating yield reductions. It occurs in all major cowpea producing areas in my country, severely restricting the development of the cowpea industry. Breeding and promoting FUL-resistant cowpea varieties is the most economical and effective strategy for controlling this disease. Discovering disease-resistant genes and developing tightly linked molecular markers are prerequisites and key factors for conducting FUL-resistant molecular marker-assisted breeding.

[0003] Currently, several genes / QTLs related to cowpea resistance to Fusarium wilt have been identified through GWAS analysis and QTL mapping. However, molecular markers closely linked to major QTLs and efficiently applied in breeding practices are lacking. Kompetitive allele-specific PCR (KASP) is a high-throughput endpoint fluorescent genotyping technique based on SNPs, enabling precise bicelestealous detection of SNP loci. It offers advantages such as flexible marker selection, high detection efficiency, and accurate results, and has been widely used in plant molecular breeding to effectively accelerate the breeding process. Therefore, developing KASP markers closely linked to major QTLs for cowpea resistance to Fusarium wilt and establishing efficient molecular-assisted selection breeding methods are of great significance for the genetic improvement of cowpea resistance to Fusarium wilt. Summary of the Invention

[0004] To address the lack of molecular markers in existing technologies that can be efficiently applied to cowpea wilt resistance breeding, this invention provides a KASP molecular marker closely linked to the major QTL VD-6.1 for cowpea wilt resistance. It also provides a primer set, kit, and application for detecting this marker, as well as a method for breeding cowpea wilt-resistant varieties based on this primer set. This enables early, rapid, and accurate identification of cowpea wilt-resistant genotypes, improving breeding efficiency and shortening the breeding cycle.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] This invention provides a molecular marker closely linked to the major QTL VD-6.1 for resistance to Fusarium wilt in cowpea. The molecular marker is of the KASP type and is named 3_00159. Its SNP site is located at 43673168 bp on chromosome 6 of the reference genome of cowpea variety G98, and its polymorphism is A / T. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1.

[0007] This invention identified a major QTLVD-6.1 for resistance to Fusarium wilt on chromosome 6 of cowpea using QTL mapping analysis, and developed a closely linked KASP molecular marker 3_00159. The SNP site of this marker is located at 43,673,168 bp on chromosome 6 of the reference genome of cowpea variety G98, with an A / T polymorphism. The nucleotide sequence is shown in SEQ ID NO.1, where the SNP site is the A / T variation at position 101. The linkage disequilibrium coefficient r² between this molecular marker and the major QTLVD-6.1 is 0.96, and the typing accuracy is over 99%.

[0008] Another aspect of the present invention provides a KASP primer set for detecting the aforementioned molecular markers, the primer set comprising 3_00159 Primer1, 3_00159 Primer2, and 3_00159 Primer_Common; wherein,

[0009] The nucleotide sequence of Primer1 3_00159 is shown in SEQ ID NO.2, corresponding to the disease-susceptible allele A;

[0010] The nucleotide sequence of Primer2 3_00159 is shown in SEQ ID NO.3, corresponding to the disease resistance allele T;

[0011] The nucleotide sequence of 3_00159 Primer_Common is shown in SEQ ID NO.4, and it is a universal reverse primer.

[0012] Preferably, the 5' end of the 3_00159 Primer1 is connected to a FAM fluorescent group, and the 5' end of the 3_00159 Primer2 is connected to a HEX fluorescent group.

[0013] In another aspect, the present invention provides a cowpea wilt resistance genotype detection kit, which includes the above-mentioned KASP primer set and 2×KASP Mastermix reagent required for KASP reaction. It can be directly used for KASP reaction, is easy to operate, and is suitable for high-throughput detection.

[0014] The aforementioned molecular marker 3_00159, KASP primer set, and kit can be used to detect the major QTLVD-6.1 for cowpea resistance to Fusarium wilt, and can also be used for molecular marker-assisted breeding of cowpea varieties resistant to Fusarium wilt, providing a reliable molecular tool for genetic improvement of cowpea resistance to Fusarium wilt.

[0015] In another aspect, the present invention provides a method for breeding cowpea varieties resistant to Fusarium wilt using the above-mentioned KASP primer set, comprising the following steps:

[0016] S1. Extract genomic DNA from the cowpea plants to be tested;

[0017] S2. Using the genomic DNA extracted in step S1 as a template, perform KASP reaction detection using the KASP primer set;

[0018] S3. Read the fluorescence signal of the KASP reaction in step S2. If the FAM fluorescent group signal is detected, determine that the cowpea to be tested carries the genotype of susceptibility to Fusarium wilt; if the HEX fluorescent group signal is detected, determine that the cowpea to be tested carries the genotype of resistance to Fusarium wilt; select cowpea plants carrying the genotype of resistance to Fusarium wilt for breeding.

[0019] As a preferred option, the PCR system for KASP reaction detection is: 0.8 μl DNA template, 0.75 μl 2×KASP Master mix, and 0.05 μl Primer mix;

[0020] Preferably, the KASP reaction procedure in step S2 is as follows: pre-denaturation at 94°C for 15 minutes; denaturation at 94°C for 20 seconds; gradient refolding at 61~55°C for 60 seconds, with the refolding temperature decreasing by 0.6°C per cycle; extension at 55°C for 60 seconds; 10 cycles; denaturation at 94°C for 20 seconds; refolding at 55°C for 60 seconds; extension at 55°C for 60 seconds; 26 cycles.

[0021] The advantages and beneficial effects of this invention are as follows:

[0022] 1. The molecular marker 3_00159 of this invention is closely linked to the major QTLVD-6.1 for cowpea resistance to Fusarium wilt (r²=0.96), with a typing accuracy of over 99%, which can accurately distinguish between cowpea resistant and susceptible genotypes of Fusarium wilt, providing a reliable molecular basis for Fusarium wilt resistance breeding;

[0023] 2. The detection method based on this molecular marker is not limited by environmental conditions and can be used for genotyping in cowpea seedlings, enabling early screening of wilt-resistant materials and shortening the cowpea wilt-resistant breeding cycle from the traditional 2-3 years to 1 month, significantly improving breeding efficiency;

[0024] 3. The KASP primer set and kit of the present invention can achieve high-throughput detection, are easy to operate and have low detection cost, and are suitable for identification of Fusarium wilt resistance genotypes in large-scale cowpea germplasm resources;

[0025] 4. The molecular markers and breeding methods of this invention provide a novel technical means for the creation and genetic improvement of cowpea wilt-resistant germplasm, which can effectively promote the breeding and promotion of cowpea wilt-resistant varieties, provide support for the green prevention and control of cowpea wilt, and promote the healthy development of the cowpea industry. Attached Figure Description

[0026] To more clearly illustrate the solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0027] Figure 1 KASP typing diagram of the 3_00159 marker site in 80 cowpea germplasms;

[0028] The horizontal axis represents the intensity of FAM fluorescence signal, and the vertical axis represents the intensity of HEX fluorescence signal. The red clusters represent susceptible genotypes (A allele) carrying FAM signals, and the blue clusters represent resistant genotypes (T allele) carrying HEX signals. The genotyping boundaries are clear and the results are stable.

[0029] Figure 2 Plot of allelic variation in resistance to Fusarium wilt at marker site 3_00159;

[0030] The horizontal axis represents allele type (A / T), and the vertical axis represents disease index. The results show that the average disease index of the line carrying the T allele (HapII, n=12) was significantly lower than that of the line carrying the A allele (HapI, n=65), and the difference was extremely significant (p<0.0001). Detailed Implementation

[0031] To further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described implementation cases are only some embodiments of the present invention, and not all implementation cases.

[0032] Unless otherwise specified, the experimental methods used in the following implementation examples are generally performed under standard conditions or as recommended by the manufacturer.

[0033] Unless otherwise specified, all materials and reagents used in the following implementation examples are commercially available.

[0034] The PCR amplification system used in the KASP reaction of this invention embodiment is as follows: DNA 0.8 μl, 2×KASP Mastermix 0.75 μl, Primer mix 0.05 μl.

[0035] The PCR amplification program used in this embodiment of the invention is as follows: pre-denaturation at 94℃ for 15 minutes, denaturation at 94℃ for 20 seconds, gradient annealing at 61~55℃ for 60 seconds, with the annealing temperature decreasing by 0.6℃ in each cycle, extension at 55℃ for 60 seconds, for 10 cycles; then denaturation at 94℃ for 20 seconds, annealing at 55℃ for 60 seconds, extension for 60 seconds, for 26 cycles.

[0036] Example 1

[0037] Location of QTLVD-6.1, the main active ingredient in cowpea's resistance to Fusarium wilt, and development of its molecular marker 3_00159.

[0038] 1. Experimental materials: Using cowpea disease-resistant material ZN016 and disease-susceptible material TZ30 as parents, an F8 generation recombinant inbred line population containing 209 materials was constructed.

[0039] 2. Phenotypic identification of resistance to Fusarium wilt: A randomized block design was used, with three replicates per material. Inoculation was carried out when the first trifoliate leaf unfolded. The soil around the plant roots was thoroughly watered the day before inoculation. The next day, the fibrous roots were cut downwards at a depth of 1 cm from the main root, approximately 4-5 cm below the soil surface, ensuring that the root system was not damaged. The final concentration of the Fusarium wilt spore suspension was adjusted to 1×10⁵ spores / mL, and 50 mL of the suspension was applied to each plant. Approximately 28 days after inoculation, the vascular bundle browning (VD) index was measured to identify resistance to Fusarium wilt. A control group without Fusarium wilt was set up for each material.

[0040] Disease index (DI) was calculated based on browning grade, and resistance levels were assigned. Table 1 shows the genotypes, disease index, and resistance levels of some of the 209 recombinant inbred lines.

[0041] - Vascular bundle browning levels: Level 0 (no browning in roots), Level 1 (10% browning), Level 2 (25%), Level 3 (50%), Level 4 (75%), Level 5 (100%).

[0042] - Disease index calculation formula: DI=∑[(r×nr) / λNt], where r is the disease level, nr is the number of individual plants of the corresponding level, Nt is the total number of plants surveyed, and λ is the highest level number;

[0043] - Sensitivity rating: High sensitivity (DI: 0-0.10), sensitivity (0.11-0.30), sensitivity (0.31-0.50), high sensitivity (0.51-1).

[0044] 3. SNP marker development and QTL mapping: 209 recombinant inbred lines were resequencing using the Illumina platform, yielding 1122 high-quality SNP markers. QTL mapping analysis was performed using Map QTL5 software and interval mapping (IM) method. A major QTL for resistance to Fusarium wilt, VD-6.1, was identified on chromosome 6 of cowpea. The peak SNP (numbered 3_00159) is located at 43673168 bp. The SNP site polymorphism of this marker is A / T, and the nucleotide sequence is shown in SEQ ID NO.1. Based on its sequence information, a KASP molecular marker was developed to indicate the major QTL for resistance to Fusarium wilt, VD-6.1.

[0045] 4. Primer design: KASP primer set was designed based on the sequence of molecular marker 3_00159, including 3_00159Primer1 (SEQ ID NO.2), 3_00159 Primer2 (SEQ ID NO.3) and 3_00159 Primer_Common (SEQ ID NO.4), where Primer1 is linked to the FAM group and Primer2 is linked to the HEX group.

[0046] Table 1 shows the genotype, haplotype, and resistance to Fusarium wilt for some of the 209 recombinant inbred lines.

[0047]

[0048] The results showed that materials carrying the A allele (HapI) were mostly highly susceptible / susceptible to the disease, with only a few being resistant; materials carrying the T allele (HapII) were mostly highly resistant / resistant to the disease, confirming that the molecular marker 3_00159 can effectively associate cowpea with the wilt resistance phenotype.

[0049] In this embodiment, the KASP marker 3_00159 was successfully screened and obtained. It is located on chromosome 6 in the reference genome of cowpea variety G98, and its SNP site is located at 43673168 bp with polymorphism A / T.

[0050] The nucleotide sequence of the KASP-tagged sequence is shown in SEQ ID NO.1, with the SNP site at position 101.

[0051] Based on this sequence, the present invention designed a series of KASP primers, the sequences of which from 5' to 3' are shown below.

[0052] The KASP primers for this molecular marker include a specific forward primer Primer1, a specific forward primer Primer2, and a universal reverse primer Primer_Common, the sequences of which are shown below.

[0053] 3_00159 Primer1 is shown as SEQ ID NO.2;

[0054] 3_00159 Primer2 is shown in SEQ ID NO.3;

[0055] 3_00159 Primer_Common is shown in SEQ ID NO.4.

[0056] In some specific implementations, Primer1 is linked to the FAM group and Primer2 is linked to the HEX group.

[0057] Example 2

[0058] Validation of the molecular marker 3_00159:

[0059] 1. Experimental materials: 80 cowpea germplasm resources were randomly selected (from the Vegetable Research Institute of Zhejiang Academy of Agricultural Sciences).

[0060] 2. Phenotypic identification: When the first trifoliate compound leaf of the cowpea seedling is fully expanded, use 1×10 5 Inoculate with a suspension of Fusarium wilt spores per mL, treat for 28 days, measure the browning rate of vascular bundles, calculate the disease index and classify the resistance and susceptibility levels, and set up a control group without pathogen stress.

[0061] 3. KASP detection:

[0062] -Genomic DNA extraction: Genomic DNA was extracted from 80 cowpea germplasms using the CTAB method;

[0063] -KASP reaction: The KASP primer set designed in Example 1 was used for detection on the IntelliQube genotyping platform. The reaction system was: DNA 0.8 μl, 2×KASP Master mix 0.75 μl, Primer mix 0.05 μl; the reaction program was: 94℃ pre-denaturation for 15 min; 10 cycles of landing PCR (94℃ for 20 s, 61-55℃ for 60 s, with a 0.6℃ decrease per cycle); 26 conventional cycles (94℃ for 20 s, 55℃ for 60 s).

[0064] - Fluorescence signal reading: Read FAM and HEX fluorescence signals to determine genotype.

[0065] 4. Results Analysis:

[0066] -Classification results: such as Figure 1 As shown, the 80 cowpea germplasm accessions can be clearly divided into two clusters: the A genotype carrying the FAM signal (HapI, n=65) and the T genotype carrying the HEX signal (HapII, n=12), with good genotyping effect and clear boundaries.

[0067] - Phenotypic association analysis: such as Figure 2 As shown in Table 2, the average disease index of the T genotype lines was significantly lower than that of the A genotype lines, and the difference was extremely significant (p<0.0001). Among the T genotype lines, the proportion of highly resistant / disease-resistant types reached 83.3%, while the proportion of highly susceptible / disease-susceptible types reached 90.8% in the A genotype lines. This confirms that the molecular marker 3_00159 can effectively distinguish the cowpea wilt resistance genotypes and is suitable for molecular marker-assisted breeding.

[0068] Table 2. Statistical data on genotypes and phenotypic characteristics of selected germplasm treatments.

[0069]

[0070] Note: The disease index and the criteria for the level of resistance to infection are consistent with those in Example 1.

[0071] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made to the present invention should be included within the scope of protection of the present invention.

Claims

1. A molecular marker tightly linked to VD-6.1, the major QTL for resistance to Fusarium wilt in cowpea, characterized in that, The molecular marker is of the KASP type and named 3_00159. Its SNP site is located at 43673168 bp on chromosome 6 of the cowpea variety G98 genome, and its polymorphism is A / T. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

1.

2. A KASP primer set for detecting the molecular marker of claim 1, characterized in that, The primer set includes 3_00159 Primer1, 3_00159 Primer2, and 3_00159 Primer_Common; wherein... The nucleotide sequence of Primer1 3_00159 is shown in SEQ ID NO.2; The nucleotide sequence of Primer2 3_00159 is shown in SEQ ID NO.3; The nucleotide sequence of 3_00159 Primer_Common is shown in SEQ ID NO.

4.

3. The KASP primer set according to claim 2, characterized in that, The 5' end of the 3_00159 Primer1 is connected to a FAM fluorescent group, and the 5' end of the 3_00159 Primer2 is connected to a HEX fluorescent group.

4. A reagent kit, characterized in that, The kit contains the KASP primer set as described in claim 2 or 3, and also includes 2×KASP Mastermix reagent.

5. The application of the KASP primer set according to claim 2 or 3 and the kit according to claim 4 in detecting the major active ingredient QTLVD-6.1 for cowpea resistance to Fusarium wilt.

6. The application of the KASP primer set as described in claim 2 or 3 and the kit as described in claim 4 in molecular marker-assisted breeding of cowpea varieties resistant to Fusarium wilt.

7. A method for breeding cowpea varieties resistant to Fusarium wilt using the KASP primer set described in claim 2 or 3, characterized in that, Includes the following steps: S1. Extract genomic DNA from the cowpea plants to be tested; S2. Using the genomic DNA extracted in step S1 as a template, perform KASP reaction detection using the KASP primer set; S3. Read the fluorescence signal of the KASP reaction in step S2. If the FAM fluorescent group signal is detected, determine that the cowpea to be tested carries the genotype of susceptibility to Fusarium wilt; if the HEX fluorescent group signal is detected, determine that the cowpea to be tested carries the genotype of resistance to Fusarium wilt; select cowpea plants carrying the genotype of resistance to Fusarium wilt for breeding.

8. The method according to claim 7, characterized in that, The PCR system for KAS reaction detection was as follows: 0.8 μl DNA template, 0.75 μl 2×KASP Master mix, and 0.05 μl Primer mix.

9. The method according to claim 7, characterized in that, The procedure for the KASP reaction in step S2 is as follows: pre-denaturation at 94°C for 15 minutes; denaturation at 94°C for 20 seconds; gradient refolding at 61~55°C for 60 seconds, with the refolding temperature decreasing by 0.6°C per cycle; extension at 55°C for 60 seconds; 10 cycles; denaturation at 94°C for 20 seconds; refolding at 55°C for 60 seconds; extension at 55°C for 60 seconds; 26 cycles.

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