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caps molecular marker, primers, detection method, detection kit and application based on tomato gray leaf spot resistance gene sm

A technology for tomato gray leaf spot and a detection method, which is applied in the field of plant molecular biology, can solve the problems of unstable molecular marker band type and weak specificity, and achieves the effects of high stability, strong specificity and high precision

Active Publication Date: 2022-01-28
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem that the existing molecular markers linked to the tomato gray leaf spot resistance gene have unstable band patterns and low specificity, the present invention provides CAPS molecular markers, primers, Detection method, detection kit and application

Method used

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  • caps molecular marker, primers, detection method, detection kit and application based on tomato gray leaf spot resistance gene sm
  • caps molecular marker, primers, detection method, detection kit and application based on tomato gray leaf spot resistance gene sm
  • caps molecular marker, primers, detection method, detection kit and application based on tomato gray leaf spot resistance gene sm

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Effect test

Embodiment 1

[0034] This example provides a CAPS molecular marker based on tomato gray leaf spot resistance gene Sm and its amplification primers.

[0035] The tomato gray leaf spot resistance gene Sm is located on chromosome 11, and the target region of the marker is located on the entire chromosome 11 in this embodiment. Using the genome resequencing data of tomato gray leaf spot disease-resistant and susceptible parents, the nucleotide sequences of the parents were compared by DNAMAN software, and the SNP sites were verified, and finally the nucleotides of tomato gray leaf spot disease-resistant materials were found The sequence has a SNP site located at the restriction site of TaqI.

[0036] like figure 1 As shown, there is a TaqI cleavage site in the nucleotide sequence of the P1 resistant parent, but there is no TaqI cleavage site in the nucleotide sequence of the P2 susceptible parent.

[0037] Select the base sequence of about 500bp-800bp before and after the SNP site located at ...

Embodiment 2

[0040] The present embodiment provides a kind of detection method of tomato gray leaf spot resistance gene Sm, comprises the steps:

[0041] Step 1, take the genomic DNA of tomato material to be tested as template, utilize the amplification primer of the CAPS molecular mark that embodiment 1 provides to carry out PCR amplification, the nucleotide sequence of described amplification primer is:

[0042] Forward primer sequence: 5'-GTCCGAGCAACATAGCTCCC-3',

[0043] Reverse primer sequence: 5'-ATGGACCAACCTTGCAAACG-3';

[0044] The reaction system for PCR amplification is: 1 μL forward primer, 1 μL reverse primer, 1 μL template DNA, 10 μL 2×TaqPCR StarMix, 7 μL dd H 2 O.

[0045] The amplification program of PCR amplification was as follows: firstly, pre-denaturation at 94°C for 2 min; secondly, denaturation at 94°C for 30 s, annealing at 57.5°C for 30 s, extension at 72°C for 40 s, and 35 cycles; finally, extension at 72°C for 5 min; storage temperature at 4°C.

[0046] Step 2:...

Embodiment 3

[0048] This embodiment provides a tomato gray leaf spot resistance gene detection kit based on the CAPS molecular marker of the tomato gray leaf spot resistance gene Sm, including the tomato gray leaf spot resistance gene Sm based on the test kit. The amplification primer of CAPS molecular mark, the nucleotide sequence of described amplification primer is:

[0049] Forward primer sequence: 5'-GTCCGAGCAACATAGCTCCC-3'

[0050] Reverse primer sequence: 5'-ATGGACCAACCTTGCAAACG-3'.

[0051] The kit also includes Taq DNA polymerase, dNTPs, MgCl 2 , reaction buffer, restriction endonuclease TaqI, TaqI Buffer, 0.1% BSA and sterilized water.

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Abstract

The invention relates to a CAPS molecular marker, primer, detection method, detection kit and application based on tomato gray leaf spot resistance gene Sm, and belongs to the technical field of plant molecular biology. In order to solve the problems of unstable band type and low specificity in the molecular markers linked to the existing tomato gray leaf spot resistance gene, the present invention provides a CAPS molecular marker based on the tomato gray leaf spot resistance gene Sm and its expansion. Amplifying primers, including detection methods for PCR amplification and enzymatic cleavage of amplification products using the amplification primers, and detection kits containing the amplification primers. The CAPS molecular marker band type provided by the invention is stable, easy to distinguish, high in accuracy and strong in specificity; the detection method is simple in operation, high in accuracy of amplification results and low in cost, and has high stability in field disease resistance identification , the coincidence rate of genotype and field phenotype is as high as 90.19%, which can be used to breed tomato gray leaf spot resistant germplasm resources.

Description

technical field [0001] The invention belongs to the technical field of plant molecular biology, and in particular relates to a CAPS molecular marker, a primer, a detection method, a detection kit and an application based on tomato gray leaf spot resistance gene Sm. Background technique [0002] Tomato is one of the important vegetables widely grown in the world, and it is often harmed by diseases and insect pests during its growth. Tomato gray leaf spot (Tomato gray leaf spot) is a popular disease in recent years. The pathogen of the disease is Stemphylium fungus, which is a member of the subphylum Hyphomycetes and can cause a variety of vegetable diseases. Tomato gray leaf spot occurs worldwide, especially in warm and humid regions. The outbreak of tomato gray leaf spot is serious, which can lead to a 20% to 80% reduction in tomato yield, affecting the yield and quality of tomato. [0003] At present, only the Sm gene has been discovered and available for resistance to gr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/13C12Q2600/156C12Q2531/113C12Q2521/301
Inventor 杨欢欢姜景彬刘明月许向阳李景富赵婷婷张贺孙要光王鹤璇
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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