SNP (Single Nucleotide Polymorphism) site related to sesame wilt resistance and application thereof

Abstract

The invention belongs to the technical field of sesame biology, and particularly relates to an SNP site related to sesame fusarium wilt resistance and application of the SNP site. The SNP loci are located on a sesame No.1 chromosome 2933576, a sesame No.1 chromosome 2938347, a sesame No.1 chromosome 2945542 and a sesame No.1 chromosome 2947129. The sesame fusarium wilt resistance can be identified and detected when the locus genotype is TAAA, and the sesame susceptible fusarium wilt can be identified when the locus genotype is CGGG. Compared with the prior art, the method provided by the invention can be used for carrying out genotype detection on seeds or seedlings, screening single plants with a breeding target, reducing the workload of links such as field planting and investigation, retaining disease-resistant offspring as much as possible, and improving the breeding efficiency.

Description

Technical Field

[0001] The invention belongs to the field of sesame biotechnology, and particularly relates to SNP sites related to sesame wilt resistance and applications thereof. Background Art

[0002] Sesame oil is the most popular oil in my country and even in the world. Sesame, its raw material, also occupies an important position in my country's traditional culture. For example, "Sesame flowers bloom higher and higher" is an auspicious phrase that everyone in my country knows and says. "Lost the watermelon to pick up sesame seeds" and "Sesame official" not only truly reflect the "smallness" of sesame seeds, but also illustrate the difficulties in sesame breeding and high-yield variety improvement. Sesame is a healthy and special oil crop that is widely planted in the world. It has an oil content of up to 55% and is known as the "Queen of Oils". my country has a history of planting sesame for more than 2,200 years and is still an important sesame producer and consumer in the world, making important contributions to the development of the global sesame industry.

[0003] Fusarium wilt is a fungal disease caused by Fusarium oxysporum. It is common and serious in sesame-growing areas in my country, with an annual incidence rate of about 15%, and can reach more than 30% in severe cases. It is one of the main diseases that currently affects sesame yield and quality and restricts sesame development. Improving sesame disease resistance and increasing sesame yield are important tasks in sesame breeding.

[0004] SNP (Single nucleotide polymorphism) is the most abundant, widely distributed and polymorphic in the genome, and is the most ideal molecular marker for constructing high-density genetic maps. High-throughput sequencing technology is the main method for developing high-quality SNPs. With the reduction of sequencing costs, SNPs have gradually become a new generation of widely used molecular markers and a powerful tool for QTL positioning and candidate gene identification. However, there are few studies on the molecular mechanisms related to sesame wilt resistance, and the available regulatory genes and molecular markers need to be further explored. Summary of the invention

[0005] To solve the above problems, the present invention provides SNP sites associated with sesame wilt resistance, wherein the SNP sites are located at 2933576, 2938347, 2945542, and 2947129 of sesame chromosome 1, which are T / C, A / G, A / G, and A / G, respectively.

[0006] The present invention also provides a primer set for amplifying the above SNP marker.

[0007] The present invention also provides a detection reagent for identifying the above SNP marker, including the above primer set.

[0008] The present invention also provides a detection kit for identifying the above SNP marker, comprising the above primer set or the above detection reagent.

[0009] The present invention also provides application of the SNP site in sesame breeding.

[0010] The present invention also provides application of the SNP site in identifying resistance to sesame wilt disease.

[0011] The invention also provides a method for identifying whether sesame is resistant to wilt disease, and the genotypes at 2933576, 2938347, 2945542 and 2947129 of chromosome 1 of sesame are detected. When the genotype is TAAA, the sample has the trait of wilt disease resistance.

[0012] The present invention also provides a method for identifying whether sesame is susceptible to wilt disease, and the genotypes at 2933576, 2938347, 2945542 and 2947129 of chromosome 1 of sesame are detected. When the genotype is CGGG, the sample is susceptible to wilt disease.

[0013] The present invention has the following beneficial effects:

[0014] Compared with the prior art, the method provided by the present invention can perform genotype detection on seeds or seedlings, screen individual plants with breeding targets, reduce the workload of field planting, investigation and other links, and at the same time retain as many disease-resistant offspring as possible to improve breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 QTL positioning result map;

[0017] Figure 2 Figure 1 shows the results of the F2 population grouping in Example 1;

[0018] Figure 3 Figure 2 shows the results of the F2 population grouping in Example 2;

[0019] Figure 4 Schematic diagram of SNP sites. DETAILED DESCRIPTION

[0020] Now, various exemplary embodiments of the present invention are described in detail. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0021] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] Example 1: Discovery of SNPs

[0026] 1. A hybrid combination was constructed with 201029-0-9-3-0-9-1 (resistant to Fusarium wilt) as the female parent and ZZM00254 (susceptible to Fusarium wilt) as the male parent. The parental materials were provided by the Hebei Agricultural Biological Resources Conservation Center. The hybridization work was completed in Shijiazhuang, Hebei in the summer of 2020. In the winter of the same year, F1 was planted in Sanya, Hainan, self-pollinated and harvested. F2 was planted in Shijiazhuang, Hebei in the summer of 2021. There were a total of 169 individual plants, and conventional field management was used. The incidence of Fusarium wilt in individual plants of the parental and F2 populations was investigated.

[0027] 2. Use the CTAB method to extract DNA from parents and F2 strains, use agarose gel electrophoresis to detect the integrity of the DNA, and use NanoDrop 2000 to determine the DNA concentration.

[0028] 3. Use resequencing technology to develop SNPs for the population. The experimental process is performed according to the standard protocol provided by Illumina, including sample quality detection, library construction, library quality detection and library sequencing.

[0029] 4. Filter the offline data according to LI and other data analysis methods, and align the high-quality sequences to the sesame reference genome using BWA (0.7.17) software. Use GATK (4.0.11.0) software to identify SNP markers.

[0030] 5. First, filter the following low-quality reads: remove the adapter sequence; remove those with an uncertain base type ratio greater than 10%; remove those containing more than 50% low-quality bases. After filtering, the high-quality reads were first detected using GATK software for SNPs, and then the genetic map was constructed using MSTMap software, with an LOD value of 4.0-20.0.

[0031] 6. Combining the genetic map and fiber quality data, the ICIM program of QTLIciMapping 4.0 software was used to locate QTL, with the parameters of Step=1cM, PIN=0.001, and the LOD value was determined by 1000 iterations. As a result, a major effect QTL with a contribution rate of 23.84% and a LOD value of 7.56 was located on chromosome 1, named qWilt-chr1( Figure 1 ).

[0032] 7. According to the genomic position, qWilt-chr1 is located at 35-45Mb of chromosome 1. The sequence at the LOD peak is chr12933576-2947129. There are 4 base differences between the maternal and paternal materials, located at 2933576, 2938347, 2945542, and 2947129 (seq1, seq2, seq3, and seq4, respectively; purple is the maternal base TAAA, and red is the paternal base CGGG).

[0033] 8. The F2 population was divided according to the parental genotypes. The results showed that the incidence of wilt disease in F2 plants (83 plants) with genotype TAAA(A) was 16.81%, and the incidence of wilt disease in F2 plants (36 plants) with genotype CGGG(B) was 39.65%, which was significantly higher than that in plants with genotype TAAA(A). The incidence of wilt disease in heterozygous (H) F2 plants (50 plants) was 16.86% ( Figure 2). This shows that TAAA is effective in identifying Fusarium wilt-resistant plants and CGGG is effective in identifying Fusarium wilt-susceptible plants.

[0034] Example 2: Verification of SNP

[0035] 1. A hybrid combination was constructed with 201029-0-9-3-0-9-1 (resistant to Fusarium wilt) as the female parent and ZZM00254 (susceptible to Fusarium wilt) as the male parent. The hybridization was completed in Shijiazhuang, Hebei in the summer of 2022. F1 was planted in Sanya, Hainan in the winter of the same year, and F2 was harvested after self-pollination. It was planted in Shijiazhuang, Hebei in the summer of 2023. There were a total of 127 individual plants, and conventional field management was used. The incidence of Fusarium wilt in individual plants of the parental and F2 populations was investigated.

[0036] 2. Use the CTAB method to extract DNA from parents and F2 strains, use agarose gel electrophoresis to detect the integrity of the DNA, and use NanoDrop 2000 to determine the DNA concentration.

[0037] 3. Use resequencing technology to develop SNPs for the population. The experimental process is performed according to the standard protocol provided by Illumina, including sample quality detection, library construction, library quality detection and library sequencing.

[0038] 4. Filter the offline data according to LI and other data analysis methods, and align the high-quality sequences to the sesame reference genome using BWA (0.7.17) software. Use GATK (4.0.11.0) software to identify SNP markers.

[0039] 5. The bases at positions 2933576, 2938347, 2945542, and 2947129 on chromosome 1 of the F2 individual plants were detected, and the F2 population was genotyped. The results showed that the incidence of wilt disease in the F2 individual plants with genotype TAAA (A) (61 plants) was 15.77%, and the incidence of wilt disease in the F2 individual plants with genotype CGGG (B) (29 plants) was 38.36%, which was significantly higher than the incidence of the individual plants with genotype TAAA (A) (37 plants). The incidence of wilt disease in the heterozygous (H) individual plants was 16.43% ( Figure 3 ). This proves that among the four SNPs, TAAA has the function of identifying sesame resistant to Fusarium wilt, and CGGG has the function of identifying sesame susceptible to Fusarium wilt.

[0040] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A SNP locus associated with resistance to sesame wilt disease, characterized in that: The SNP sites are located at 2933576, 2938347, 2945542, and 2947129 on chromosome 1 of sesame, which are T / C, A / G, A / G, and A / G, respectively.

2. A primer set for amplifying the SNP marker according to claim 1.

3. A detection reagent for identifying the SNP marker according to claim 1, characterized in that: Comprising the primer set described in claim 2.

4. A detection kit for identifying the SNP marker according to claim 1, characterized in that: It includes the primer set according to claim 2 or the detection reagent according to claim 3.

5. Use of the SNP locus as claimed in claim 1 in sesame breeding.

6. Use of the SNP locus as claimed in claim 1 in identifying resistance to sesame Fusarium wilt.

7. A method for identifying whether sesame is resistant to wilt disease, characterized in that: The genotypes at 2933576, 2938347, 2945542, and 2947129 on chromosome 1 of sesame were tested. When the genotype was TAAA, the sample was resistant to Fusarium wilt.

8. A method for identifying whether sesame is susceptible to wilt disease, characterized in that: The genotypes at 2933576, 2938347, 2945542, and 2947129 on chromosome 1 of sesame were tested. When the genotype was CGGG, the sample was susceptible to Fusarium wilt disease.

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