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SNP locus combination for detecting tomato verticillium wilt resistance and application thereof

A Verticillium wilt and disease resistance technology, applied in the field of plant biology, can solve the problems of linkage redundancy, insufficient versatility of SNP sites, invalid selection of target genes, etc., achieve accurate detection, reduce manual inoculation identification and field transplanting work Quantity, the effect of improving breeding efficiency

Pending Publication Date: 2021-12-03
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

Based on tomato genome variation information, multiple SNP loci that can be used for rapid and effective detection of tomato Verticillium wilt resistance were obtained through direct analysis and identification of a large number of variation group data in the adjacent regions of the upstream and downstream of the known Ve1 and Ve2 genes and within the genes. Accurate identification and selection of target gene regions can be achieved to solve the lack of versatility of SNP sites in existing technologies, or functional molecular markers that are not directly related to target traits / genes (only linkage markers), resulting in invalid selection of target genes, or linkage cumbersomeness Bringing problems such as other non-target unfavorable gene fragments

Method used

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  • SNP locus combination for detecting tomato verticillium wilt resistance and application thereof
  • SNP locus combination for detecting tomato verticillium wilt resistance and application thereof
  • SNP locus combination for detecting tomato verticillium wilt resistance and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] The screening of embodiment 1 SNP site combination

[0075] 1. Experimental materials

[0076] The variant group data of 660 representative tomato germplasm resources from different sources worldwide were selected for SNP site screening in this embodiment. The source of the tomato variant group data is mainly based on the previous work of the project team where the inventor belongs. (Lin,T.,Zhu,G.,Zhang,J.et al.Genomic analyzes 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 Ve1 and Ve2 genes come from the existing public database (https: / / solgenomics.net / ).

[0077] 2. Screening of SNP site combinations

[0078] Use target genes Ve1 (Solyc09g005090, database source: https: / / solgenomics.net / locus / 1531 / vie...

Embodiment 2

[0082] Example 2 Primer Synthesis and Kit Preparation

[0083] 1. Primer design and screening

[0084] According to the flanking sequences of the Ve1-SNP01 site to Ve1-SNP04 site and the Ve2-SNP01 site to Ve2-SNP05 site provided in Example 1, for each SNP site, the principle of design and development according to the KASP marker (target product 80-150bp, the primers specifically match the target region on the reference genome and are located in non-SNP dense regions, avoiding complex sequence regions with high A\T or G\C content, etc.), use Primer3.0 software to design two upstream SNP sites A forward primer and a reverse primer were designed downstream.

[0085] Tomato samples to be tested include some germplasm resources of known genotypes and Verticillium wilt resistance phenotypes and 21 samples randomly selected from breeding materials as resistant, susceptible or heterozygous controls, and finally added 3 ddH 2 O is used as the NTC blank control, a total of 24 copies. ...

Embodiment 3

[0098] Example 3 Verification and Breeding Application of Tomato Verticillium Wilt Resistance Genes Ve1 and Ve2 Highly Efficient KASP Markers

[0099]In this example, based on the SNP loci provided in Example 1, the kit (including primer combinations) provided in Example 2 was used to carry out the verification and breeding application of tomato Verticillium wilt resistance genes Ve1 and Ve2 high-efficiency KASP markers.

[0100] Select 381 representative tomato samples including mainstream commercial varieties, core public germplasm of the National Resource Bank, breeding intermediate materials, new hybrid combinations, etc., involving large fruit tomatoes, cherry tomatoes, cluster tomatoes, fresh tomatoes, processed tomatoes and Various types of cultivated tomatoes such as farm species (local varieties). Among them, 177 have obtained Ve1 and Ve2 genotype data through third-party commercial institutions using closely linked SCAR markers (including 15 tomato germplasm resource...

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Abstract

The invention relates to the technical field of plant biology, and particularly relates to an SNP locus combination for detecting tomato verticillium wilt resistance and application thereof. Based on this, the invention develops a primer combination, a kit and a detection method capable of quickly, intuitively and effectively identifying the genotype state of a target SNP locus. By applying the SNP locus combination, the quick, accurate and high-throughput detection of haplotypes of Ve1 and Ve2 sections of tomato verticillium wilt resistance genes can be realized; the SNP locus combination has the advantages of simple operation, low cost, automation, high throughput efficiency, stable marking, safety, no toxicity, no harm and the like; the tomato verticillium wilt resistance can be quickly and accurately identified at high throughput in a tomato seedling stage; the workload of artificial inoculation identification and field transplantation is reduced; the breeding efficiency is improved; the breeding cost is reduced; the breeding process is accelerated; and the SNP locus combination is very suitable for modern commercial breeding application 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 tomato verticillium wilt resistance and its application. Background technique [0002] Tomato is an important vegetable economic crop in the world, which has important production application and basic research value. With the gradual expansion of tomato planting area around the world, the impact of tomato diseases and insect pests is increasing day by day. Verticillium wilt (Verticillium Wilt) is a worldwide fungus caused by Verticillium dahliae and Verticillium alboatrum Reinke and Berth in soil. disease. So far, two pathogenic physiological races have been found, corresponding to two disease resistance genes Ve1 and Ve2, both of which are fully dominant genes derived from Peruvian tomato. Ve1 and Ve2 are located on Chr.09S, and both have been cloned (Kawchuk et al., 2001), corresponding to Solyc09g005090 and Solyc09g005080, respecti...

Claims

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Application Information

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