A molecular marker closely linked to a main-effect qtl of salt tolerance of vigna unguiculata, a primer and application thereof

Abstract

The application discloses a molecular marker closely linked with a main-effect QTL of salt tolerance of cowpea and primer sequences of the molecular marker. The main-effect QTL of salt tolerance of cowpea disclosed by the application is named SC3_00126, and a KASP type molecular marker 3_00126 closely linked with the main-effect QTL is located at a position of 43816166 bp on chromosome 11. The marker can be used for amplifying to-be-tested materials, and whether the sample carries a key allelic variation for controlling salt tolerance can be judged according to the genotype. The molecular marker and the primer provided by the application can be used for rapidly screening the main-effect QTL of salt tolerance of cowpea, and compared with a traditional phenotype identification method, the application has the advantages of high detection flux, accurate and reliable results and the like, and the genotyping accuracy can reach more than 99%. The technology can significantly improve the breeding efficiency of salt-tolerant varieties 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] The application identifies a salt tolerance major QTL (SC3_00126) located at the position of 43816166 bp of chromosome 11 of Vigna unguiculata by genome-wide association analysis (GWAS), and develops a KASP molecular marker closely linked thereto. The development of the marker provides an effective technical means for salt tolerance molecular breeding of Vigna unguiculata, and will significantly improve the breeding efficiency and shorten the breeding period.

[0007] To achieve the above object, the application provides the following technical solutions.

[0008] In one aspect, the application provides a molecular marker closely linked to the salt tolerance major QTL of Vigna unguiculata, which is 3_00126, located at the position of 43816166 bp of chromosome 11 of the genome of Vigna unguiculata variety G98, and is a KASP type, with SNP site polymorphism being C / T. In some preferred embodiments, the nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1.

[0009] In another aspect, the application also provides a KASP primer for detecting the above molecular marker, which comprises a specific forward primer 3_00126 Primer1, a specific forward primer 3_00126 Primer2 and a common reverse primer 3_00126 Primer_Common.

[0010] In some preferred embodiments, the detection system of the application uses a complementary strand as a primer binding template for reaction; the SNP polymorphism is C / T, and the corresponding G / A on the complementary strand.

[0011] 3_00126 Primer1 is shown in SEQ ID NO. 2;

[0012] 3_00126 Primer2 is shown in SEQ ID NO. 3;

[0013] Generally, 3_00126 Primer1 and 3_00126 Primer2 are two specific primers, respectively connected with different fluorescent sequences; in some preferred embodiments, 3_00126 Primer1 is connected with a FAM group, and 3_00126 Primer2 is connected with a HEX group.

[0014] 3_00126 Primer_Common is shown in SEQ ID NO. 4.

[0015] In another aspect, the application also provides a use of the above KASP primer, or a reagent or kit containing the above KASP primer in detecting the salt tolerance major QTL of Vigna unguiculata.

[0016] The application also provides a method for breeding different salt-tolerant varieties of cowpea using the KASP primer, which specifically comprises the following steps:

[0017] S1, extracting genomic DNA of cowpea plant samples;

[0018] S2, using 3_00126 KASP primer for KASP reaction detection with the genomic DNA of cowpea plant samples as a template;

[0019] S3, reading the fluorescence signal of KASP reaction detection, if the genomic DNA of cowpea plant samples shows the signal of the fluorescent group FAM group connected by 3_00126 Primer1, it is judged that the sample carries the salt-tolerant trait genotype; if the genomic DNA of cowpea plant samples shows the signal of the fluorescent group HEX group connected by 3_00126 Primer2, it is judged that the sample carries the salt-sensitive trait genotype.

[0020] As preferred, the KASP reaction detection further comprises 2X KASP Master mix reagent.

[0021] As preferred, the PCR system of KASP reaction detection is: DNA 0.8 μl, 2x KASP Master mix 0.75 μl, Primer mix 0.05 μl. The Primer mix is the mixture of Primer1 and Primer_Common, or Primer2 and Primer_Common, each primer solution is 0.025 μl, and the concentration is 10 μM / L.

[0022] As preferred, 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; 94℃ denaturation for 20 seconds, 55℃ recombination for 60 seconds, extension for 60 seconds, 26 cycles.

[0023] The name terms involved in the application are:

[0024] Quantitative trait loci (QTL) refers to the locus in the genome that controls quantitative traits.

[0025] Major QTL refers to the quantitative trait locus that has a greater impact or a more significant effect on the quantitative traits studied.

[0026] Kompetitive Allele Specific PCR (KASP) is a kind of end-point fluorescence genotyping technology based on known SNP (Single Nucleotide Polymorphism), which can accurately detect the SNP and indel (insertion and deletion) at specific loci in DNA samples.

[0027] Single Nucleotide Polymorphism (SNP) refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level.

[0028] The advantages and beneficial effects of the present application are as follows:

[0029] (1) The molecular marker 3_00126 disclosed in the present application is closely linked to the salt tolerance major QTL (r²=0.999), and the typing accuracy is more than 99.9%, which can realize early and accurate identification of cowpea salt tolerance.

[0030] (2) Compared with the traditional salt tolerance identification method, the present method is not limited by environmental conditions, and can be detected at the seedling stage, significantly shortening the breeding period (from 2-3 years to 1 month) and reducing the breeding cost.

[0031] (3) The marker can effectively distinguish salt-tolerant and salt-sensitive genotypes, and provides a reliable molecular tool for cowpea salt-tolerant variety breeding, and is particularly suitable for cowpea breeding work in coastal and saline-alkali areas. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiment or prior art description will be briefly introduced as follows.

[0033] Figure 1 Figure 3 is a KASP marker genotyping diagram of the 3_00126 marker site related gene;

[0034] Figure 2 Figure 4 is a salt tolerance allelic variation difference diagram of the 3_00126 marker site. DETAILED DESCRIPTION

[0035] In order to further understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments.

[0036] The experimental methods used in the following examples are generally performed according to the conventional conditions or the conditions recommended by the manufacturer, unless otherwise specified.

[0037] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0038] The PCR amplification system used in the KASP reaction of the embodiment of the present application is: DNA 0.8 μl, 2x KASP Master mix 0.75 μl, Primer mix 0.05 μl. The primer mix is a mixture of Primer 1 and Primer_Common, or Primer 2 and Primer_Common, each primer solution being 0.025 μl, and the concentration being 10 μM / L.

[0039] The PCR amplification procedure adopted in the embodiment of the present application is: pre-denaturation at 94℃ for 15 minutes, denaturation at 94℃ for 20 seconds, gradient recombination at 61-55℃ for 60 seconds, recombination temperature decreasing by 0.6℃ for each cycle, extension at 55℃ for 60 seconds, 10 cycles; then denaturation at 94℃ for 20 seconds, recombination at 55℃ for 60 seconds, extension for 60 seconds, 26 cycles.

[0040] Example 1: Screening of SNP sites closely linked to the main QTL of salt tolerance of cowpea

[0041] The present application collects and screens a cowpea natural population containing 344 materials for salt tolerance phenotype identification. A randomized block design is adopted, each material is planted in 3 replicates, the aboveground fresh weight is measured under salt stress conditions (final concentration 150 mM NaCl), and a control group without salt stress is set for each material, and the rest of the conditions are consistent. The aboveground fresh weight ratio is calculated according to the percentage of the average aboveground fresh weight of single plant of the treatment group and the control group.

[0042] The population materials are resequenced using the Illumina platform, and 1,856,342 high-quality SNP markers are obtained. Combined with the aboveground fresh weight ratio of various qualities, whole genome association analysis is carried out by mixed linear model (MLM), and a main SNP linkage site (SC3_00126) is identified at 43816166 bp on chromosome 11, which is closely linked to an unknown function gene containing a CRS1 (RNA binding domain) structure domain. Based on this, KASP marker 3_00126 is developed, and its polymorphism is C / T.

[0043] The genotype of part of the materials in the 344 germplasms and the statistical results of the aboveground fresh weight ratio thereof are shown in Table 1.

[0044] Table 1 Genotype of part of the materials in the 344 and aboveground fresh weight ratio thereof

[0045]

[0046] The KASP marker 3_00126 screened in the embodiment is located on chromosome 11 of the genome of the cowpea variety G98, and the SNP site is located at 43816166 bp, and the polymorphism is C / T.

[0047] The nucleotide sequence of the KASP marker is shown as SEQ ID NO. 1, and the SNP site is the 101st site in the sequence.

[0048] The present application designs a series of KASP primers according to the sequence, and the KASP primers of the molecular marker include specific forward primer 3_00126 Primer1, specific forward primer 3_00126 Primer2 and universal reverse primer 3_00126 Primer_Common, and the sequences from 5' to 3' are as follows. The sequences are as follows.

[0049] 3_00126 Primer1 is shown as SEQ ID NO. 2;

[0050] 3_00126 Primer2 is shown as SEQ ID NO. 3;

[0051] 3_00126 Primer_Common is shown as SEQ ID NO. 4.

[0052] 3_00126 Primer1 is connected with FAM group, and 3_00126 Primer2 is connected with HEX group.

[0053] Example 2:

[0054] In this embodiment, 70 cowpea germplasms obtained from the Institute of Vegetable Research, Zhejiang Academy of Agricultural Sciences are randomly selected.

[0055] The salt tolerance phenotype of 70 randomly selected cowpea germplasms is identified. The salt treatment adopts the water culture method, and the aboveground fresh weight is measured after 150 mM NaCl treatment for 7 days at the three-leaf stage of seedlings. Each germplasm is set with a control group without salt stress, and the rest of the conditions are consistent. The aboveground fresh weight ratio of each material is calculated.

[0056] Genomic DNA was extracted by CTAB method, and KASP analysis was performed by using IntelliQube genotyping platform, KASP primer of SC3_00126 designed in Example 1 to amplify genomic DNA of the above population. Amplification system: DNA 0.8 μl, 2x KASP Master Mix 0.75 μl, primer mix 0.05 μl. PCR program: 94℃ pre-denaturation for 15 min; 10 cycles of descending PCR (94℃ 20 s, 61-55℃ 60 s); 26 cycles of normal PCR (94℃ 20 s, 55℃ 60 s).

[0057] The primer mix was obtained by mixing Primer 1 and Primer Common, or Primer 2 and Primer Common, and the volume of each primer was 0.025 μl, and the concentration was 10 μM / L.

[0058] The fluorescence data reading and analysis were performed by using IntelliQube machine, and the analysis results are shown in Figure 1 . Wherein, when the fluorescence signal is red (FAM signal), it indicates that the cowpea carries salt-tolerant allele C (Hap I). When the fluorescence signal is blue (HEX signal), it indicates that the cowpea carries salt-sensitive allele T (Hap II).

[0059] As shown in Figure 1 , the KASP molecular marker of the application obtains a highly consistent and clear population typing effect in the cowpea population, and the two genotypes can be obviously distinguished and clustered, and the typing effect is good.

[0060] Combined with the salt tolerance performance (see Figure 2 , and part of the data is shown in Table 2), it is found that the average salt-tolerant aboveground fresh weight ratio of the strain carrying C genotype is higher, while the average salt-tolerant aboveground fresh weight ratio of the strain carrying T genotype is relatively lower. Data analysis shows that the average salt tolerance of the strain carrying T genotype is significantly higher than that of the strain carrying C genotype (p<0.0001), which confirms that the marker can effectively distinguish the salt tolerance difference of cowpea, and finally achieves the purpose of molecular marker screening.

[0061] Table 2 Part of the selected germplasm genotype and salt treatment phenotype data statistics

[0062]

[0063] 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.

[0064] The above merely illustrates the preferred embodiments of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made to the present application should be included in the protection scope of the present application.

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 FAM group, and 3_00126 Primer2 is connected with a HEX group.

3. A KASP primer according to claim 2, or a kit containing the KASP primer of claim 2, for use in detecting a salt-tolerant 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 the KASP primer of claim 2 to perform KASP reaction detection with the cowpea plant sample genomic DNA as a template; S3, reading the fluorescence signal of the KASP reaction detection, if the cowpea plant sample genomic DNA shows the signal of the FAM group connected with the fluorescence group of 3_00126 Primer1, it is judged that the sample carries a salt-tolerant trait genotype; if the cowpea plant sample genomic DNA shows the signal of the HEX group connected with the fluorescence group of 3_00126 Primer2, it is judged that the sample carries a salt-sensitive trait genotype.

5. The method for breeding cowpea varieties with different salt tolerance according to claim 4, characterized in that, The KASP reaction detection further comprises a 2X KASP Master mix reagent.

6. The method for breeding cowpea varieties with different salt tolerance according to claim 5, characterized in that, The PCR system of the 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 the KASP reaction detection is: 94℃ pre-denaturation for 15 minutes, 94℃ denaturation for 20 seconds, 61-55℃ gradient annealing for 60 seconds, each cycle of annealing temperature decreases by 0.6℃, 55℃ extension for 60 seconds, 10 cycles; then 94℃ denaturation for 20 seconds, 55℃ annealing for 60 seconds, extension for 60 seconds, 26 cycles.

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