Molecular marker related to cowpea fusarium wilt resistance major QTL (quantitative trait loci) as well as primer and application thereof

Abstract

The invention discloses a molecular marker related to cowpea fusarium wilt resistance major QTL (quantitative trait loci) as well as primers and application thereof, and belongs to the technical field of plant molecular breeding. A KASP type molecular marker 300160 related to the KASP type molecular marker 300160 is located at the position 46659973 bp on a chromosome 6, the marker can be used for amplifying a to-be-detected material, and whether a sample carries allelic variation for controlling wilt resistance or not is judged according to the genotype of the to-be-detected material. The KASP labeled primer combination comprises the following three sequences: a forward primer Primer1 as shown in a sequence 2, a forward primer Primer2 as shown in a sequence 3 and a universal reverse primer PrimerCommon as shown in a sequence 4. By utilizing the molecular marker and the primer thereof provided by the invention, rapid screening of the main effect QTL for resisting the fusarium wilt of the cowpea can be realized, and the molecular marker and the primer thereof are of great significance for breeding new disease-resistant varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology and relates to a molecular marker (3_00160) related to the major QTL LFD-6.2 for cowpea resistance to Fusarium wilt and its application. Background Technology

[0002] Cowpea wilt is mainly caused by Fusarium oxysporum var. trachomatis (a type of fungus). Fusarium oxysporum f.sp. Tracheiphilum, Fusarium wilt (Fot) is a soil-borne fungal disease, often referred to as the "cancer" of cowpeas, seriously threatening safe cowpea production. This disease is widespread, the pathogen is highly resilient, and extremely difficult to control. Therefore, identifying disease-resistant genes in cowpeas and developing molecular markers for diagnosis and breeding assistance to accelerate the breeding of Fusarium wilt-resistant cowpea varieties is the most economical, effective, and environmentally friendly control measure.

[0003] As a novel technology, molecular marker-assisted breeding combines molecular biology and conventional breeding methods, enabling the selection of target plants at various stages of plant development and greatly improving breeding efficiency. Among these methods, competitive allele-specific PCR technology based on SNP detection has become one of the important techniques.

[0004] Reports on cowpea resistance to Fusarium wilt indicate that some corresponding resistance QTLs and SNP sites have been identified for existing races such as race 3, race 4, CN-1, and FW-HZ, including sites like Fot 3-1, Fot 4-1, 1_0075, and 2_51816. However, the number of these sites remains relatively small, and information on KASP markers is still very limited. Therefore, discovering new resistance QTLs and developing related molecular markers is of great significance for advancing disease-resistant breeding and providing novel solutions for disease control. Summary of the Invention

[0005] The technical problem solved by this invention is to utilize KASP markers for assisted breeding of cowpea resistance to Fusarium wilt. This invention provides a molecular marker 3_00160 and its KASP primers associated with the major QTL (LFD-6.2) for cowpea resistance to Fusarium wilt, and establishes a Fusarium wilt-assisted breeding method based on this marker. This method identifies SNP loci associated with Fusarium wilt resistance through genome-wide association analysis (GWAS), converts them into KASP markers, and verifies them. This can be used to rapidly identify the Fusarium wilt-resistant genotype in cowpea germplasm, providing molecular breeding technical support for the genetic improvement of cowpea resistance to Fusarium wilt.

[0006] This invention identified the major-effect QTL LFD-6.2 for resistance to Fusarium wilt at position 46659973 bp on chromosome 6 of cowpea through genome-wide association analysis, and developed the associated KASP molecular marker 3_00160. The development of this marker provides an effective technical means for molecular breeding of cowpea for resistance to Fusarium wilt, which will significantly improve breeding efficiency and shorten the breeding cycle.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In one aspect, this invention provides a molecular marker associated with the major QTL LFD-6.2 for resistance to Fusarium wilt in cowpea. The molecular marker is 3_00160, located at 46659973 bp on chromosome 6 of the cowpea variety G98 genome. The molecular marker is of the KASP type and has an SNP site polymorphism of A / G.

[0008] Preferably, the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1.

[0009] In another aspect, the present invention provides a KASP primer for detecting the above-mentioned molecular marker, wherein the KASP primer includes a specific forward primer Primer1, a specific forward primer Primer2, and a universal reverse primer Primer_Common; 3_00160 Primer1 is shown as SEQ ID NO.2; 3_00160 Primer2 is shown in SEQ ID NO.3; 3_00160 Primer1 and 3_00160 Primer2 are two specific primers, each linked to a different fluorescent sequence; 3_00160 Primer_Common is shown in SEQ ID NO.4.

[0010] Preferably, 3_00160 Primer1 is connected to the FAM group, and 3_00160 Primer2 is connected to the HEX group.

[0011] In another aspect, the present invention provides the application of the above-mentioned KASP primers, or reagents or kits containing the above-mentioned KASP primers, in detecting the major QTLs for resistance to Fusarium wilt in cowpeas.

[0012] In another aspect, the present invention provides a method for breeding different cowpea varieties resistant to Fusarium wilt using the above-mentioned KASP primers, the method specifically including the following steps: S1. Extract genomic DNA from cowpea plant samples; S2. Using cowpea plant sample genomic DNA as a template, KASP reaction detection was performed using 3_00160 KASP primers; S3. Read the fluorescence signal detected by the KASP reaction. If the genomic DNA of the cowpea plant sample shows the signal of the fluorescent group attached to 3_00160Primer1, then the sample is determined to carry the genotype of resistance to Fusarium wilt; if the genomic DNA of the cowpea plant sample shows the signal of the fluorescent group attached to 3_00160Primer2, then the sample is determined to carry the genotype of susceptibility to Fusarium wilt.

[0013] Preferably, Primer1 is linked to the FAM group and Primer2 is linked to the HEX group.

[0014] Preferably, the KASP reaction assay also includes 2×KASP Master mix reagent.

[0015] As a preferred option, the PCR system for KASP reaction detection is: DNA 0.8 μl, 2× KASP Master mix 0.75 μl, Primer mix 0.05 μl.

[0016] As a preferred method, the PCR reaction program for KASP reaction detection 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℃ per 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.

[0017] The terminology involved in this invention includes: Quantitative trait loci (QTLs) are loci in the genome that control quantitative traits.

[0018] Major effect QTLs refer to QTL loci that have a significant impact on the quantitative trait being studied or whose effect is relatively significant.

[0019] Kompetitive alle specific PCR (KASP) is a endpoint fluorescent genotyping technique based on known SNPs (single nucleotide polymorphisms) that can accurately detect biallelic alleles at specific sites in DNA samples.

[0020] Single nucleotide polymorphism (SNP) refers to DNA sequence polymorphism caused by a single nucleotide variation at the genomic level.

[0021] The advantages and beneficial effects of this invention are as follows: (1) The molecular marker 3_00160 disclosed in this invention is associated with the main QTL for resistance to Fusarium wilt, LFD-6.2 (r²=0.95), with a typing accuracy of over 99%, which can enable early and accurate identification of cowpea resistance to Fusarium wilt.

[0022] (2) Compared with traditional methods for identifying resistance to Fusarium wilt, this method is not limited by environmental conditions and can be tested during the seedling stage, significantly shortening the breeding cycle (from 2-3 years to 1 month) and reducing breeding costs.

[0023] (3) This marker can effectively distinguish between resistant and susceptible genotypes of Fusarium wilt, providing a reliable molecular tool for breeding cowpea varieties resistant to Fusarium wilt, and is particularly suitable for cowpea breeding in coastal and saline-alkali areas. Attached Figure Description

[0024] To more clearly illustrate the technical 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.

[0025] Figure 1 Genotyping diagram of the KASP gene associated with the 3_00160 marker site in 80 cowpea germplasms; Figure 2 Allelic variation differences in resistance to Fusarium wilt at the 3_00160 marker site. Detailed Implementation

[0026] 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 embodiments are only some embodiments of the present invention, and not all embodiments.

[0027] Unless otherwise specified, the experimental methods used in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] The PCR amplification system used for the KASP reaction in this embodiment of the invention is as follows: DNA 0.8 μl, 2×KASP Master mix 0.75 μl, Primer mix 0.05 μl. The Primer mix is ​​a mixture of Primer1 and Primer_Common, or Primer2 and Primer_Common, with 0.025 μl of each primer solution at a concentration of 10 μM / L.

[0030] 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.

[0031] Example 1: Screening of SNP sites associated with LFD-6.2, the major QTL for cowpea resistance to Fusarium wilt. This invention identified resistance to Fusarium wilt in 344 core cowpea germplasm materials from the Zhejiang Academy of Agricultural Sciences. A randomized block design was used, with three replicates per material. Inoculation was performed when the second trifoliate leaf had fully expanded. 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 no damage to the root system. The final concentration of the Fusarium wilt spore suspension was adjusted to 1 × 10⁻⁶. 5 The inoculum concentration was 50 mL / mL, and each plant was irrigated with 50 mL of bacterial solution. Approximately 28 days after inoculation, leaf damage (LDF) index was measured to identify resistance to Fusarium wilt. A control group was set up for each material without Fusarium wilt stress conditions and with all other conditions being the same.

[0032] Simultaneously, the population material was resequencing using the Illumina platform, yielding 1,856,342 high-quality SNP markers. Genome-wide association analysis was performed using a mixed linear model (MLM), identifying a major-effect QTL LFD-6.2 associated with Fusarium wilt resistance on chromosome 6. Based on this, the KASP marker 3_00160 was developed, with a polymorphism of A / G.

[0033] Table 1 shows the genotypes, disease index (DI), and susceptibility grades of some materials from 344 germplasm accessions. The susceptibility grade was calculated based on the disease index, which was determined by the degree of leaf damage. The leaf damage grading standards were as follows: Grade 0, no symptoms; Grade 1, mild symptoms, 1-2 leaves yellowing and falling off, but normal growth; Grade 2, obvious lesions or obvious wilting, more than 3 leaves yellowing and falling off, and growth inhibited; Grade 3, stunted growth, lodging, or severe wilting and death. For each accession, after statistically analyzing the disease grade of all inoculated plants, the disease index (DI) was calculated using the following formula: DI = ∑[ ( r × n r ) / λN t ].in, r Disease severity level n rThis refers to the number of individual plants at this level. N t The total number of plants surveyed for each material. λ The highest level is indicated by the disease index. The susceptibility level of the materials is classified according to the disease index, namely: high susceptibility (DI: 0-0.10), susceptibility (DI: 0.11-0.30), susceptibility (DI: 0.31-0.50), and high susceptibility (DI: 0.51-1).

[0034] Table 1. Genotypes and Fusarium wilt resistance identification of some materials from 344 samples. In this embodiment, the KASP marker 3_00160 was successfully screened and obtained. It is located on chromosome 6 in the genome of cowpea variety G98, and its SNP site is located at 46659973bp with polymorphism A / G.

[0035] The nucleotide sequence of the KASP-tagged sequence is shown in SEQ ID NO.1, with the SNP site at position 101.

[0036] Based on this sequence, the present invention designed a series of KASP primers, the sequences of which from 5' to 3' are shown below.

[0037] 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.

[0038] 3_00160 Primer1 is shown as SEQ ID NO.2; 3_00160 Primer2 is shown in SEQ ID NO.3; 3_00160 Primer_Common is shown in SEQ ID NO.4.

[0039] In some specific implementations, Primer1 is linked to the FAM group and Primer2 is linked to the HEX group.

[0040] Example 2: In this embodiment, 80 cowpea germplasm samples were randomly selected from the Vegetable Research Institute of Zhejiang Academy of Agricultural Sciences.

[0041] Phenotypic identification of resistance to Fusarium wilt was performed on 80 randomly selected cowpea germplasms. When the second trifoliate compound leaf of the seedling was fully expanded, a final concentration of 1×10⁻⁶ was applied. 5Inoculation was performed using a spore suspension of 1 spore / mL. After 28 days of treatment, leaf damage characteristics were measured to determine the disease index and resistance level. A control group was set up for each germplasm sample without the stress of Fusarium wilt, with all other conditions being the same.

[0042] Genomic DNA was extracted using the CTAB method, and KASP analysis was performed using the IntelliQube genotyping platform. The PCR reaction mixture consisted of 0.8 μl DNA, 0.75 μl 2×KASP Master Mix, and 0.05 μl primer mixture (primer preparation ratios were the same as in Example 1). The PCR program was as follows: 94℃ pre-denaturation for 15 min; 10 cycles of landing PCR (94℃ for 20 s, 61-55℃ for 60 s); and 26 conventional cycles (94℃ for 20 s, 55℃ for 60 s).

[0043] Analysis results as follows Figure 1 As shown in the diagram, when the fluorescence signal is red (FAM signal), it indicates that the cowpea carries the wilt resistance allele A (HapI). When the fluorescence signal is blue (HEX signal), it indicates that the cowpea carries the wilt susceptibility allele G (HapII).

[0044] Depend on Figure 1 As shown, the KASP molecular marker of this invention achieved highly consistent and well-defined population genotyping results in cowpea populations. The two genotypes could be clearly distinguished and clustered, and the genotyping effect was good.

[0045] Combined with its resistance to wilt disease (see...) Figure 2 (Partial data are shown in Table 2). It was found that lines carrying genotype A (n=18) had higher average resistance to Fusarium wilt, while lines carrying genotype G (n=57) had relatively lower average resistance. Data analysis showed that the average resistance to Fusarium wilt in lines carrying genotype A was significantly higher than that in lines carrying genotype G (p<0.0001), confirming that this marker can effectively distinguish differences in Fusarium wilt resistance in cowpeas, ultimately achieving the purpose of molecular marker screening.

[0046] Table 2. Statistical data on genotypes and phenotypic characteristics of selected germplasm treatments. Note: The disease index and the criteria for the level of resistance to infection are consistent with those in Example 1.

[0047] 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 associated with a major QTL for cowpea resistance to Fusarium wilt, characterized in that, The molecular marker is 3_00160, and the molecular marker is of the KASP type; the SNP site of the molecular marker is located at 46659973 bp on chromosome 6 of the cowpea variety G98 genome, and the polymorphism is A / G; the nucleotide sequence of the molecular marker is shown in SEQ ID NO.

1.

2. A KASP primer for detecting the molecular marker as described in claim 1, characterized in that, The KASP primers include 3_00160 Primer1, 3_00160 Primer 2, and 3_00160 Primer_Common; 3_00160 Primer1 is shown as SEQ ID NO.2; 3_00160 Primer2 is shown in SEQ ID NO.3; 3_00160 Primer_Common is shown in SEQ ID NO.4; 3_00160 Primer1 is linked to the FAM group, and 3_00160 Primer2 is linked to the HEX group.

3. The application of the KASP primer according to claim 2, or a kit containing the KASP primer according to claim 2, in detecting the major QTL for resistance to Fusarium wilt in cowpea.

4. A method for breeding different cowpea varieties resistant to Fusarium wilt using the KASP primers described in claim 2, characterized in that, The method specifically includes the following steps: S1. Extract genomic DNA from cowpea plant samples; S2. Using cowpea plant sample genomic DNA as a template, KASP reaction detection was performed using the KASP primers described in claim 2; S3. Read the fluorescence signal detected by the KASP reaction. If the genomic DNA of the cowpea plant sample shows the signal of the FAM group of the fluorescent group attached to 3_00160 Primer1, then the sample is determined to carry the genotype of resistance to Fusarium wilt; if the genomic DNA of the cowpea plant sample shows the signal of the HEX group of the fluorescent group attached to 3_00160 Primer2, then the sample is determined to carry the genotype of susceptibility to Fusarium wilt.

5. The method for breeding different cowpea varieties resistant to Fusarium wilt according to claim 4, characterized in that, The KASP reaction assay also includes 2×KASP Master mix reagents.

6. The method for breeding different cowpea varieties resistant to Fusarium wilt according to claim 5, characterized in that, The PCR system for KASP reaction detection was as follows: DNA 0.8 μl, 2×KASP Master mix 0.75 μl, Primer mix 0.05 μl.

7. The method for breeding different cowpea varieties resistant to Fusarium wilt according to claim 4, characterized in that, The PCR reaction program for KASP reaction detection 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℃ per 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.

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