InDel marker for detecting tomato gray leaf spot resistance and application of InDel marker

Abstract

The invention provides an InDel marker for detecting tomato gray leaf spot disease resistance. The InDel marker is closely linked with a gene of a sixth chromosome SL4.0 ch06: 33855052-33933438 of a tomato. The InDel marker developed by the invention can realize accurate, rapid and high-throughput detection of the tomato gray leaf spot disease resistant sm6.1 gene, and can be applied to commercial breeding of tomatoes and genetic improvement application research of gray leaf spot disease resistance; important clues can be provided for research on group evolution, gene chips and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biological technology, and in particular relates to an InDel marker for detecting resistance to tomato gray leaf spot disease and its application. Background Art

[0002] Gray leaf spot, a major tomato disease caused by the fungus Stemphylium spp., was first reported in 1930 and has recently caused widespread outbreaks in countries around the world. Gray leaf spot spreads rapidly, making it difficult to control. In my country, the disease affects over 3 million mu (approximately 1.5 million hectares) of crops annually, resulting in losses of nearly 1 billion yuan. Identifying disease-resistant genes and cultivating high-quality, resistant varieties are the most economical, effective, and safe prevention and control measures.

[0003] my country is a major tomato producer worldwide. Currently, the Sm gene from the currant tomato (Solanum pimpinellifolium) is widely used in tomato production. It is also the only reported gene for resistance to tomato gray leaf spot disease. The long-term, large-scale use of a single disease-resistance gene is highly susceptible to being overcome by mutated pathogens, posing significant future risks. Therefore, the discovery of new gray leaf spot resistance genes and the development of novel molecular markers are urgently needed to ensure the sustainable development of the tomato industry. Summary of the Invention

[0004] To solve the above problems in the prior art, the present invention provides an InDel marker for detecting resistance to tomato gray leaf spot and its application.

[0005] To achieve the purpose of the present invention, the present invention provides an InDel marker for detecting resistance to tomato gray leaf spot disease, wherein the InDel marker is tightly linked to the gene on tomato chromosome 6 SL4.0ch06:33855052-33933438.

[0006] In a preferred embodiment of the present application, the InDel marker includes InDel-1, InDel-2 and / or InDel-3, the InDel-1 marker is based on the insertion and deletion site at base 33,849,589 of tomato SL4.0ch06, the InDel-2 marker is based on the insertion and deletion site at base 33,869,105 of tomato SL4.0ch06, and the InDel-3 marker is based on the insertion and deletion site at base 33,933,304 of tomato SL4.0ch06.

[0007] In a preferred embodiment of the present application, the InDel-1 marker comprises a deletion of the nucleotide sequence shown in SEQ ID NO. 1, the InDel-2 marker comprises an insertion of the nucleotide sequence shown in SEQ ID NO. 2, and the InDel-3 marker comprises an insertion of the nucleotide sequence shown in SEQ ID NO. 3.

[0008] The present invention also provides the use of the InDel marker in screening tomato varieties resistant to gray leaf spot disease.

[0009] The present invention also provides the InDel-tagged primer pair, which includes an InDel-1 primer pair, an InDel-2 primer pair, and / or an InDel-3 primer pair, wherein the InDel-1 primer pair includes an upstream primer sequence shown in SEQ ID NO.4 and a downstream primer sequence shown in SEQ ID NO.5; The InDel-2 primer pair includes the upstream primer sequence shown in SEQ ID NO.8 and the downstream primer sequence shown in SEQ ID NO.9; The InDel-3 primer pair includes an upstream primer sequence shown in SEQ ID NO.12 and a downstream primer sequence shown in SEQ ID NO.13.

[0010] In a preferred embodiment of the present application, PCR amplification is performed using the genomic DNA of the tomato material to be tested as a template using the primer pair.

[0011] In a preferred embodiment of the present application, when the DNA fragment amplified by the InDel-1 primer is 182 bp, or the DNA fragment amplified by the InDel-2 primer is 124 bp, or the DNA fragment amplified by the InDel-3 primer is 135 bp, the tomato to be tested is susceptible to gray leaf spot disease; When the DNA fragment amplified by the InDel-1 primer is 165 bp, or the DNA fragment amplified by the InDel-2 primer is 140 bp, or the DNA fragment amplified by the InDel-3 primer is 172 bp, the tomato to be tested is a material resistant to gray leaf spot disease.

[0012] The present invention also provides a kit for screening gray leaf spot-resistant tomato varieties, comprising the primer pair.

[0013] The present invention also provides application of the kit in screening gray leaf spot-resistant tomato varieties.

[0014] The beneficial effects of the present invention are: The InDel marker developed in this invention can realize the accurate, rapid and high-throughput detection of the sm6.1 gene for resistance to tomato gray leaf spot disease, and can be applied to commercial tomato breeding and applied research on genetic improvement of gray leaf spot disease resistance; it can also provide important clues for research on population evolution, gene chips, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The leaf phenotypes of the gray leaf spot pathogen Stemphylium solani inoculated with the LA1777 introgression line were analyzed. Figure 2 To analyze the sm6.1 locus for resistance to tomato gray leaf spot using the LA1777 introgression line; Figure 3 This is the fine positioning map of sm6.1; Figure 4 PCR fragments amplified for the co-segregating markers InDel-1, InDel-2, and InDel-3. DETAILED DESCRIPTION

[0016] The present invention is further described in detail below through specific implementation cases.

[0017] Example 1 Fine Mapping of the Recessive Locus sm6.1 for Tomato Gray Leaf Spot Resistance and Development of Co-segregating Markers 1) Identification of gray leaf spot resistance phenotype S. solani is a dominant pathogenic strain of tomatoes in my country. Tomato seedlings are inoculated with the fungus at the 4th to 6th leaf stage, with a mycelial suspension OD600 of approximately 1.6. Environmental conditions after inoculation include a temperature of approximately 25°C and a humidity of above 80% for 48-72 hours. Plants are then managed normally, and leaf lesions are observed 7 days after inoculation. Leaves with lesions are considered susceptible and designated S, while leaves without lesions are designated resistant and designated R. This method was used to inoculate and identify the LA1777 introgression line. LA1777 was resistant, while LA3945, LA3946, LA3949, and LA4009 were resistant. The recurrent parent, E6203, was susceptible ( Figure 1 ).

[0018] 2) Resequencing of the LA1777 introgression population: DNA from the LA1777 introgression population was extracted using the CTAB method and high-depth whole-genome resequencing was performed using the BGI platform, with an average sequencing depth of >10×. Based on the sequencing results and the development of molecular markers, the gray leaf spot resistance introgression fragment from LA1777 was determined to be located in a 300 kb region on chromosome 6 ( Figure 2 ).

[0019] 3) Fine positioning of sm6.1 The disease-resistant introgression line LA3946 was crossed with the susceptible recurrent parent E6203 to obtain the F1 and F2 generations.

[0020] After inoculation with S. solani, the F1 plants were susceptible, a recessive phenotype. Among the 416 F2 segregating plants, 113 showed resistance, while 303 showed susceptibleness. The segregation ratio of resistant to susceptible plants was 1:2.68, approaching the Mendelian ratio of 1:3. A chi-squared test (χ²=1.04, P=0.25) indicated that the resistance trait is controlled by a single recessive locus, which we named sm6.1.

[0021] To fine-map the sm6.1 locus, polymorphic markers were developed within the 35-Mb introgressed region on chromosome 6 of LA3946. Genotyping of 416 F2 plants was performed, and genetic linkage analysis was performed using QTL IciMapping (V4.1). Linkage analysis identified a resistance QTL (LOD > 25) that overlaps with the previously analyzed resistance introgressed region and contributes over 65% to the phenotypic contribution.

[0022] Using flanking molecular markers, 30 recombinant strains were identified from a population of 2,400 F2 strains. Further marker development based on resequencing data from both parents was used to genotype these recombinant strains.

[0023] Fourteen important recombinant plants were screened and their progeny were identified at the seedling stage. Based on the genotypes and phenotypes of these recombinant plants and their corresponding F2:3 progeny, the sm6.1 locus was located within a 240 kb region ( Figure 3 To further fine-tune the locus, molecular markers were used to screen for new recombinant plants from 11,080 F2:3 segregating populations (progeny of F2 recombinant plants). A total of 40 recombinant plants were identified, and through self-pollination, F3:4 populations were generated for progeny identification. Phenotypic characterization of the progeny of 16 key recombinant plants ultimately led to the precise localization of the sm6.1 locus to a 78.4 kb interval between markers PC-235 and PC-261 ( Figure 3 c). According to the annotation information of the tomato reference genome annotation database (ITAG4.0), there are 9 genes in this candidate region. Among them, the sm6.1 fine-mapping interval flanking markers PC-233, PC-237, and PC-261 are closely linked to sm6.1 ( Figure 3 d), named InDel-1, InDel-2, and InDel-3 respectively.

[0024] The physical location is the SL4.0 version of tomato Heinz1706, the base at position 33,849,589 on chromosome 6, the variant type is G / GCCAAAAACAGTTGAAATA (SEQ ID NO.1); the base at position 33,869,105 on chromosome 6, the variant type is C / CTCCGGATAAGTCTCTC (SEQ ID NO.2); the base at position 33,933,304 on chromosome 6, the variant type is T / AACAAGCTAGTTTAGTTGTACTATAAATTTGGACTTGT (SEQ ID NO.3).

[0025] The three mutation sites are closely linked to the tomato gray leaf spot sm6.1 gene.

[0026] 4) sm6.1 co-segregated InDel marker sequences The sequences of the markers InDel-1, InDel-2, and InDel-3 flanking the sm6.1 fine-mapping interval are as follows: InDel-1 F primer (5'-3'): TAGCTAGATCTCATAAGTTGGTCGT (SEQ ID NO. 4) InDel-1 R primer (5'-3'): CTAAGAGGGTGTCTGGATTGG (SEQ ID NO. 5) InDel-1 amplifies the susceptible sequence, 182 bp in length: TAGCTAGATCTCATAAGTTGGTCGTATGAAACTTTTGCTTTTACTTATTTTTGTACTTCTTTGGCCTAAATTAAGTGCTTTAAAACACTTTTTATCTTTCCTAAACACCACAAAAAATAGATAGAGAGCTTTAATGCCAAAAACAGTTGAAATAAGCTAAACCAATCCAGACACCCTCTTAG (SEQ ID NO. 6) InDel-1 amplifies the disease resistance sequence, 165 bp in length: TAGCTAGATCTCATAAGTTGGTCGTATGAAACTTTAGCTTTACTTATTTTTGTACTTCTTTGGCCTAAAAGTAAGTGATTCAAAACACTTCTTATTCTTTCCTAAACACCACAAAAAATAGATAAAGAGCTTTAATGAGCTAAACCAATCCAGACACCCTCTTAG (SEQ ID NO. 7) InDel-2 F primer (5'-3'): GCTCAAACACTCTGAAAGCAGC (SEQ ID NO. 8) InDel-2 R primer (5'-3'): GTGCAAATCTTAATGTAAACGAATG (SEQ ID NO. 9) InDel-2 amplifies the susceptible sequence, 124 bp in length: GCTCAAACACTCTGAAAGCAGCAGATTCTTTAAGCATGATATCTTAGGCCTCATTTGTTTACACTTAACAAAGAGACTCCAGATCTTAGGTCATTAATGCATTCGTTTACATTAAGATTTGCAC (SEQ ID NO. 10) InDel-2 amplifies the disease resistance sequence, 140 bp in length: GCTCAAACACTCTGAAAGCAGCTCCGGATAAGTCTCTCAGATTCTTTAAGTATGATATCTTAGGCCTCATTTATTTACACTTAACAAAGAGTCTCCAGATCTTAGGTCATTAATGCATTCGTTTACATTAAGATTTGCAC (SEQ ID NO.11) InDel-3 F primer (5'-3'): TTGGACGAACACAACAATTAAGT (SEQ ID NO. 12) InDel-3 R primer (5'-3'): CATTGGAGAAGGAAGGCAGAAC (SEQ ID NO. 13) InDel-3 amplifies the susceptible sequence, 135 bp in length: TTGTACGAACACAACAATTAAGTTGTCATTGGTTGTGGATTTTAGGTTGTTTGCTTAACACGTGTATACTTCTTTTTTCCAGTAAAACGATTGTATGTCAAAACACATCAAAGTTCTGCCTTCCTTCTCCAATG (SEQ ID NO. 14) InDel-3 amplifies the disease resistance sequence, 172 bp in length: TTGTACGAACACAACAATTAAGTAACAAGCTAGTTTAGTTGTACTATAAATTTGGACTTGTGTCATTGGTTGTGGATTTTAGGTTGTTTGCTTAACACGTGTATACTTCTTTTTTCCAGTAAAACAATTGTATGTCAAAACACATCAAAGTTCTGCCTTCCTTCTCCAAATG (SEQ ID NO. 15) Example 2 Application of InDel-1, InDel-2 and InDel-3 DNA was extracted from leaves of the susceptible parent E6203, the resistant introgression line LA3946, the F1 plants of these two materials (LA3946×E6203) and 32 F2 plants using the CTAB method.

[0027] The reaction was carried out using 10 μL PCR reaction system.

[0028] 10 μL PCR reaction system: 1 μL DNA template (100 ng / μL), 0.25 μL each of upstream and downstream primers, 5 μL 2× Taqmaster mix, and 3.5 μL ddH2O.

[0029] PCR amplification program: pre-denaturation at 95°C for 4 min (1 cycle); denaturation at 95°C for 30 s (34 cycles), annealing at 56°C for 30 s (34 cycles), extension at 72°C for 30 s (34 cycles), and final extension at 57°C for 5 min (1 cycle).

[0030] Use a pipette to draw 7 μL of PCR product for gel electrophoresis. After the electrophoresis is completed, take a picture with a gel imager ( Figure 4 The leftmost column shows a 100 bp DNA marker. Lanes 1-32 represent F2 individual lines. The three lanes on the right represent typing results for the susceptible parent (P1) E6203, the F1 line (LA3946×E6203), and the resistant parent (P2) LA3946. The amplified fragment for InDel-1 is 182 bp long for susceptible lines, and 165 bp long for resistant lines; the amplified fragment for InDel-2 is 124 bp long for susceptible lines, and 140 bp long for resistant lines; and the amplified fragment for InDel-3 is 135 bp long for susceptible lines, and 172 bp long for resistant lines.

[0031] The above describes in detail an InDel marker for detecting resistance to tomato gray leaf spot and its application provided by the present invention. Specific examples are used herein to illustrate the principles and implementations of the present invention. The above examples are only intended to help understand the method and core concept of the present invention.

[0032] It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made to the present invention without departing from the principles of the present invention. These improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. An InDel marker for detecting resistance to tomato gray leaf spot, characterized in that: The InDel marker is closely linked to the gene on tomato chromosome 6 SL4.0ch06: 33855052-33933438.

2. The InDel marker according to claim 1, characterized in that The InDel markers include InDel-1, InDel-2 and / or InDel-3, The InDel-1 marker is based on the insertion and deletion site at tomato SL4.0ch06: 33,849,589 bases, the InDel-2 marker is based on the insertion and deletion site at tomato SL4.0ch06: 33,869,105 bases, and the InDel-3 marker is based on the insertion and deletion site at tomato SL4.0ch06: 33,933,304 bases.

3. The InDel marker according to claim 2, characterized in that The InDel-1 marker includes a deletion of the nucleotide sequence shown in SEQ ID NO. 1, the InDel-2 marker includes an insertion of the nucleotide sequence shown in SEQ ID NO. 2, and the InDel-3 marker includes an insertion of the nucleotide sequence shown in SEQ ID NO.

3.

4. Use of the InDel marker according to any one of claims 1 to 3 in screening tomato varieties resistant to gray leaf spot disease.

5. An InDel-labeled primer pair according to any one of claims 1 to 3, characterized in that: The primer pair includes an InDel-1 primer pair, an InDel-2 primer pair and / or an InDel-3 primer pair, wherein the InDel-1 primer pair includes an upstream primer sequence shown in SEQ ID NO.4 and a downstream primer sequence shown in SEQ ID NO.5; The InDel-2 primer pair includes the upstream primer sequence shown in SEQ ID NO.8 and the downstream primer sequence shown in SEQ ID NO.9; The InDel-3 primer pair includes an upstream primer sequence shown in SEQ ID NO.12 and a downstream primer sequence shown in SEQ ID NO.

13.

6. A method for screening tomatoes resistant to gray leaf spot disease, characterized in that: The genomic DNA of the tomato material to be tested is used as a template and PCR amplification is performed using the primer pair described in claim 5.

7. The screening method according to claim 6, characterized in that When the DNA fragment amplified by the InDel-1 primer is 182 bp, or the DNA fragment amplified by the InDel-2 primer is 124 bp, or the DNA fragment amplified by the InDel-3 primer is 135 bp, the tomato to be tested is susceptible to gray leaf spot disease; When the DNA fragment amplified by the InDel-1 primer is 165 bp, or the DNA fragment amplified by the InDel-2 primer is 140 bp, or the DNA fragment amplified by the InDel-3 primer is 172 bp, the tomato to be tested is resistant to gray leaf spot disease.

8. A kit for screening tomato varieties resistant to gray leaf spot disease, characterized in that: Comprising the primer pair described in claim 5.

9. Use of the kit according to claim 8 in screening tomato varieties resistant to gray leaf spot disease.

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