Special primer for identifying gummy stem blight resistance of muskmelon and molecular marking method

A molecular marker, vine blight technology, applied in the field of molecular biology, can solve the problems of inaccurate identification of vine blight resistance, heavy workload, etc., and achieve the goal of improving breeding efficiency, speeding up breeding, saving time and labor costs. Effect

Inactive Publication Date: 2017-05-24
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the problems existing in the current muskmelon blight-resistant breeding work, such as the artificial inoculation of pathogenic bacteria, the identification of the resistance to vine blight is inaccurate and the shortcomings of heavy workload, the present invention provides a method for identifying the resistance to vine blight of muskmelon. Sexual SSR molecular markers and their special primers, through polymerase chain reaction (Polymerase Chain Reaction, PCR), in the early stage of muskmelon plants, the purpose of screening muskmelon materials that are resistant to vine blight and not resistant to vine blight can be completed. Accurate identification, not limited by disease conditions, convenient to obtain materials, etc.

Method used

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  • Special primer for identifying gummy stem blight resistance of muskmelon and molecular marking method
  • Special primer for identifying gummy stem blight resistance of muskmelon and molecular marking method
  • Special primer for identifying gummy stem blight resistance of muskmelon and molecular marking method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: the extraction of test material and genomic DNA

[0028] Melon material for test

[0029] All of them are melon high-generation inbred strain materials, which are the offspring of the melon material PI482398 and Baipicrisp, and the hybridization of the two. Among them, PI482398 is derived from the National Plant Seed Resource System of the United States Department of Agriculture. Melon planting resources; Baipicris is native to Xinjiang, and it is a high-quality and conventional variety that is susceptible to vine blight. Crossbreed PI482398 and Baipicria as the female parent and male parent respectively, and select and breed among the hybrid offspring to obtain high-generation inbred lines A, B, C, D, E, F, G with different resistance to vine blight , H, used together with PI482398 and Baipicui to detect the resistance of muskmelon materials to vine blight by molecular markers.

[0030] Extraction of Genomic DNA from the Tested Melon Materials

[0031]...

Embodiment 2

[0032] Example 2: Screening of molecular markers and design of special primers

[0033] Using high-throughput sequencing technology, two muskmelon lines identified as PI482398, which are resistant to creeping blight and Baipicriu, which are susceptible to creeping blight, were resequenced and compared, and the obtained SSR molecular markers were compared with those of Previously, the germplasm resources of muskmelon resistant to creeping blight and related molecular markers were compared to find markers at similar chromosomal positions. At both ends of the molecular markers, special primers were designed. The length of the primers was selected to be 20-26 bp, and the annealing temperature was 58-60 °C, the GC content is between 40% and 60%.

Embodiment 3

[0034] Embodiment 3: PCR amplification obtains target fragment

[0035] Using the genomic DNA of the sample to be tested extracted in Example 1 as a template and the molecular marker-specific primers designed in Example 2 as primers, perform PCR amplification to obtain the target fragment of the molecular marker. The reaction system of PCR amplification adopts the reaction system of 10 μ L, and the composition of described mixed solution is as follows:

[0036]

[0037] The reaction conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, denaturation at 57°C for 30 s, extension at 72°C for 30 s, 35 cycles; final extension at 72°C for 5 min; storage at 4°C.

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Abstract

The invention provides a special primer for identifying gummy stem blight resistance of muskmelon and a molecular marking method. Specific to the problems existing in gummy stem blight resisting breeding work of the muskmelon, such as the defects of inaccuracy and large workload in manual inoculation of pathogenic bacteria and identification of gummy stem blight resistance, the method comprises the following steps: extracting a genome DNA (Deoxyribonucleic Acid) of a muskmelon sample to be detected to serve as a template; performing polymerase chain reaction (PCR) amplification by using a primer special for molecular marking; detecting an amplification product through electrophoresis. By adopting the SSR (Simple Sequence Repeat) molecular marker for identifying the gummy stem blight resistance and the special primer thereof, the aim of screening muskmelon materials resisting to the gummy stem blight and not resisting to the gummy stem blight can be fulfilled at the early growth period of muskmelon plants, and the molecular marker and the special primer have the advantages of accurate identification, freeness from limitations by infection conditions, adoption of readily-available raw materials and the like. Muskmelon auxiliary breeding is performed, so that the special primer has the advantages of definite target selection, saving in breeding time and labor cost, and increase of the breeding efficiency.

Description

technical field [0001] The invention relates to a special primer and a molecular marker method for identifying the resistance of muskmelon blight, and belongs to the technical field of molecular biology. Background technique [0002] Melon (Cucumis melo L.) is a herbaceous plant belonging to Cucurbitaceae Cucurbitaceae. Its fruit is sweet, fragrant and crisp, and is deeply loved by consumers. It is an important economic crop in my country. Various diseases and insect pests have a serious impact on the growth and development of melons, reducing the yield and quality of melons. In order to prevent the occurrence of diseases and insect pests, the production costs of melons have been increased, and the ecological environment has been destroyed. [0003] Gummy stem blight (GSB) is a worldwide soil-borne fungal disease caused by the ascomycete Didymelabryoniae (Auersw) Rehm. Disease, pathogenic bacteria without the discovery of physiological races. The disease causes great harm ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 华德平刘莉付金玉李三培杨旭辉
Owner TIANJIN UNIV
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