KASP primers for detecting tomato yellow leaf curl virus disease-resistant gene Ty-1 and application of KASP primers

A technology of tomato yellowing and curved leaves and disease-resistant genes, applied in the fields of molecular biology and crop breeding, can solve the problems of undiscovered KASP markers, etc., and achieve the effects of improving breeding efficiency, saving breeding costs, and reducing workload

Pending Publication Date: 2020-11-20
河北省农林科学院经济作物研究所
View PDF5 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no KASP marker applied to the

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • KASP primers for detecting tomato yellow leaf curl virus disease-resistant gene Ty-1 and application of KASP primers
  • KASP primers for detecting tomato yellow leaf curl virus disease-resistant gene Ty-1 and application of KASP primers
  • KASP primers for detecting tomato yellow leaf curl virus disease-resistant gene Ty-1 and application of KASP primers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Development of KASP markers

[0068] By comparing and analyzing the sequences of the two alleles of Ty-1 disease resistance and disease susceptibility, the alleles of Ty-1 disease resistance were named Ty-1 / Ty-1, and the alleles of Ty-1 disease resistance were named ty-1 / ty-1; and both of them have a SNP site at the 3461bp position. This SNP site meets the requirements for KASP marker development: the genotypes are different at 3461 bp; there are no other SNP sites nearby, and it is located in a non-SNP-intensive area; and complex sequences with high content of continuous AT and GC are avoided.

[0069] Therefore, the 3461st T / C site is used as the only selection target; the genotype of Ty-1+ is T at this site, and the genotype of Ty-1 is C.

[0070] KASP primers were designed according to the above SNP sites, and the primer sequences were:

[0071] No. 3461 T / C:

[0072] Forward primer 1: 5'- GAAGGTGACCAAGTTCATGCT TCAATGAACCTTCATCTCCATGT-3';

[0073] Forward prim...

Embodiment 2

[0078] Amplification of molecular markers

[0079] The materials used in this example are the high-generation homozygous disease-resistant material P808-1 (Ty-1 / Ty-1) and the susceptible material P802-1 (ty-1 / ty-1) selected by the applicant. Field identification shows resistance and susceptibility (the same below), and P808-1-P802-1-F1 hybrid disease-resistant material (Ty-1 / ty-1) prepared by crossing the two as parents. Leaf tissues of plants were collected for genomic DNA extraction.

[0080] 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.

[0081] Configure the PCR reaction system according to the requirements of the KASP-PARMS kit (Wuhan Jingpeptide Biotechnology Co., Ltd.), with a total reaction volume of 10 μl, including: 2X PARMS PCR Mix: 5 μl; DNA ex...

Embodiment 3

[0084] Detection and Analysis of Amplified Products

[0085] 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.

[0086] 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 sign...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to the technical field of molecular biology and crop breeding, in particular to KASP primers for detecting typing of a tomato yellow leaf curl virus disease-resistant gene Ty-1 and an application of the KASP primers. The tomato yellow leaf curl virus disease-resistant gene Ty-1 has an SNP site at the position of the 3461st < base, and the mutation of the base of the SNP sitecauses the change of the resistance of the Ty-1 gene. A group of KASP primers capable of being used for genotyping and a kit containing the KASP primers are designed on the basis of the SNP site. TheKASP primers and the kit can be used for identifying whether tomatoes contain the tomato yellow leaf curl virus disease-resistant gene Ty-1 or not, and distinguishing tomatoes which contain Ty-1 and resist tomato yellow leaf curl virus diseases from tomatoes which do not contain the Ty-1 and are infected with the tomato yellow leaf curl virus diseases. Compared with the prior art, rapid, accurateand high-throughput disease resistance identification can be performed on tomato plants in the seedling stage, the workload of manual inoculation in the seedling stage and field disease resistance identification is reduced, the tomato breeding efficiency can be improved through the application of the KASP primers, the breeding cost is saved, and the breeding process is accelerated.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and crop breeding, in particular to a KASP primer for detecting tomato yellow leaf curl virus disease resistance gene Ty-1 and an application 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 yellow 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 at the b...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156C12Q2600/13
Inventor 吴志明王鹏李亚栋康忱田哲娟王洪乐邙光伟杨超沙王美娥
Owner 河北省农林科学院经济作物研究所
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap