Molecular marker closely linked with cowpea salt tolerance major QTL (quantitative trait loci) as well as primer and application of molecular marker

Abstract

The invention discloses a molecular marker closely linked with a cowpea salt tolerance major QTL (quantitative trait loci) and a primer sequence of the molecular marker. The name of the cowpea salt tolerance major QTL is SC300126, a KASP type molecular marker 300126 closely linked with the cowpea salt tolerance major QTL is located at the position of 43816166 bp on a chromosome 11, the marker can be used for amplifying a material to be detected, and whether a sample carries key allelic variation for controlling salt tolerance or not is judged according to the genotype of the marker. The molecular marker and the primer provided by the invention can be used for quickly screening the cowpea salt tolerance major QTL, and compared with a traditional phenotype identification method, the molecular marker and the primer have the advantages of high detection flux, accurate and reliable result and the like, and the typing accuracy can reach 99% or above. The technology can significantly improve the breeding efficiency of the salt-tolerant variety of cowpea and shorten the breeding period.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant molecular breeding, and relates to a molecular marker closely linked to a main-effect QTL (SC3_00126) of salt tolerance of cowpea, primers thereof and application. BACKGROUND

[0002] Cowpea (Vigna unguiculata (L.) Walp.) is an important crop of the genus Vigna Savi of the family Fabaceae, with a genome size of about 620 Mb and a chromosome number of 2n=2x=22. As a legume crop widely cultivated in tropical and subtropical regions, cowpea is one of the important edible legumes and vegetable crops.

[0003] Salt stress is an important environmental factor affecting the growth and yield of cowpea, especially in coastal areas and inland saline soils, where soil salinization seriously restricts the planting and yield of cowpea. Studies have shown that there is significant difference in salt tolerance among cowpea varieties, which is mainly determined by genetic factors. As a complex quantitative trait, salt tolerance is controlled by multiple genes and is easily affected by the environment, and traditional breeding methods have low efficiency and long cycle through phenotypic screening.

[0004] With the completion of cowpea genome sequencing and the development of molecular marker technology, SNP (single nucleotide polymorphism)-based molecular marker-assisted selection provides a new technical means for cowpea salt tolerance breeding. Among them, the Kompetitive Allele Specific PCR (KASP) technology has become an important tool for crop molecular breeding due to its high throughput, low cost and high accuracy. KASP technology has achieved remarkable results in salt tolerance breeding of crops such as rice and wheat, but its application in cowpea salt tolerance breeding is still in its infancy. SUMMARY

[0005] The technical problem solved by the present application is to use KASP markers for assisted breeding of cowpea salt tolerance, to provide a molecular marker closely linked to a main-effect QTL (SC3_00126) of cowpea salt tolerance and KASP primers thereof, and to establish a salt tolerance assisted breeding method based on the marker. The method identifies SNP sites related to salt tolerance through genome-wide association analysis, converts them into KASP markers for verification, and can be used for rapid identification of salt tolerance genotypes of cowpea germplasm, providing molecular breeding technical support for genetic improvement of cowpea salt tolerance.

[0006] This invention identified a major salt-tolerance QTL (SC3_00126) located at 43816166 bp on chromosome 11 of cowpea using genome-wide association analysis (GWAS), and developed a KASP molecular marker closely linked to it. The development of this marker provides an effective technical means for molecular breeding of salt tolerance in cowpea, 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: This invention provides a molecular marker tightly linked to a major QTL for salt tolerance in cowpea, the marker being 3_00126, located at 43816166 bp on chromosome 11 of the cowpea variety G98. The marker is of the KASP type, with a C / T SNP polymorphism. In some preferred embodiments, the nucleotide sequence of the marker is shown in SEQ ID NO.1.

[0008] In another aspect, the present invention provides a KASP primer for detecting the above-mentioned molecular marker, the KASP primer comprising a specific forward primer 3_00126 Primer1, a specific forward primer 3_00126 Primer2, and a universal reverse primer 3_00126 Primer_Common; In some preferred embodiments, the detection system of the present invention uses complementary strands as primers to bind to templates for the reaction; the SNP polymorphism is C / T, while the corresponding polymorphism on the complementary strand is G / A.

[0009] 3_00126 Primer1 is shown in SEQ ID NO.2; 3_00126 Primer2 is shown in SEQ ID NO.3; Typically, 3_00126 Primer1 and 3_00126 Primer2 are two specific primers, each linked to a different fluorescent sequence; in some preferred embodiments, 3_00126 Primer1 is linked to the FAM group, and 3_00126 Primer2 is linked to the HEX group.

[0010] 3_00126 Primer_Common is shown in SEQ ID NO.4.

[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 major QTLs of salt tolerance in cowpeas.

[0012] In another aspect, this invention provides a method for breeding different salt-tolerant cowpea varieties using the above-mentioned KASP primers, the method specifically comprising 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_00126 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 FAM group of the fluorescent group attached to 3_00126Primer1, then the sample is determined to carry the salt tolerance genotype; if the genomic DNA of the cowpea plant sample shows the signal of the HEX group of the fluorescent group attached to 3_00126Primer2, then the sample is determined to carry the salt sensitivity genotype.

[0013] As a preferred option, the KASP reaction assay also includes 2X KASP Master mix reagent.

[0014] As a preferred option, the PCR system for KASP reaction detection is as follows: 0.8 μl DNA, 0.75 μl 2x KASP Master mix, and 0.05 μl Primer mix. 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.

[0015] 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.

[0016] The terminology involved in this invention includes: A quantitative trait locus (QTL) is a gene locus in the genome that controls a quantitative trait.

[0017] Major effect QTLs refer to QTL loci that have a significant impact on the quantitative trait being studied or whose effect is relatively significant.

[0018] Kompetitive Allele Specific PCR (KASP) is a endpoint fluorescent genotyping technique based on known SNPs (single nucleotide polymorphisms). It can accurately detect biallelic alleles at specific sites in DNA samples.

[0019] Single nucleotide polymorphism (SNP) refers to DNA sequence polymorphism caused by a single nucleotide variation at the genomic level.

[0020] The advantages and beneficial effects of this invention are as follows: (1) The molecular marker 3_00126 disclosed in this invention is closely linked to the major QTL of salt tolerance (r²=0.999), and the typing accuracy rate is over 99.9%, which can realize the early and accurate identification of salt tolerance of cowpea.

[0021] (2) Compared with traditional salt tolerance identification methods, this method is not limited by environmental conditions and can be tested during the seedling stage, which significantly shortens the breeding cycle (from 2-3 years to 1 month) and reduces breeding costs.

[0022] (3) This marker can effectively distinguish between salt-tolerant and salt-sensitive genotypes, providing a reliable molecular tool for the breeding of salt-tolerant cowpea varieties, and is particularly suitable for cowpea breeding work in coastal and saline-alkali areas. Attached Figure Description

[0023] 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.

[0024] Figure 1 KASP marker genotyping diagram of genes associated with the 3_00126 marker site; Figure 2 A graph showing the differences in salt tolerance allelic variation at the 3_00126 marker site. Detailed Implementation

[0025] 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.

[0026] Unless otherwise specified, the experimental methods used in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] The PCR amplification system used for the KASP reaction in this embodiment of the invention is as follows: DNA 0.8 μl, 2x 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.

[0029] 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.

[0030] Example 1: Screening of SNP sites closely linked to the major QTL for salt tolerance in cowpea This invention collected and screened natural cowpea populations comprising 344 accessions for salt tolerance phenotypic identification. A randomized block design was used, with three replicates per accession. Aboveground fresh weight was measured under salt stress conditions (final concentration 150 mM NaCl). A control group was set up for each accession, without salt stress but with all other conditions identical. The aboveground fresh weight percentage for each accession was calculated. The aboveground fresh weight percentage was calculated as the percentage of average aboveground fresh weight per plant in the treatment group and the control group.

[0031] Resequencing of the population material using the Illumina platform yielded 1,856,342 high-quality SNP markers. Genome-wide association analysis (GWAS) was performed using a mixture linear model (MLM) and considering the aboveground fresh weight ratios of various qualities. A major-effect SNP linkage site (SC3_00126) was identified at 43,816,166 bp on chromosome 11. This site is tightly linked to an unknown functional gene encoding a CRS1 (RNA-binding domain) structure. Based on this, the KASP marker 3_00126 with a C / T polymorphism was developed.

[0032] Table 1 shows the statistical results of genotypes and aboveground fresh weight ratios of some materials from 344 germplasm accessions.

[0033] Table 1. Genotypes and aboveground fresh weight percentages of some materials from 344 accessions The KASP marker 3_00126 obtained in this embodiment is located on chromosome 11 of the genome of cowpea variety G98. Its SNP site is located at 43816166bp, and its polymorphism is C / T.

[0034] The nucleotide sequence of the KASP-tagged sequence is shown in SEQ ID NO.1, with the SNP site at position 101.

[0035] Based on this sequence, this invention designed a series of KASP primers, including a specific forward primer 3_00126Primer1, a specific forward primer 3_00126Primer2, and a universal reverse primer 3_00126Primer_Common, whose sequences from 5' to 3' are shown below. Their sequences are shown below respectively.

[0036] 3_00126 Primer1 is shown in SEQ ID NO.2; 3_00126 Primer2 is shown in SEQ ID NO.3; 3_00126 Primer_Common is shown in SEQ ID NO.4.

[0037] 3_00126 Primer1 is linked to the FAM group, and 3_00126 Primer2 is linked to the HEX group.

[0038] Example 2: In this embodiment, 70 cowpea germplasm samples were randomly selected from the Vegetable Research Institute of Zhejiang Academy of Agricultural Sciences.

[0039] Salt tolerance phenotypes were identified in 70 randomly selected cowpea germplasms. Salt treatment was performed using hydroponics. After 7 days of treatment with 150 mM NaCl at the three-leaf stage of seedlings, the aboveground fresh weight was measured. A control group without salt stress and with all other conditions identical was set up for each germplasm. The aboveground fresh weight ratio of each material was calculated.

[0040] Genomic DNA was extracted using the CTAB method. The genomic DNA of the above population was amplified using the IntelliQube genotyping platform and the KASP primers (SC3_00126) designed in Example 1. KASP analysis was then performed. Amplification system: DNA 0.8 μl, 2×KASP Master Mix 0.75 μl, primer mixture 0.05 μl. PCR program: 94℃ pre-denaturation for 15 min; 10 cycles of landing PCR (94℃ 20 s, 61-55℃ 60 s); 26 conventional cycles (94℃ 20 s, 55℃ 60 s).

[0041] The primer mix was prepared by mixing Primer1 with Primer_Common or Primer2 with Primer_Common. The volume of each primer was 0.025 μl and the concentration was 10 μM / L.

[0042] Fluorescence data were read and analyzed using an IntelliQube machine. The analysis results are as follows: Figure 1 As shown in the diagram, a red fluorescence signal (FAM signal) indicates that the cowpea carries the salt-tolerant allele C (HapI). A blue fluorescence signal (HEX signal) indicates that the cowpea carries the salt-sensitive allele T (HapII).

[0043] 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.

[0044] Based on its salt tolerance performance (see...) Figure 2 (Partial data are shown in Table 2). It was found that lines carrying the C genotype had a higher average aboveground fresh weight percentage of salt tolerance, while lines carrying the T genotype had a relatively lower average aboveground fresh weight percentage of salt tolerance. Data analysis showed that the average salt tolerance of lines carrying the T genotype was significantly higher than that of the C genotype (p < 0.0001), confirming that this marker can effectively distinguish differences in salt tolerance in cowpeas, ultimately achieving the purpose of molecular marker screening.

[0045] Table 2. Statistical data of genotypes and salt treatment phenotypes of selected germplasm. Note: The aboveground fresh weight ratio standard is the same as that in Example 1, which is the aboveground fresh weight ratio of the same germplasm treatment group and the control group.

[0046] 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 salt tolerance in Vigna unguiculata, characterized in that, The molecular marker is 3_00126, which is a KASP type; the SNP site of the molecular marker is located at 43816166 bp on chromosome 11 of the G98 genome of the cowpea variety, and the polymorphism of the SNP site is C / T; the nucleotide sequence of the molecular marker is shown as SEQ ID No.

1.

2. A KASP primer for detecting the molecular marker of claim 1, characterized in that, The KASP primer comprises a specific forward primer 3_00126 Primer1, a specific forward primer 3_00126 Primer2 and a common reverse primer 3_00126 Primer_Common. 3_00126 Primer1 is shown as SEQ ID No.

2. 3_00126 Primer2 is shown as SEQ ID No.

3. 3_00126 Primer_Common is shown as SEQ ID No.

4. 3_00126 Primer1 is connected with a HEX group, and 3_00126 Primer2 is connected with a SAM group.

3. A KASP primer according to claim 2, or a reagent or kit containing the KASP primer of claim 2, for use in detecting a salt tolerance major QTL in cowpea.

4. A method for breeding cowpea varieties with different salt tolerance using the KASP primer of claim 2, characterized in that, The method specifically comprises the following steps: S1, extracting cowpea plant sample genomic DNA; S2, using 3_00126 KASP primer for KASP reaction detection with cowpea plant sample genomic DNA as a template; S3, reading the fluorescence signal of KASP reaction detection, if the cowpea plant sample genomic DNA shows 3_00126 Primer1 connected fluorescence group FAM group signal, it is judged that the sample carries salt tolerance trait genotype; if the cowpea plant sample genomic DNA shows 3_00126 Primer2 connected fluorescence group HEX group signal, it is judged that the sample carries salt sensitive trait genotype.

5. The method of breeding varieties of cowpea with different salt tolerance as claimed in claim 4, wherein, The KASP reaction detection further comprises 2X KASP Master mix reagent.

6. The method of breeding cowpea varieties with different salt tolerance as claimed in claim 5, wherein, The PCR system of KASP reaction detection is: DNA 0.8 μl, 2x KASP Master mix 0.75 μl, Primer mix 0.05 μl.

7. The method for breeding cowpea varieties with different salt tolerance according to claim 4, characterized in that, The PCR reaction program of KASP reaction detection is: 94℃ pre-denaturation for 15 minutes, 94℃ denaturation for 20 seconds, 61-55℃ gradient recombination for 60 seconds, each cycle recombination temperature decreases by 0.6℃, 55℃ extension for 60 seconds, 10 cycles; then 94℃ denaturation for 20 seconds, 55℃ recombination for 60 seconds, extension for 60 seconds, 26 cycles. The PCR system of KASP reaction detection is: DNA 0.8 μl, 2x KASP Master mix 0.75 μl, Primer mix 0.05 μl.

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