A molecular marker tightly linked to a major QTL in cowpea resistance to Fusarium wilt and its application

Abstract

This invention discloses a molecular marker tightly linked to a major QTL for resistance to Fusarium wilt in cowpea and its application, belonging to the field of plant molecular breeding technology. The KASP-type molecular marker 3_00139, tightly linked to this major QTL, is located at 5729658 bp on chromosome 3. This marker can be used to amplify test materials, and the genotype can be used to determine whether the sample carries the allelic variant controlling Fusarium wilt resistance. Using the molecular marker and primers provided by this invention, rapid screening of major QTLs for Fusarium wilt resistance in cowpea can be achieved, significantly improving the efficiency of disease-resistant variety breeding and shortening the breeding cycle.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology and relates to a molecular marker (3_00139) that is tightly linked to a major QTL for cowpea resistance to Fusarium wilt and its application. Background Technology

[0002] cowpea( Vigna unguiculata L. Walp is an important legume crop. Fusarium wilt is one of the most devastating soil-borne diseases in cowpea production, causing crop rotation problems in the field. It is mainly caused by *Fusarium oxysporum* var. *hygroscopicum*. Fusarium oxysporum f. sp. Tracheiphilum, This disease is caused by *Fot*. Under high temperature and humidity conditions, it can lead to yield losses of up to 70%, or even total crop failure. Conventional control measures are difficult to achieve effective control. Breeding and promoting disease-resistant varieties is the most economical, effective, and sustainable approach. However, traditional breeding methods are time-consuming, labor-intensive, and slow.

[0003] Kompetitive allele-specific PCR (KASP) is a novel genotyping technique that can accurately genotype single nucleotide polymorphisms (SNPs) and insertion and deletion polymorphisms (InDels) at the genome level, featuring high throughput, low cost, and high accuracy. This technique can be used to assist molecular breeding to accelerate the breeding process, providing a novel technical means for breeding cowpea for resistance to Fusarium wilt. Currently, this technology has been successfully applied to crops such as tomatoes and watermelons, and molecular markers for diagnosis and breeding have been obtained. However, the number of KASP markers for cowpea resistance to Fusarium wilt is relatively limited; the completion of genome sequencing for this species provides favorable conditions for the successful implementation of this technology. Summary of the Invention

[0004] 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_00139) closely linked to the major QTL (VD-3.1) for cowpea resistance to Fusarium wilt, along with its KASP primers, and establishes a Fusarium wilt-assisted breeding method based on this marker. This method identifies SNP loci associated with Fusarium wilt resistance through quantitative trait locus mapping analysis, 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.

[0005] This invention, through quantitative trait locus (QTL) mapping analysis of the trait vascular discoloration (VD), identified the major-effect QTL VD-3.1 for resistance to Fusarium wilt located at 5729658 bp on chromosome 3 of cowpea, and developed the closely linked KASP molecular marker 3_00139. 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.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In one aspect, this invention provides a molecular marker closely linked to VD-3.1, the major QTL for resistance to Fusarium wilt in cowpea. The molecular marker is 3_00139, located at 5729658 bp on chromosome 3 of the cowpea variety G98 genome. The molecular marker is of the KASP type, and the SNP site polymorphism is T / A.

[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;

[0010] 3_00139 Primer1 is shown in SEQ ID NO.2;

[0011] 3_00139 Primer2 is shown in SEQ ID NO.3;

[0012] 3_00139 Primer1 and 3_00139 Primer2 are two specific primers, each linked to a different fluorescent sequence;

[0013] 3_00139 Primer_Common is shown in SEQ ID NO.4.

[0014] Preferably, 3_00139 Primer1 is linked to the FAM group, and 3_00139 Primer2 is linked to the HEX group.

[0015] 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.

[0016] 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:

[0017] S1. Extract genomic DNA from cowpea plant samples;

[0018] S2. Using cowpea plant sample genomic DNA as a template, KASP reaction detection was performed using 3_00139 KASP primers;

[0019] 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 FAM group attached to 3_00139Primer1, then the sample is determined to carry the genotype of susceptibility to Fusarium wilt. If the genomic DNA of the cowpea plant sample shows the signal of the fluorescent group HEX group attached to 3_00139Primer2, then the sample is determined to carry the genotype of resistance to Fusarium wilt.

[0020] Preferably, Primer1 is linked to the FAM group and Primer2 is linked to the HEX group.

[0021] Preferably, the KASP reaction assay also includes 2×KASP Master mix reagent.

[0022] 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.

[0023] 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.

[0024] The terminology involved in this invention includes:

[0025] Quantitative trait loci (QTLs) are loci in the genome that control quantitative traits.

[0026] Major effect QTLs refer to QTL loci that have a significant impact on the quantitative trait being studied or whose effect is relatively significant.

[0027] 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.

[0028] Single nucleotide polymorphism (SNP) refers to DNA sequence polymorphism caused by a single nucleotide variation at the genomic level.

[0029] The advantages and beneficial effects of this invention are as follows:

[0030] (1) The molecular marker 3_00139 disclosed in this invention is closely linked to the main QTL VD-3.1 for resistance to Fusarium wilt (r²=0.93), with a typing accuracy of over 99%, which can realize early and accurate identification of cowpea resistance to Fusarium wilt.

[0031] (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.

[0032] (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

[0033] 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.

[0034] Figure 1 Genotyping diagram of the KASP gene associated with the 3_00139 marker site in 120 cowpea germplasms;

[0035] Figure 2 Allelic variation differences in resistance to Fusarium wilt at the 3_00139 marker site. Detailed Implementation

[0036] 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.

[0037] Unless otherwise specified, the experimental methods used in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] 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.

[0040] 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.

[0041] Example 1: Screening of SNP sites closely linked to VD-3.1, the major QTL for resistance to Fusarium wilt in cowpea.

[0042] This invention uses disease-resistant cowpea material (ZN016) and disease-susceptible material (Zhijiang 282) as parents to construct a recombinant inbred line population of F8 generation cowpea containing 209 materials for phenotypic identification of resistance to Fusarium wilt. A randomized block design was used, with three replicates planted for each material. Inoculation was carried out 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 plant roots. 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, vascular bundle browning indices were measured to assess resistance to Fusarium wilt. A control group was established for each material without Fusarium wilt stress, with all other conditions identical. Resequencing of the population was performed using the Illumina platform, yielding 1122 high-quality SNP markers. Quantitative trait locus mapping analysis was performed using MapQTL5 software and the Interval Mapping (IM) method. A major-effect QTL VD-3.1 associated with Fusarium wilt resistance was identified on chromosome 3, explaining 21.30% of the phenotypic variation. Based on this, the KASP marker 3_00139 was developed, with a polymorphism of T / A.

[0043] Table 1 shows the genotypes, disease index (DI), and resistance / susceptibility grades of some of the 209 materials. The resistance / susceptibility grade was calculated based on the disease index, which was determined by the degree of browning of the vascular bundles.

[0044] The criteria for judging the browning level of vascular bundles are as follows: Level 0, no browning of plant roots; Level 1, browned tissue accounts for about 10%; Level 2, browned tissue accounts for about 25%; Level 3, browned tissue accounts for about 50%; Level 4, browned tissue accounts for about 75%; Level 5, browned tissue accounts for about 100%.

[0045] After statistically analyzing the disease severity of all inoculated plants for each data set, the disease index (DI) was calculated using the following formula: DI = ∑[ ( r × n r ) / λN t ].in, r Disease severity level n r This 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).

[0046] Table 1. Genotypes and Fusarium wilt resistance identification of some materials from 209 samples.

[0047]

[0048] In this embodiment, the KASP marker 3_00139 was successfully screened and obtained. It is located on chromosome 3 in the genome of cowpea variety G98, and its SNP site is located at 5729658 bp with a polymorphism of T / A.

[0049] The nucleotide sequence of the KASP-tagged sequence is shown in SEQ ID NO.1, with the SNP site at position 101.

[0050] Based on this sequence, the present invention designed a series of KASP primers, the sequences from 5' to 3' of which are shown below.

[0051] 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.

[0052] 3_00139 Primer1 is shown in SEQ ID NO.2;

[0053] 3_00139 Primer2 is shown in SEQ ID NO.3;

[0054] 3_00139 Primer_Common is shown in SEQ ID NO.4.

[0055] In some specific implementations, Primer1 is linked to the FAM group and Primer2 is linked to the HEX group.

[0056] Example 2:

[0057] In this embodiment, 120 cowpea germplasm samples were randomly selected from the Vegetable Research Institute of Zhejiang Academy of Agricultural Sciences.

[0058] Phenotypic identification of resistance to Fusarium wilt was performed on 120 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. 5 Inoculation was performed using a spore suspension of 1 spore / mL. After 28 days of treatment, the browning rate of the vascular bundle tissue was 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, and all other conditions were the same.

[0059] 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).

[0060] 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 susceptibility allele A (HapII). When the fluorescence signal is blue (HEX signal), it indicates that the cowpea carries the wilt resistance allele T (HapI).

[0061] Depend on Figure 1As 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.

[0062] Combined with its resistance to wilt disease (see...) Figure 2 (Partial data are shown in Table 2). It was found that lines carrying the T genotype (n=35) had higher average resistance to Fusarium wilt, while lines carrying the A genotype (n=80) had relatively lower average resistance. Data analysis showed that the average resistance to Fusarium wilt in lines carrying the T genotype was significantly higher than that in lines carrying the A genotype (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.

[0063] Table 2. Statistical data on genotypes and phenotypic characteristics of selected germplasm treatments.

[0064]

[0065] Note: The disease index and the criteria for the level of resistance to infection are consistent with those in Example 1.

[0066] 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 closely linked to a major QTL for resistance to Fusarium wilt in Vigna unguiculata, characterized in that, The molecular marker is 3_00139, and the molecular marker is of the KASP type; the SNP site of the molecular marker is located at 5729658 bp on chromosome 3 of the cowpea variety G98 genome, and the polymorphism is T / A; the nucleotide sequence of the molecular marker is shown in SEQ ID NO.

1.

2. A KASP primer for detecting the molecular marker of claim 1, characterized in that, The KASP primers include 3_00139 Primer1, 3_00139 Primer 2, and 3_00139 Primer_Common; 3_00139 Primer1 is shown in SEQ ID NO.2; 3_00139 Primer2 is shown in SEQ ID NO.3; 3_00139 Primer_Common is shown in SEQ ID NO.4; 3_00139 Primer1 is linked to the FAM group, and 3_00139 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_00139 Primer1, then the sample is determined to carry the genotype of susceptibility 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_00139 Primer2, then the sample is determined to carry the genotype of resistance 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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