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Acquisition of closely linked SNP loci and development of markers for tomato spotted wilt resistance gene sw-5b

A technology for sw-5b and spotted wilt, which is applied in the biological field, can solve the problems of low detection efficiency, affect the breeding process, and not suitable for high-throughput molecular detection platforms, and achieve the effect of high detection efficiency and simple detection methods

Active Publication Date: 2018-05-08
北京通州国际种业科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the traditional breeding process of spotted wilt-resistant varieties, the genotype of the intermediate material needs to be identified by inoculating the pathogen in the field for each generation, and the environmental conditions will affect the selection of the correct intermediate material, which will inevitably affect the breeding process; at the same time, if Improper operation of the inoculation process will also cause the spread of the virus, which is not conducive to the comprehensive control of spotted wilt. Therefore, the problem of difficult breeding of disease-resistant varieties has not been completely solved
Although the above-mentioned Sw-5 gene-linked markers that have been developed can be used for molecular marker-assisted selection, the detection efficiency is relatively low, and it is not suitable for high-throughput molecular detection platforms

Method used

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  • Acquisition of closely linked SNP loci and development of markers for tomato spotted wilt resistance gene sw-5b
  • Acquisition of closely linked SNP loci and development of markers for tomato spotted wilt resistance gene sw-5b
  • Acquisition of closely linked SNP loci and development of markers for tomato spotted wilt resistance gene sw-5b

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Acquisition and identification of closely linked SNP sites and SNP markers with tomato spotted wilt resistance Sw-5b gene

[0038] After a large number of sequence analysis and discovery of SNP sites by the inventors, and correlation analysis between SNP sites and multiple traits, a SNP site related to tomato spotted wilt resistance was found, named SNP724.

[0039] The SNP724 marker sequence is as follows, and the polymorphic site is the 59th nucleotide of the following sequence 1, which is A or G;

[0040] 5'-CTACTTTTTTCCAATGGATAAAGCTTTGGAATGGAATCATGGACACAACTGGAGTTATT[A / G]CTATATATCTTTGAAACTTGTACAAGTAAAATTTGAAGAATGACTGTTG-3' (SEQ ID NO: 1)

[0041] According to SNP724 primer design, pre-experimental comparison effect, select the specific primer combination of SNP724 site:

[0042] FP1SNP724: 5'-GAAGGTCGGAGTCAACGGATTCATGGACACAACTGGAGTTATTA-3' (SEQ ID NO: 2)

[0043] FP2SNP724: 5'-GAAGGTGACCAAGTTCATGCTCATGGACACAACTGGAGTTATTG-3' (SEQ ID NO: 3)

[0044] RPSNP...

Embodiment 2

[0046] Example 2, the application of SNP sites and their specific primer combinations in identifying whether tomato contains the Sw-5b gene for resistance to spotted wilt

[0047] 1. Identify whether tomato contains the spotted wilt resistance Sw-5b gene

[0048] 1. Genomic DNA extraction

[0049] Genomic DNA of 48 tomato materials shown in Table 1 was extracted.

[0050] Table 1 Identification table of 48 tomato material phenotypes and marker genotypes

[0051]

[0052] Note: 1. The samples with DNA number 1-36 in the table are registered samples of Tomato Germplasm Resource Center (Tomato GeneticsResource Center http: / / tgrc.ucdavis.edu / index.aspx); the samples with DNA number 37-48 are commodity type;

[0053] 2. In column Sw-5-2, 'R' indicates the homozygous resistant genotype of spotted wilt, 'S' indicates the homozygous susceptible genotype of spotted wilt, and 'H' indicates the heterozygous resistant gene of spotted wilt type;

[0054] 3. In the SNP724 column, 'A...

Embodiment 3

[0071] Example 3, Application of SNP724 site and its specific primer combination in identification of resistance to spotted wilt of tomato

[0072] 1. Phenotypic detection of tomato resistance to spotted wilt

[0073] The 48 tomato materials from different sources shown in Table 1 were selected, and after planting in the field, the phenotype observation was carried out. 10 plants were planted in each material, and the disease resistance phenotype statistics were carried out during the high-incidence period of spotted wilt. If there were more than 7 disease-resistant plants in each material, this material was recorded as disease-resistant. The statistical results are shown in Table 1.

[0074] 2. Identification of tomato resistance to spotted wilt by SNP loci and their specific primer combinations

[0075] 1. Obtaining genomic DNA

[0076] Genomic DNA of 48 tomatoes from different sources was extracted.

[0077] 2. PCR amplification

[0078] The above genomic DNA was amplif...

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Abstract

The invention discloses tomato spotted wilt resistance gene Sw-5b close linkage SNP site acquisition and marker development. The invention provides application of substances for detecting polymorphismor genotype of SNP724 sites in a tomato genome in identification or auxiliary identification on whether spotted wilt resistant Sw-5b genes exist in the tomato genome or not, or application of substances for detecting polymorphism or genotype of SNP724 sites in a tomato genome in preparation identification or auxiliary identification on whether spotted wilt resistant Sw-5b gene products exist in the tomato genome or not. Experiments prove that an SNP marker SNP724 which is interlocked with the tomato spotted wilt resistance gene Sw-5b is developed, the SNP marker can be used for a high-throughput molecule detection platform, and compared with a second-generation molecular marker, the SNP maker has the advantages that the detection mode is simple, convenient and accurate, and the detectionefficiency is high.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the field of genome sequence and plant biotechnology, and in particular to the acquisition of SNP sites closely linked to tomato spotted wilt resistance gene Sw-5b and the development of markers. Background technique [0002] Tomato spotted wilt is a serious disease caused by tomato spotted wilt virus, which threatens the normal production of tomato. Breeding tomato varieties resistant to spotted wilt is one of the most effective methods to control tomato spotted wilt. In the traditional breeding process of spotted wilt-resistant varieties, the genotype of the intermediate material needs to be identified by inoculating the pathogen in the field for each generation, and the environmental conditions will affect the selection of the correct intermediate material, which will inevitably affect the breeding process; at the same time, if Improper operation of the inoculation process will a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 刘宝平王喜萍李常保王铭堂张东峰王亚楠马俊召周明刘海燕杜敏敏
Owner 北京通州国际种业科技有限公司
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