Fusarium wilt resistant gene scadir40 and use thereof

Abstract

The present invention relates to a gene for regulating and controlling a resistance trait of Fusarium wilt. A Fusarium wilt resistant gene ScaDIR40 is derived from chromosome 16 of a wild species Sesamum calycinum genome having high Fusarium wilt resistance, and belongs to a dominant control gene; and the alleles of the Fusarium wilt resistant gene ScaDIR40 in a susceptible wild species Sesamum angustifolium and in a susceptible cultivated species are respectively Saudir32 and Sindir37. By means of deep research on the Fusarium wilt resistant gene and the alleles thereof, a theoretical basis can be provided for research on a Fusarium wilt resistance regulation and control development mechanism of crops such as sesame, and material and technical bases can also be provided for creating an excellent sesame disease-resistant germplasm, accelerating the development of sesame disease-resistant molecule-assisted breeding technology, and breeding new disease-resistant varieties; and the present invention has extremely important scientific research value and economic application value and particularly has important significance on accelerating breeding of new sesame varieties and improving production stability.

Description

Fusarium wilt resistance gene ScaDIR40 and its application Technical Field

[0001] The invention belongs to the technical field of sesame molecular genetic breeding, and in particular relates to a gene for controlling and regulating wilt disease resistance traits. Background Art

[0002] Sesame (Sesamum indicum L.), belonging to the Pedaliaceae family and the genus Sesamum, is an ancient, high-quality, and specialty oilseed crop worldwide, playing a vital role in food processing. During sesame cultivation and management, pest and disease control is essential for ensuring high yields. Sesame wilt is one of the most common and difficult sesame diseases to control.

[0003] Sesame wilt is a fungal disease caused by Fusarium oxysporum f.sp. sesami (FOS), which infects the plant through the roots in the soil. Infection damages the root system and stems, ultimately causing plant death and yield loss. Conventional chemical control agents are ineffective due to the lack of highly resistant varieties. Therefore, identifying and breeding new sesame varieties with high resistance to sesame wilt is of great technical significance for ensuring stable and high sesame yields.

[0004] Summary of the Invention

[0005] Combined with the mining of relevant information on wild germplasm resources with high resistance to wilt, the purpose of this application is to provide a wilt-resistant gene ScaDIR40 that can regulate the wilt resistance trait, thereby laying a certain technical foundation for the breeding of new sesame varieties.

[0006] The specific technical solutions claimed in this application are as follows.

[0007] A wilt-resistance gene, ScaDIR40, originates from chromosome 16 of the genome of the wild species Sesamum calycinum, which is highly resistant to wilt. It is a dominant gene (compared to the highly susceptible wilt allele) and explains 100% of the wilt resistance trait (i.e., this gene controls the expression of high wilt resistance). The gene is 570 bp long and has no introns. Its base sequence is shown in SEQ ID No. 1, specifically:

[0008] Correspondingly, the anti-blight protein encoded by the wilt-resistant gene ScaDIR40 contains 189 amino acids, and the amino acid sequence is as follows:

[0009] The alleles of the wilt resistance gene ScaDIR40 are the highly susceptible wilt gene Saudir32 and the highly susceptible wilt gene Sindir37 in the highly susceptible wild species Sesamum angustifolium and the highly susceptible cultivated species respectively;

[0010] The highly susceptible wilt gene Saudir32 is derived from chromosome 14 of the highly susceptible wild species Sesamum angustifolium. Compared with the wilt-resistant gene ScaDIR40, the highly susceptible wilt gene Saudir32 has an insertion mutation of a 1411 bp Copia_LTR_retrotransposon sequence at -210 bp in the 5'UTR promoter region of the gDNA sequence. This SV mutation leads to the inactivation of gene expression, ultimately causing the plant phenotype to mutate from a highly wilt-resistant type to a highly susceptible type.

[0011] The highly susceptible wilt gene Saudir32 is 528 bp in length and has no introns. The base sequence is shown in SEQ ID No. 2, specifically:

[0012] Correspondingly, the protein encoded by the highly susceptible Fusarium wilt gene Saudir32 contains 175 amino acids, and the amino acid sequence is as follows:

[0013] The highly susceptible wilt gene Sindir37 is derived from chromosome 9 of the highly susceptible wilt sesame cultivar Sesamum indicum (Yuzhi No. 11). Compared with the wilt-resistant gene ScaDIR40, the gDNA sequence of the highly susceptible wilt gene Sindir37 has a loss of -1 bp to -1400 bp sequence in the 5'UTR promoter region and some exon sequences, resulting in gene expression inactivation and ultimately changing the phenotype from a highly wilt-resistant type to a highly susceptible type. The highly susceptible wilt gene Sindir37 is 237 bp long and has no introns. The base sequence is shown in SEQ ID No. 3, specifically:

[0014] Correspondingly, the protein encoded by the highly susceptible Fusarium wilt gene Sindir37 contains 78 amino acids, and the amino acid sequence is as follows:

[0015] The primer pair for preparing the wilt resistance gene ScaDIR40 or its allele by PCR amplification is specifically designed as follows:

[0016] Targeting the wilt resistance gene ScaDir40, the specific design is:

[0017] Forward primer Dir40 Primer F: 5'-ATATCTATGGCATACATAAAACAC-3',

[0018] Reverse primer Dir40 Primer R: 5′-CCTAATCATCTTGATTGAAGG-3′;

[0019] Targeting the highly susceptible wilt gene Saudir32, the specific design is:

[0020] Forward primer dir32 Primer F: 5'-GAGGATGAGATTTTATTACACC-3',

[0021] Reverse primer dir32 Primer R: 5′-GGCTAGAATTTTCCCTTACAA-3′;

[0022] Targeting the highly susceptible Fusarium wilt gene Sindir37, the specific design is:

[0023] Forward primer dir37 Primer F: 5'-TTTTATACCACCTCCCAAAG-3',

[0024] Reverse primer dir37 Primer R: 5′-CCATCCACGAGTAGTTCCA-3′.

[0025] The PCR amplification method for preparing the wilt-resistant gene ScaDIR40 or its allele using the primer pair for PCR amplification comprises the following steps:

[0026] (1) Extracting genomic DNA from Sesamum calycinum (var. KEN8), a highly resistant germplasm to Fusarium wilt, Sesamum angustifolium (var. G01), or a highly susceptible cultivar of Sesamum indicum (Yuzhi No. 11);

[0027] (2) using the genomic DNA extracted in step (1) as a template and performing PCR amplification using a PCR amplification primer pair;

[0028] The wilt resistance gene ScaDir40 was obtained by amplifying the Dir40 F / R primer pair using DNA from the highly wilt-resistant Sesamum calycinum (var. KEN8) germplasm as a template.

[0029] When DNA from the highly susceptible Fusarium wilt Sesamum angustifolium (var. G01) accession was used as a template and amplified using the dir32 F / R primer pair, the allele (Fusarium wilt susceptible gene) Sadir32 was obtained;

[0030] Using DNA from a highly susceptible wilt sesame variety, Sesamum indicum (Yuzhi No. 11), as a template and the dir37 F / R primer pair as amplification, the allele (susceptibility to wilt) Sadir37 was obtained.

[0031] The primer set for detecting and distinguishing the wilt resistance gene ScaDir40 and its alleles is specifically designed as follows:

[0032] DIR1 F1: 5'-TCTTGGCTCCTGAAAATAGT-3',

[0033] DIR1 R1: 5'-CGAGCACGTCCTGGAAATAGAAA-3';

[0034] DIR1 F2: 5'-TTTTATAACCACCTCCCAAAG-3',

[0035] DIR1 R2: 5'-CCATCCACGAGTAGTTCCA-3';

[0036] When using this primer set for detection and differentiation:

[0037] When PCR amplification was performed using the DIR1 F1 / R1 primer pair, if the PCR amplification template contained the genome of the highly wilt-resistant Sesamum calycinum (containing the wilt-resistant gene ScaDir40), the electrophoresis band after PCR amplification contained a 1572 bp long band;

[0038] When PCR amplification was performed using the DIR1 F1 / R1 primer pair, if the PCR amplification template contained the genome of the highly susceptible Sesamum angustifolium (containing the susceptible wilt gene Saudir32), the electrophoresis band after PCR amplification contained a 2952 bp long band;

[0039] When PCR amplification is performed using the DIR1 F1 / R2 primer pair, if the PCR amplification template contains the genome of Sesamum indicum (containing the susceptible wilt gene Sindir37), the electrophoresis band after PCR amplification contains a 624 bp long band.

[0040] The method for detecting and determining the wilt resistance phenotype using the primer set for detecting and distinguishing the wilt resistance gene ScaDir40 and its alleles comprises the following steps:

[0041] (1) extracting genomic DNA from the germplasm of the plant to be tested (specifically, sesame);

[0042] (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using primer pairs DIR1 F1 / R1 and DIR1 F2 / R2, respectively;

[0043] (3) performing electrophoresis on the PCR amplification product in step (2), and making a determination based on the electrophoresis results; the specific determination criterion is: after amplification using DIR1 F1 / R1, if there is only one electrophoresis band of 1572 bp in length, it indicates that the germplasm resource to be identified has a dominant homozygous high resistance to wilt disease phenotype;

[0044] After amplification using DIR1 F1 / R1, if there is only one electrophoretic band of 2952 bp in length, it indicates that the germplasm to be identified is recessive homozygous and highly susceptible to Fusarium wilt disease;

[0045] After amplification using DIR1 F1 / R1, if two electrophoretic bands with lengths of 1572 and 2952 bp were present at the same time, it indicated that the germplasm to be identified was heterozygous for the high wilt resistance phenotype;

[0046] After amplification using DIR1 F2 / R2, if there is only one electrophoretic band of 624 bp in length, it indicates that the germplasm to be identified is recessive homozygous and highly susceptible to Fusarium wilt disease;

[0047] If the target electrophoresis band is absent after amplification using DIR1 F1 / R1 or DIR1 F2 / R2, it indicates that the germplasm to be identified is not Sesamum calycinum, Sesamum angustifolium, cultivars or their progeny, and other methods are required to identify the wilt resistance of the germplasm to be identified.

[0048] Prior art testing of Fusarium wilt resistance in existing sesame cultivars using the highly pathogenic FOS strain has shown that all existing sesame cultivars are highly susceptible to Fusarium wilt. Therefore, from the perspective of developing new sesame cultivars resistant to Fusarium wilt, the discovery of relevant wilt-resistant germplasm resources, particularly the identification of wilt-resistant genes, is of great technical significance for developing new wilt-resistant sesame varieties.

[0049] In the previous research work, when screening and analyzing the germplasm resources related to the genus Sesamum, the inventors found that some wild species showed obvious differences in resistance to wilt disease, and further constructed a fine map of the genome of the relevant wild germplasm. Based on this, the inventors carried out the construction of interspecific hybrid populations of the wild species S.calycinum with high resistance to wilt disease and the wild species S.angustifolium with high sensitivity to wilt disease, identification of offspring disease resistance and genetic analysis of resistance to wilt disease. The results showed that under the infection of the highly pathogenic wilt strain HSFO08027, the F1 of the S.calycinum×S.angustifolium population showed disease resistance. Among its F2 offspring, the ratio of high disease-resistant germplasm to highly susceptible germplasm is 3.31:1 (X 2 =0.04), indicating that the phenotypic segregation ratio of wilt resistance trait conforms to Mendel's law, indicating that wilt resistance trait in sesame is controlled by a pair of dominant genes.

[0050] To further clone sesame wilt resistance genes, the inventors conducted a genome-wide association analysis of wilt resistance in F2 progeny of an interspecific hybrid population of S. calycinum × S. angustifolium based on a detailed map of the sesame genome. Genome-wide association analysis revealed that in the genome of the highly wilt-resistant wild species S. calycinum var. KEN8, the SNP most significantly associated with wilt resistance, SNP106805, is located at Backbone_267 on chromosome 16, explaining 70.73% of the trait's variation. In the genome of the highly susceptible wild species S. angustifolium var. G01, the SNP most significantly associated with wilt resistance, SNP131559, is located at Backbone_197 on chromosome 14, explaining 62% of the trait's variation. Genome homology comparison analysis showed that chromosome 16, where the significant association region Backbone_267 of the highly disease-resistant wild species S. calycinum is located, is a homologous chromosome to chromosome 14, where SNP131559 of the highly susceptible wild species S. angustifolium is located.

[0051] Subsequently, through genomic sequence analysis, collinear gene and collinear fragment analysis, and SV (Structural variation) analysis of the 400Kb interval upstream and downstream of the most significantly associated SNP sites in S. calycinum and S. angustifolium, it was finally determined that the wilt resistance target gene was located on chromosome 16 of S. calycinum and belonged to the plant DIR (Dirigent) disease resistance-related gene. Therefore, it was named the wilt resistance gene ScaDir40.

[0052] Furthermore, collinearity analysis and structural alignment analysis of the target gene were performed using the genomes of the disease-resistant wild species S. calycinum (var. KEN8) and the susceptible wild species S. angustifolium (var. G01). The allele of the gene in the susceptible sample S. angustifolium was identified and named: the wilt-resistant gene allele (susceptibility gene) Saudir32;

[0053] Furthermore, by combining the genomes of the disease-resistant wild species S. calycinum (var. KEN8), the susceptible wild species S. angustifolium (var. G01) and the susceptible cultivated species S. indicum (var. Yuzhi No. 11), collinearity analysis and structural alignment analysis were performed on the target gene, and the allele of this gene in the genome of the susceptible sample S. indicum (var. Yuzhi No. 11) was determined, and it was named the wilt-resistant gene allele (susceptible wilt gene) Saudir37.

[0054] In general, the innovations of the present invention are mainly reflected in the following aspects:

[0055] (1) The present invention locates wilt-resistance genes by using interspecific hybrid populations of wild germplasms with high resistance and high susceptibility to wilt, and clones the wilt-resistance gene ScaDir40 and its alleles Saudir32 and Sindir37. Based on this, a PCR identification method for identifying the wilt-resistance phenotype and genotype of germplasm is provided. This method can conveniently and quickly preliminarily determine the wilt-resistance phenotype of the germplasm sample to be tested, thereby facilitating the development of new wilt-resistant varieties.

[0056] (2) The wilt-resistant gene involved in the present invention has an important impact on accelerating the research on the genetic mechanism of disease resistance in crops such as sesame, and is of great significance for accurately breeding new disease-resistant sesame varieties, improving the disease resistance level of sesame varieties, and promoting the development of sesame production. Therefore, the successful cloning of the wilt-resistant gene will inevitably accelerate the construction of a molecular-assisted breeding system for sesame and the application of efficient disease-resistant breeding;

[0057] (3) The gene marker detection method for the wilt resistance gene ScaDir40 and its alleles Saudir32 and Sindir37 provided by the present invention has good detection result stability, which is of great significance for improving the efficiency of sesame disease resistance breeding and enhancing the level of sesame genetic breeding research in my country.

[0058] In summary, through in-depth research on the wilt-resistant gene ScaDir40 and its alleles Saudir32 and Sindir37 involved in this application, a theoretical basis can be provided for the study of the regulation and development mechanism of wilt resistance in crops such as sesame. At the same time, it can also provide materials and technical basis for creating excellent disease-resistant sesame germplasm, accelerating the development of sesame disease-resistant molecular assisted breeding technology and breeding new disease-resistant varieties. It has extremely important scientific research value and economic application value, especially for accelerating the breeding of new sesame varieties in my country and improving production stability. It has important technical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 is a comparison of the typical phenotypes of wild sesame species S.calycinum, which is highly resistant to Fusarium wilt, and wild sesame species S.angustifolium, which is highly susceptible to Fusarium wilt, under FOS infection;

[0060] Left: S. angustifolium plants inoculated with the highly pathogenic strain HSFO 08027 for 14 days (susceptible) (lower row) and water control (upper row);

[0061] Right: S. calycinum plants inoculated with the highly pathogenic strain HSFO 08027 for 14 days (resistant) (lower row) and water control (upper row);

[0062] Figure 2 is a Manhattan plot of genome-wide association analysis of resistance to Fusarium wilt in an interspecific hybrid population of highly resistant S. calycinum × highly susceptible S. angustifolium;

[0063] Upper panel: Manhattan plot of genome-wide association analysis of wilt resistance using the S. calycinum genome as the reference genome;

[0064] Bottom: Manhattan plot of genome-wide association analysis of wilt resistance using the S. angustifolium genome as the reference genome;

[0065] Figure 3 is a schematic diagram showing the structural comparison of the sesame wilt resistance gene ScaDir40 and its alleles Saudir32 and Sindir37; in color view: black horizontal lines represent promoters; green represents exons; blue represents the inserted Copia_LTR sequence; red dotted lines represent deleted promoter sequences; and green hollow boxes represent lost exons;

[0066] Figure 4 shows the comparison of the protein sequences encoded by the sesame wilt resistance gene ScaDir40 and its alleles Saudir32 and Sindir37;

[0067] FIG5 shows the PCR amplification results of molecular markers (primer pairs) DIR1 F1 / R1 and DIR1 F2 / R2 of sesame wilt resistance genes in the S.calycinum×S.angustifolium F2 validation population and some sesame cultivated germplasm; wherein:

[0068] Lane M is the DL 5000 marker, and the bands shown are 5000 bp, 3000 bp, 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp from top to bottom;

[0069] Lanes 1, 2, and 61: S. calycinum (HR), S. angustifolium (HS), and cultivar Yuzhi 11;

[0070] Lanes 3, 6, 8, 12, 19, 20, 24, 29, 33, 44, 50, and 60: 12 homozygous F2 plants carrying the Saudir32 locus, showing disease susceptibility when inoculated with HSFO08027;

[0071] Lanes 4, 5, 7, 9, 15, 36, 37, 43, 46, 52, 53, and 55: 11 homozygous F2 individuals carrying the ScaDIR40 locus, appearing healthy under inoculation with HSFO 08027;

[0072] Lanes 10, 11, 13, 14, 16-18, 21-23, 25-28, 30-32, 34, 35, 38-42, 45, 47-49, 51, 54, 56-59: 34 heterozygous F2 plants carried both the ScaDIR40 and Saudir32 loci and appeared healthy when inoculated with HSFO08027; Lanes 62-72: 11 cultivated sesame germplasm resources carried the Sidir37 locus and appeared susceptible to disease when inoculated with HSFO08027. DETAILED DESCRIPTION

[0073] The present application will be further explained below with reference to the following examples. Before introducing the specific examples, a brief description of some experimental backgrounds in the following examples is given below.

[0074] Biomaterials:

[0075] Germplasm resources such as the wild species S. calycinum (var. KEN8) with high resistance to wilt, the wild species S. latifolium (var. KEN1) with high resistance to wilt, the wild species S. angustifolium (var. G01) with high susceptibility (susceptibility) to wilt, and Yuzhi No. 11 (Sesamum indicum) are all commonly used and reported germplasm resources in existing research and are available through public channels. As a professional scientific research institution, the applicant owns the germplasm resource bank of the Sesame Research Center of the Henan Academy of Agricultural Sciences, a public scientific research infrastructure, which retains relevant germplasm resources all year round. Therefore, the relevant germplasm materials used in the examples are all from this germplasm resource bank.

[0076] Sesame Fusarium wilt pathogen HSFO08027 is a highly pathogenic strain that is commonly used and reported in existing sesame wilt resistance research and is publicly available. It should be emphasized that this strain was selected in this application solely for the convenience of experimental design and experimental conditions. In actual research and application, other wilt strains may be selected as needed, and the technical solution claimed in this application does not necessarily depend on the selection or use of this strain.

[0077] The relevant primer synthesis and sequencing analysis were provided by Tianjin Gene Chip Biological Company.

[0078] Example 1

[0079] In previous research, the inventors discovered that, when infected with a highly pathogenic strain of the sesame Fusarium wilt pathogen (HSFO08027), the wild S. calycinum (var. KEN8) exhibited high resistance to wilt (infected plant rate = 0), while germplasms such as S. angustifolium (var. G01) and the cultivated sesame cultivar Yuzhi 11 were highly susceptible to wilt (infected plant rate = 100%). Furthermore, the F2 progeny of the S. calycinum × S. angustifolium population segregated for resistance and susceptibility. Based on these findings, the inventors conducted a detailed mapping analysis of the gene to accurately identify the regulatory gene for wilt resistance, as briefly described below.

[0080] (1) Phenotypic investigation of wilt resistance traits

[0081] Based on the previous work (i.e., on the basis of completing the identification of wilt resistance of germplasms such as S. calycinum (var. KEN8), S. angustifolium (var. G01), S. latifolium (var. KEN1), and S. radiatum (var. G02)), the inventors further configured a hybrid combination of S. calycinum × S. angustifolium, constructed interspecific hybrid population offspring, and identified wilt resistance of the two parents, F1, F2, and other offspring (using the highly pathogenic sesame wilt fungus strain HSFO08027 for infection, with 3 replicates set for each test sample, and 5-10 strains tested in each replicate).

[0082] In the experimental design, the water treatment was used as the control, and the disease resistance of F2 plants was based on the F 2-3 The results of strain detection showed that (i.e., F 2-3 The disease incidence rate of a strain indicates the disease resistance phenotype of its F2 individual plants).

[0083] When identifying resistance to Fusarium wilt, the disease rate of the plants was 0-50% on the 28th day after inoculation. 2-3 strains, and its F2 samples were identified as disease-resistant samples; F2 samples with a disease rate of >50% 2-3 strains, whose F2 samples were identified as susceptible samples, carrying the susceptible allele; the F2 samples with disease incidence rates > 0% and < 100% 2-3 strains, and its F2 is considered a heterozygous genotype sample; the incidence rate of the strain is equal to 0% and 100% of the F 2-3 strains, and their F2 were considered to be samples of highly disease-resistant homozygous and highly susceptible homozygous genotypes.

[0084] A comparison of typical phenotypic traits of wild species with high resistance to Fusarium wilt and high susceptibility to Fusarium wilt is shown in Figure 1. The phenotypic statistics of the population and its F2 progeny are shown in Table 1 below.

[0085] Table 1 Statistical results of phenotypic resistance to Fusarium wilt in interspecific sesame populations *X 2 0.05,1 =3.84.

[0086] The statistical results showed that among the 70 S.calycinum×S.angustifolium hybrid F2 samples tested, the F 2:3 There were 54 F2 samples with a disease rate of ≤50% (disease resistant), 2:3 There were 16 F2 samples with a disease rate > 50% (susceptible). The ratio of resistance to susceptible was 3.38:1 (X 2 =0.0), and the segregation ratio of the trait conformed to Mendel's law of 3:1. This result indicated that the wilt resistance trait was controlled by a pair of dominant genes.

[0087] (II) Genome-wide association analysis

[0088] After initially confirming that the wilt resistance trait is controlled by a pair of dominant genes, the inventors used the whole gene relationship analysis method to further locate the genes. The specific work is briefly described as follows.

[0089] (1) Whole genome sequencing of interspecific hybrid populations

[0090] Leaf samples from wild S. calycinum (var. KEN8) and S. angustifolium (var. G01) parents and 126 F2 sample plants were collected and DNA was extracted using the CTAB method (for specific procedures, see: Method for Simultaneous DNA and RNA Extraction in Sesame, 2008, Molecular Plant Breeding). The extracted DNA samples were then subjected to genome resequencing using Illumina sequencing, with a sequencing coverage of ≥10×.

[0091] (2) Genome association analysis of the highly susceptible wild species S. angustifolium

[0092] Combined with the previously constructed genome information of the highly susceptible wild species S. angustifolium (Miao et al., The Sesame Genome, Springer Nature publisher, 2021), the genome resequencing data of 126 F2 individuals in the interspecific population were aligned and spliced ​​using BWA (Burrows-WheelerAligner) technology.

[0093] Subsequently, VCFs of 126 samples were extracted and analyzed using a SNP matrix. A total of 180,977 SNPs were identified, distributed across 1,968 scaffolds. Of these, 99,716 SNPs were distributed across all 16 chromosomes of the wild S. angustifolium species. Simultaneously, GLM (generalized linear model) association analysis was performed using individual plant incidence data as phenotypes.

[0094] Finally, combined with the comparative analysis and synthesis of the above population data, the variants and P values ​​closely associated with the wilt disease resistance trait were determined. Some of the results are shown in Figure 2.

[0095] (3) Genome association analysis of wild sesame species S.calycinum with high resistance to Fusarium wilt

[0096] Combined with the previously constructed genome information of the wild sesame species S. calycinum, which is highly resistant to Fusarium wilt, the BWA (Burrows-WheelerAligner) technology was used to align and splice the genome resequencing data of 126 F2 individuals in the interspecific population.

[0097] Subsequently, the vcf of 126 samples were extracted and analyzed by SNP matrix, which identified a total of 1,197,494 SNP sites distributed on 476 scaffolds. Among them, 933,362 SNPs were distributed on all 16 chromosomes of the wild species S. calycinum. 2:3 The disease incidence data of the lines represented the disease resistance phenotype of F2 individual plants, and GLM association analysis was performed.

[0098] Finally, combined with the comparative analysis and synthesis of the above population data, the variants and P values ​​closely associated with the wilt disease resistance trait were determined. Some of the results are shown in Figure 2.

[0099] (4) Data comparison and synthesis

[0100] Combined with the above association analysis results in the highly resistant and highly susceptible genomes, the distribution characteristics of the SNPs with the most significant P values ​​located in the two genomes were analyzed. The results showed:

[0101] In the S. angustifolium genome, there were 10 SNP sites with P values ​​less than E-7, all located on chromosome 14, with the lowest P value reaching 1.56E-14. The corresponding sites explained 62.41% of the variation in wilt resistance.

[0102] In the S. calycinum genome, a total of 2531 SNPs had P values ​​less than E-10, located on chromosomes 5, 15, and 16, respectively. The lowest P value reached 8.43E-18, and the corresponding site had a maximum explanation rate of 70.73% for the variation in wilt resistance traits. Meanwhile, the lowest P value on chromosome 16 reached 3.53E-17, and the corresponding site had a maximum contribution rate of 69.39% to the variation in traits.

[0103] Further combined with the results of homologous alignment of the genomes of wild species of sesame S. angustifolium and S. calycinum and the sesame pan-genome map, the most significant SNP sites located above are located on chromosomes 14 and 16 of the genomes of S. angustifolium and S. calycinum, which are homologous chromosome pairs.

[0104] In view of the large differences in genome and gene sequences among different species of Sesame, the inventors further performed homologous comparisons on the upstream and downstream regions of the above-mentioned preliminary located SNP sites. At the same time, in order to further determine the sequence characteristics of homologous chromosomes and homologous genes, the inventors used the high-quality genomes of another wild species with high resistance to Fusarium wilt, S. latifolium (var. KEN1), and the highly susceptible cultivated species S. indicum (var. Yuzhi No. 11) (Miao et al., The Sesame Genome, Springer Nature publisher, 2021) as references, and compared the homologous genes in the disease-resistant linkage target region in the genomes of the highly resistant wild species S. calycinum and the highly susceptible wild species S. angustifolium. The statistical information of some homologous genes is shown in Table 2 below.

[0105] Table 2. Statistics of homologous genes in the target regions of the genomes of four species “ / ” means that no homologous gene was found in the compared genome.

[0106] Statistical results showed that across the four species genomes, a total of 52 genes (pairs) were involved within the 400 kb homologous region upstream and downstream of the most significant correlation P value for wilt resistance between the highly resistant wild species S. calycinum and the highly susceptible wild species S. angustifolium, five of which were species-specific. Simultaneously, genomic SV analysis was performed on genes within this region of S. calycinum and S. angustifolium, in conjunction with the reference genome, ultimately identifying 40 SVs. Subsequently, genome resequencing data from six highly resistant and six highly susceptible F2 plants were selected, and SVs within this target region were identified using various genomic analysis software. The results are shown in Table 3 below.

[0107] Table 3. SV analysis statistics within the target region in F2 material

[0108] The analysis showed that, using S. angustifolium as the reference genome, a total of 10 SVs were detected in the F2 materials tested, involving a total of 5 genes. Using S. calycinum as the reference genome, a total of 13 SVs were identified, involving a total of 5 genes.

[0109] Furthermore, PCR verification analysis was performed on the above-mentioned target genes and SVs using verification populations such as BC1 and F2. The final results showed that only SV_297664 was consistent with the variation of the population's wilt-resistance phenotype. The SV is located on Sangu_2112300 of S. angustifolium; correspondingly, the allele in S. calycinum is Scaly_2470000. Therefore, it is believed that the homologous gene Scaly_2470000 in the disease-resistant wild species S. calycinum is a wilt-resistance target gene, corresponding to a highly wilt-resistant phenotype; and Sangu_2112300 of S. angustifolium is a disease-resistant allele (susceptible, highly susceptible to wilt gene), corresponding to a highly susceptible (susceptible) wilt phenotype. The two genes belong to the DIR class of disease-resistance-related proteins.

[0110] Further structural analysis of the target gene DIR was performed based on the reference genome. The results showed that the gene and protein structures of Scaly_2470000 and Sangu_2112300 were similar. Compared with Scaly_2470000, there was a 1411bp LTR fragment inserted in the 5'UTR region of Sangu_2112300. According to the statistical number of the DIR gene family in each genome, we named the wilt-resistant gene Scaly_2470000 of the highly wilt-resistant wild species S.calycinum ScaDir40, and the allele Sangu_2112300 in the highly susceptible wild species S.angustifolium Saudir32. Furthermore, the allele Sindi_2089700 in the highly susceptible cultivated species S.indicum was named Sindir37.

[0111] Example 2

[0112] Based on the gene mapping in Example 1, the inventors further cloned and sequenced the located wilt-resistance genes and alleles. The specific process is briefly described as follows.

[0113] Based on the region and target gene of the tightly linked SNP for wilt resistance obtained in Example 1 above, and after preliminarily determining the sequence information of the target gene ScaDir40 and its alleles, the inventors designed a primer pair for PCR amplification, and used PCR amplification to obtain the specific wilt resistance gene ScaDir40, the highly susceptible wilt gene Saudir32, and the highly susceptible wilt gene Saudir37. The specific PCR amplification primer pair was designed as follows:

[0114] Dir40 F: 5'-ATATCTATGGCATACATAAAACAC-3';

[0115] Dir40 R: 5'-CCTAATCATCTTGATTGAAGG-3';

[0116] dir32 F: 5'-GAGGATGAGATTTTATTACACC-3';

[0117] dir32 R: 5'-GGCTAGAATTTTCCCTTACAA-3';

[0118] dir37 F: 5'-TTTTATAACCACCTCCCAAAG-3';

[0119] dir37 R: 5'-CCATCCACGAGTAGTTCCA-3';

[0120] During PCR amplification, genomic DNA of the corresponding wild species of sesame S.calycinum (var.KEN8) with high resistance to Fusarium wilt, wild species of sesame S.angustifolium (var.G01) with high sensitivity to Fusarium wilt, and cultivated species of sesame S.indicum Yuzhi No. 11 with high sensitivity to Fusarium wilt were used as templates for PCR amplification.

[0121] The reference design of the PCR amplification program (PTC-100 thermal cycler, a product of MJ research company) is as follows: pre-denaturation at 98°C for 3 min; followed by denaturation at 98°C for 15 sec, annealing at 58°C for 15 sec, and extension at 72°C for 1 min, for 35 cycles; and a final extension at 72°C for 5 min. The amplified products were directly detected by electrophoresis or stored at 4°C for later use.

[0122] After electrophoresis detection and recovery of the amplified product, sequencing analysis was performed. The results showed:

[0123] The full-length genomic sequence of the sesame wilt-resistance gene ScaDir40 is 570 bp long and has no introns, and the sequence is shown in SEQ ID.1; the sequence of the Saudir32 gene corresponding to the highly susceptible wilt phenotype is 528 bp long and has no introns; the base sequence is shown in SEQ ID No.2; the sequence of the Sindir37 gene corresponding to the highly susceptible wilt phenotype is 237 bp long and has no introns; the base sequence is shown in SEQ ID No.3.

[0124] Furthermore, the gene sequence and amino acid sequence of the wilt resistance gene ScaDir40 and its alleles Saudir32 and Sindir37 were compared, as shown in Figures 3 and 4. Comparative analysis shows that the differences between the sequences of the ScaDir40 gene and its alleles Saudir32 and Sindir37 are:

[0125] The promoter region of the Saudir32 allele contains a 1411-bp insertion of the Copia_LTR_retrotransposon sequence. The Sindir37 allele loses the -1 to -1400-bp promoter region and some exon sequences. While the amino acid sequence encoding the gene is relatively conserved, it is preliminarily speculated that these changes in the DIR gene's promoter and exon sequences are the root cause of the high susceptibility to Fusarium wilt in some wild and cultivated sesame varieties.

[0126] Example 3

[0127] Based on the aforementioned experimental analysis, to further confirm that ScaDir40 is the gene regulating wilt resistance, the inventors designed a primer set for detection and differentiation, combining the sequence differences of ScaDir40 and its alleles between cultivated and susceptible wild species. This was initially validated using 58 F2 plants from an interspecific hybrid population of S. calycinum × S. angustifolium and 12 cultivated varieties. This facilitates the screening, detection, and application of this gene in breeding. The details are briefly described below.

[0128] First, based on the differences in related gene sequences and PCR amplification technology, the primer sets designed are as follows:

[0129] DIR1 F1: 5'-TCTTGGCTCCTGAAAATAGT-3',

[0130] DIR1 R1: 5'-CGAGCACGTCCTGGAAATAGAAA-3';

[0131] DIR1 F2: 5'-TTTTATAACCACCTCCCAAAG-3',

[0132] DIR1 R2: 5'-CCATCCACGAGTAGTTCCA-3';

[0133] According to the design concept, when using this primer set for PCR amplification:

[0134] After amplification of the highly wilt-resistant sample (containing the wilt-resistant gene ScaDir40) using the DIR1 F1 / R1 primer pair, the electrophoresis band was 1572 bp;

[0135] After amplification using the DIR1 F1 / R1 primer pair in a highly susceptible Fusarium wilt type sample (containing the allele Saudir32 of the Fusarium wilt resistance gene), the electrophoretic band was 2952 bp;

[0136] The DIR1 F1 / R1 primer pair amplified the highly wilt-resistant heterozygous sample (containing both the wilt-resistant gene ScaDir40 and the wilt-resistant gene allele Sadir32), resulting in two electrophoretic bands of 1572 bp and 2952 bp, respectively.

[0137] After amplification of the cultivated variety (highly susceptible to Fusarium wilt) sample (containing the Fusarium wilt resistance gene allele Sindir37) using the DIR1 F2 / R2 primer pair, the electrophoresis band was 624 bp.

[0138] Preliminary testing was performed on the highly pathogenic FOS strain HSFO08027 to assess the resistance of individual F2 plants to Fusarium wilt. During testing, individual plants were surveyed every three days for 7-28 days after inoculation to assess disease morbidity and mortality. Samples showing wilt symptoms were considered susceptible, while healthy, asymptomatic samples were considered resistant. Furthermore, on the seventh day after inoculation, a small sample of young leaves from each plant was collected and the genome extracted (see above) for phenotypic verification by PCR.

[0139] When PCR technology is used for verification, the extracted genomic DNA is used as a template and the above primer set (DIR1 F1 / R1, DIR1 F2 / R2) is used for PCR amplification. During PCR amplification, the reference setting of the 25 μL reaction system is as follows:

[0140] Template DNA (50 ng / μL), 1.0 μL;

[0141] 2×PrimeSTAR Max Premix, 12.5μL;

[0142] Forward Primer 1 (10μM), 1.0μL;;

[0143] Reverse Primer (10μM), 1.0μL;

[0144] Add ddH2O to 25 μL;

[0145] The PCR reaction program (PTC-100 Thermal Cycler, MJ Research) was as follows: initial denaturation at 98°C for 3 min, followed by 35 cycles of denaturation at 95°C for 15 sec, annealing at 55°C for 15 sec, and extension at 72°C for 3 min. A final extension at 72°C for 5 min was performed. Amplified products were directly detected by agarose gel electrophoresis (1% gel concentration, 150 V, electrophoresis for 20 min) or stored at 4°C until further use.

[0146] The electrophoresis patterns of some samples are shown in Figure 5. The analysis shows that:

[0147] When the germplasm to be identified as highly resistant to Fusarium wilt, the length of the electrophoresis band amplified by the DIR1 F1 / R1 primer pair was 1572 bp, which was completely consistent with the actual phenotypic result.

[0148] When the germplasm resource was identified as highly susceptible to Fusarium wilt, the length of the electrophoresis band amplified by the DIR1 F1 / R1 primer pair was 2952 bp, which was completely consistent with the actual phenotypic result.

[0149] When the germplasm resources were to be identified as heterozygous and highly susceptible to Fusarium wilt, the electrophoresis bands amplified using the DIR1 F1 / R1 primer pair showed two bands of 1572 bp and 2952 bp, and this pre-set result was completely consistent with the actual phenotypic result.

[0150] When the germplasm resource to be identified is a highly susceptible wilt cultivar, the length of the electrophoresis band after amplification using the DIR1 F2 / R2 primer pair is 624 bp. This preset result is completely consistent with the actual phenotypic result.

[0151] The above results indicate that the designed primer set is reasonable and can be used to distinguish and identify wilt resistance genes and their alleles, thereby laying a good technical foundation for molecular-assisted breeding of sesame.

Claims

1. A wilt resistance gene ScaDIR40, characterized in that: This gene is derived from chromosome 16 of the genome of the wild species Sesamum calycinum, which is highly resistant to Fusarium wilt, and is a dominant control gene; its base sequence is shown in SEQ ID No.1; correspondingly, the amino acid sequence of the anti-wilt protein encoded by the wilt-resistant gene ScaDIR40 is as follows: MAMGGAYAAIDNVIQMPMSDAENPKPVDEWFHSLDQAKETRTKLHFYFQDVLGGDSPTV WKVAESAVTSTSATTFGQINMVDDLLTAGPEPGSEVVGRAQGTIGFADLHNTAIQMGLNIVFTEGKYKGSTISVVGRNPIFEKDRELPIAGGTGVFRMARGIAVSNTHSFDVNTNYGVLEYTLYITHYSL.

2. The allele of the wilt resistance gene ScaDIR40 according to claim 1, characterized in that: The alleles in the susceptible wild species Sesamum angustifolium and the susceptible cultivated species are respectively the highly susceptible Fusarium wilt gene Saudir32 and the highly susceptible Fusarium wilt gene Sindir37; The highly susceptible Fusarium wilt gene Saudir32 is derived from chromosome 14 of the highly susceptible wild species Sesamum angustifolium, and the base sequence is shown in SEQ ID No. 2; Correspondingly, the amino acid sequence of the protein encoded by the highly susceptible Fusarium wilt gene Saudir32 is as follows: MPPLSDAENPKPVDEWFHSLDQAKETRTKLHFYFQDVLGGESPTVWKVAESAITSTSATTF GKINMVDDLLTAGPEPCSEVVGRAQGTIGFADLHNTAIHMGLNIVFTEGKYKGSTISVVGRNPIFEKDRELPIVGGTGVFRMARGIAVSNTHSFDVNTNYGVLEYTLYITHYSL; The highly susceptible Fusarium wilt gene Sindir37 is derived from chromosome 9 of Sesamum indicum, a sesame plant that is highly susceptible to Fusarium wilt; the base sequence is shown in SEQ ID No. 3; Correspondingly, the amino acid sequence of the protein encoded by the highly susceptible Fusarium wilt gene Sindir37 is as follows: MRLNIEFTEGKYNNTTINILRRNPIIEKDSELPIVGGTGIFKMACGVVAGNTYLFNVDTNYSV LQYTLALFTLIYKPI.

3. The primer pair for preparing the wilt resistance gene ScaDIR40 according to claim 1 by PCR amplification is characterized in that: The specific design is as follows: Forward primer Dir40 Primer F: 5'-ATATCTATGGCATACATAAAACAC-3', Reverse primer Dir40 Primer R: 5′-CCTAATCATCTTGATTGAAGG-3′.

4. A primer pair for preparing the allele of the wilt resistance gene ScaDIR40 according to claim 2 by PCR amplification, characterized in that: Targeting the highly susceptible Fusarium wilt gene Saudir32, the specific design is: Forward primer dir32 Primer F: 5'-GAGGATGAGATTTTATTACACC-3', Reverse primer dir32 Primer R: 5′-GGCTAGAATTTTCCCTTACAA-3′; Targeting the highly susceptible Fusarium wilt gene Sindir37, the specific design is: Forward primer dir37 Primer F: 5'-TTTTATACCACCTCCCAAAG-3', Reverse primer dir37 Primer R: 5′-CCATCCACGAGTAGTTCCA-3′.

5. A PCR amplification method for preparing the wilt resistance gene ScaDIR40 using the primer pair described in claim 3, characterized in that: The specific steps include: (1) Extracting genomic DNA from Sesamum calycinum var.KEN8 germplasm with high resistance to Fusarium wilt; (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification is performed using a PCR amplification primer pair.

6. A PCR amplification method for preparing an allele of the wilt resistance gene ScaDIR40 using the primer pair of claim 4, characterized in that: The specific steps include: (1) Extracting genomic DNA from Sesamum angustifolium var.G01 germplasm that is highly susceptible to Fusarium wilt, or Sesamum indicum Yuzhi No. 11 that is highly susceptible to Fusarium wilt; (2) using the genomic DNA extracted in step (1) as a template and performing PCR amplification using a PCR amplification primer pair; The allele Saudir32 was prepared by using the DNA of Sesamum angustifolium var.G01 germplasm, which is highly susceptible to Fusarium wilt, as a template and amplified using the dir32 F / R primer pair; The DNA of Sesamum indicum var. Yuzhi 11 germplasm, which is highly susceptible to Fusarium wilt, was used as a template and the dir37 F / R primer pair was used to amplify the allele Saudir37.

7. A primer set for detecting and distinguishing the wilt resistance gene ScaDir40 and its alleles according to claim 1, characterized in that: The specific design is: DIR1 F1: 5'-TCTTGGCTCCTGAAAATAGT-3', DIR1 R1: 5'-CGAGCACGTCCTGGAAATAGAAA-3'; DIR1 F2: 5'-TTTTATAACCACCTCCCAAAG-3', DIR1 R2: 5'-CCATCCACGAGTAGTTCCA-3'; When using this primer set for detection and differentiation: When the DIR1 F1 / R1 primer pair was used for PCR amplification, if the PCR amplification template contained the genome of Sesamum calycinum with high resistance to Fusarium wilt, the electrophoresis band after PCR amplification contained a 1572 bp long band; When the DIR1 F1 / R1 primer pair was used for PCR amplification, if the PCR amplification template contained the genome of Sesamum angustifolium, which was highly susceptible to Fusarium wilt, the electrophoresis band after PCR amplification contained a 2952 bp long band; When PCR amplification is performed using the DIR1 F1 / R2 primer pair, if the PCR amplification template contains the genome of Sesamum indicum, which is highly susceptible to Fusarium wilt, the electrophoresis band after PCR amplification contains a 624 bp long band.

8. A method for detecting and determining the phenotypic resistance to wilt disease using the primer set for detecting and distinguishing the wilt disease resistance gene ScaDir40 and its alleles according to claim 6, characterized in that: The steps include: (1) Extracting genomic DNA from the plant germplasm to be tested; (2) using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using primer pairs DIR1 F1 / R1 and DIR1 F2 / R2 respectively; (3) performing electrophoresis on the PCR amplification product in step (2), and making a determination based on the electrophoresis results; the specific determination standard is: after amplification using DIR1 F1 / R1, if there is only one electrophoresis band with a length of 1572 bp, it indicates that the germplasm resource to be identified has a dominant homozygous high resistance to Fusarium wilt phenotype; After amplification using DIR1 F1 / R1, if there is only one electrophoresis band of 2952 bp in length, it indicates that the germplasm resource to be identified has a recessive homozygous high susceptibility to Fusarium wilt phenotype; After amplification using DIR1 F1 / R1, if there are two electrophoretic bands with lengths of 1572 and 2952 bp respectively, it indicates that the germplasm to be identified is a heterozygous high resistance to Fusarium wilt; After amplification using DIR1 F2 / R2, if there is only one electrophoretic band of 624 bp in length, it indicates that the germplasm resource to be identified has a recessive homozygous high susceptibility to Fusarium wilt phenotype; If there is no electrophoretic band after amplification using DIR1 F1 / R1 or DIR1 F2 / R2, it indicates that the germplasm to be identified is not Sesamum calycinum, Sesamum angustifolium, cultivated species or their progeny, and other methods are required to identify the wilt resistance of the germplasm to be identified.

9. The use of the wilt resistance gene ScaDIR40 according to claim 1 in plant breeding, characterized in that: Used to breed new plant varieties resistant to Fusarium wilt.

10. The use of the allele of the wilt resistance gene ScaDIR40 according to claim 2 in plant breeding, characterized in that: Used to breed new varieties of plants that are highly susceptible to Fusarium wilt.

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