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KASP primers for detecting anti-disease genes Ty-3 and Ty-3a of tomato yellow leaf curl virus disease and application of KASP primer

A technology for tomato yellowing leaf curl and disease resistance gene, applied in the fields of molecular biology and crop breeding, can solve the problem of not finding KASP markers, and achieve the effects of improving breeding efficiency, reducing workload, and speeding up the breeding process

Active Publication Date: 2020-11-20
河北省农林科学院经济作物研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no KASP marker applied to the detection of tomato yellow leaf curl virus disease

Method used

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  • KASP primers for detecting anti-disease genes Ty-3 and Ty-3a of tomato yellow leaf curl virus disease and application of KASP primer
  • KASP primers for detecting anti-disease genes Ty-3 and Ty-3a of tomato yellow leaf curl virus disease and application of KASP primer
  • KASP primers for detecting anti-disease genes Ty-3 and Ty-3a of tomato yellow leaf curl virus disease and application of KASP primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Development of KASP markers

[0078] Compare and analyze the sequences of the disease-resistant and susceptible alleles of Ty-3 and Ty-3a respectively, and name the Ty-3 disease-resistant alleles as Ty-3 / Ty-3, Ty-3 susceptible, etc. The bit gene is named ty-3 / ty-3; and there is a SNP site at the 3723bp position of the two. The Ty-3a resistant allele was named Ty-3a / Ty-3a, and the Ty-3a susceptible allele was named ty-3a / ty-3a. And both of them have a SNP site at the 8914bp position. It was verified that the SNP sites of the two genes met the requirements for KASP marker development: the genotypes were different at the SNP site; there were no other SNP sites nearby, and they were located in non-SNP-intensive areas; complex sequences with high content of continuous AT and GC were avoided.

[0079] Therefore, the Ty-3 gene is the only selection target at the 3723rd G / A site, the genotype of Ty-3 at this site is G, and the genotype of ty-3 is A. The 8914th A / G site of Ty...

Embodiment 2

[0090] Amplification of molecular markers

[0091] The materials used in this example are the high-generation homozygous disease-resistant material P808-1 (Ty-3 / Ty-3, Ty-3a / Ty-3a) bred by the applicant, and the susceptible material P802-1 (ty- 3 / ty-3, ty-3a / ty-3a) Field identification of these materials showed resistance and susceptibility (the same below), and the P808-1-P802-1-F1 hybrid prepared by crossing the two as parents Combined with disease-resistant materials (Ty-3 / ty-3, Ty-3a / ty-3a). Leaf tissues of plants were collected for genomic DNA extraction.

[0092] According to the instructions of the Plant DNA Isolation Kit (Chengdu Fuji Biotechnology Co., Ltd.), the genomic DNA in the leaves of the above materials was extracted, and the concentration of the extracted DNA solution was diluted to 50-100ng / μl, and stored at -20°C.

[0093] Configure the PCR reaction system according to the requirements of the KASP-PARMS kit (Wuhan Jingpeptide Biotechnology Co., Ltd.), with...

Embodiment 3

[0096] Detection and Analysis of Amplified Products

[0097] Use the software that comes with Applied Biosystems 7500Real-Time PCR System to perform genotyping and data analysis on PCR products, where the value on the ordinate is set to represent the FAM fluorescence signal value, and the value on the abscissa to represent the HEX fluorescence signal value.

[0098] Ty-3 genotyping results see figure 1 . Among them, the dot I is the amplification signal of the disease-resistant material P808-1, and only the FAM fluorescence signal is detected; the dot II is the amplification signal of the heterozygous disease-resistant material P808-1-P802-1-F1, which was detected simultaneously FAM fluorescence and HEX fluorescence; the dot at III is the amplification signal of the susceptible material P802-1, and only the HEX fluorescence signal is detected; the black dot at IV near the origin represents the amplification signal of the negative control (without adding DNA sample). increase...

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Abstract

The invention relates to the technical fields of molecular biology and tomato breeding, and specifically relates KASP primers for detecting genetic types of anti-disease genes Ty-3 and Ty-3a of a tomato yellow leaf curl virus disease and application of the KASP primers. The gene Ty-3 has an SNP site at a 3723th basic group, and the resistance of the gene Ty-3 is changed due to mutation of the basic group at the SNP site; and the gene Ty-3a has an SNP site at an 8914th basic group, and the resistance of the gene Ty-3a is changed due to mutation of the basic group at the SNP site. The inventionrespectively develops and designs a group of the KASP primers for genetic typing based on the SNP sites of the two genes as well as kits containing the KASP primers. Compared with the prior art, the KASP primers have the advantages that rapid, accurate and high-flux disease resistance authentication can be carried out on tomato plants in a seedling stage, and the workloads of artificial inoculation and field disease resistance authentication in the seedling stage are greatly reduced; and by virtue of the application of the KASP primers, the breeding efficiency of tomatoes can be improved, thebreeding cost can be saved, and the breeding progress can be accelerated.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and crop breeding, in particular to KASP primers for detecting tomato yellow leaf curl virus disease resistance genes Ty-3 and Ty-3a and applications thereof. Background technique [0002] Tomato (Lycopersicon esculentum Mill.) is an annual or perennial herbaceous plant belonging to the genus Solanaceae, which originated in South America. It is a worldwide vegetable crop and one of the main cultivated vegetables in my country. With the expansion of tomato cultivation area, the diversification of cultivation methods and the obstacles of continuous cropping, tomato diseases and insect pests are also intensifying. In recent years, tomato yellow leaf curl virus disease caused by tomato yellow leaf curl virus (Tomato yell ow leaf curl virus, TYLCV) has become one of the most serious diseases in tomato production worldwide. The upper leaves of the susceptible plants show yellow mosaic leaves...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156Y02A50/30
Inventor 康忱吴志明王鹏李亚栋田哲娟王美娥
Owner 河北省农林科学院经济作物研究所
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