Combination of SNP loci for detection of tomato gray leaf spot resistance and its application

A tomato gray leaf spot and locus technology, applied in the field of plant biology, can solve problems such as linkage burden, insufficient SNP site versatility, ineffective target gene selection, etc., achieve accurate detection, reduce field transplanting workload, and improve Effects of Breeding Efficiency

Active Publication Date: 2022-06-07
AGRI GENOME INST OF SHENZHEN CHINESE ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Through the analysis and identification of a large number of variant group data directly in the adjacent regions of the upstream and downstream of the known Sm gene and within the gene, multiple SNP sites that can quickly and effectively detect the resistance of tomato gray leaf spot can be obtained, which can realize accurate identification of the target gene region and selection, so as to solve the lack of versatility of SNP sites in the prior art, or not functional molecular markers directly related to target traits / genes (only linkage markers), resulting in invalid selection of target genes, or other non-target disadvantages caused by linkage redundancy genetic fragments etc.

Method used

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  • Combination of SNP loci for detection of tomato gray leaf spot resistance and its application
  • Combination of SNP loci for detection of tomato gray leaf spot resistance and its application
  • Combination of SNP loci for detection of tomato gray leaf spot resistance and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 Screening of SNP site combinations

[0085] 1. Experimental materials

[0086] Select the variation group data of 660 representative tomato germplasm resources in the world, different source types, for SNP site screening in this example, and this tomato variation group data is mainly based on the preliminary work of the project team where the inventor is located ( Lin, T., Zhu, G., Zhang, J. et al. Genomic analyses provide insights into the history of tomato breeding[J]. Nat Genet, 2014, 46: 1220~1226; Tieman D, Zhu G, Resende M F R, et al.A chemical genetic roadmap to improved tomato flavor[J].Science,2017,355(6323):391). Among them, the genotype data of some tomato germplasm resources and the resistance phenotype data of Sm gene were obtained from the existing public database (https: / / solgenomics.net / ).

[0087] 2. Screening of SNP site combinations

[0088] Use the target gene Sm (Solyc11g018710, Solyc11g018720 and Solyc11g018730, database source: https:...

Embodiment 2

[0092] Example 2 Primer synthesis and kit preparation

[0093] 1. Primer design and screening

[0094] According to the flanking sequences of the SNP01 site to the SNP11 site provided in Example 1, for each SNP site, the principle of design and development according to KASP markers (target product 80-150bp, primers that specifically match the target region on the reference genome and are located in non- SNP-intensive regions, avoid complex sequence regions with high A\T or G\C content, etc.), use Primer3.0 software to design two forward primers upstream of the SNP site, and a reverse primer downstream.

[0095] The tomato samples to be tested include 21 randomly selected samples from germplasm resources and breeding materials with known genotypes and phenotypes as resistance, sensitivity or heterozygous controls, and finally add 3 ddH 2 O as the NTC blank control, a total of 24 copies.

[0096] DNA extraction Using conventional CTAB method or domestic magnetic bead kit to ex...

Embodiment 3

[0111] Example 3 Validation and breeding application of high-efficiency KASP marker of tomato resistance gene Sm to gray leaf spot

[0112] Based on the SNP loci provided in Example 1, this example uses the kit (including primer combination) provided in Example 2 to carry out the verification and breeding application of the high-efficiency KASP marker of the tomato gray leaf spot resistance gene Sm.

[0113] Selected 381 representative tomato samples including mainstream commercial varieties, core public germplasm of the national resource bank, intermediate breeding materials, new hybrid combinations, etc., involving large fruit tomatoes, cherry tomatoes, bunch tomatoes, fresh tomatoes, processed tomatoes and Farm varieties (landraces) and other cultivated tomatoes of different types and origins, 162 of which have obtained Sm genotype data through third-party commercial agencies using closely linked InDel markers (including 15 known phenotypes of gray leaf spot resistance) The...

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Abstract

The invention relates to the field of plant biotechnology, in particular to a combination of SNP sites for detecting tomato gray leaf spot resistance and an application thereof. Based on this, the present invention develops a primer combination, a kit and a detection method that can quickly, intuitively and effectively identify the genotype status of the target SNP site. The application of the present invention can realize rapid, accurate and high-throughput detection of the haplotype of the Sm segment of the tomato gray leaf spot resistance gene, and has the advantages of simple operation, low cost, automation, high throughput efficiency, stable labeling, safety and non-toxicity. It can quickly, accurately and high-throughput identify the resistance of tomato gray leaf spot at the tomato seedling stage, reduce the workload of manual inoculation identification and field transplanting, improve breeding efficiency, reduce breeding costs, and accelerate breeding Process, very suitable for modern commercial breeding applications and large-scale genetic improvement research.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to a combination of SNP sites for detecting resistance to tomato gray leaf spot and its application. Background technique [0002] Tomato is an important vegetable economic crop in the world, with important production application and basic research value. With the gradual expansion of tomato planting area around the world, the impact of tomato pests and diseases is increasing day by day. Among them, gray leaf spot disease (GLSD) of tomato is a new and popular fungal disease in recent years, and the incidence rate is increasing year by year. The causative bacteria are fungi of the genus Stemphylium of the subphylum Hypophysae, mainly caused by the infection of Stemphylium solani and S. lycopersici. The disease occurs worldwide, especially in warm and humid regions. In recent years, it has generally occurred in spring and summer in southern and northern my country, especially in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/13C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 黄三文吴坤练群张金喆吕亚清
Owner AGRI GENOME INST OF SHENZHEN CHINESE ACADEMY OF AGRI SCI
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