Cucumber male sterility gene related SNP (Single Nucleotide Polymorphism) marker and application

A technology of male sterility and cucumber, which is applied in the field of molecular genetic breeding, can solve the problems of no development of cucumber male sterility genes, rare discovery of cucumber male sterility genes, and inability to finely position genes, and achieve low cost and cycle time. Short, low-cost effects

Active Publication Date: 2016-03-23
TIANJIN RES INST OF VEGETABLE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The male sterility gene has been found in many crops, but in the literature reports at home and abroad, there are very few reports about the cucumber male sterility gene, and there is no development of any markers related to the cucumber male sterility gene.
[0003] In the spring of 2012, we first discovered a male sterile mutant of cucumber in a high-generation inbred line. It was verified that the sterility trait is controlled by a recessive gene in the nucleus. Male sterility has been reported in many other crops, but male sterility in cucumbers is rare. education report
In the past, the method of locating a new gene usually uses the primers developed by the existing molecular markers such as RAPD, SCAR, AFLP, SSR, etc., in the genome of plants with different traits, a large number of amplification, gel running, and screening of different bands require It consumes a lot of manpower, material resources and time, and it can only preliminarily locate the gene in a very wide area, and cannot precisely locate a gene

Method used

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  • Cucumber male sterility gene related SNP (Single Nucleotide Polymorphism) marker and application
  • Cucumber male sterility gene related SNP (Single Nucleotide Polymorphism) marker and application
  • Cucumber male sterility gene related SNP (Single Nucleotide Polymorphism) marker and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The acquisition of SNP molecular markers related to cucumber male sterility traits comprises the following steps:

[0039] (1) The male sterile line "YL-5" was used as the female parent and the distant variety "D37-1" was used as the male parent to obtain a hybrid F1, and the F2 generation population was obtained by selfing the hybrid F1, and the F2 generation segregation population There are two phenotypes, male fertile and male sterile (see Henan Agricultural Sciences, 2012, (41) 11:14-18 for methods, Hou Leilei).

[0040] (2) Genomic DNA of each individual plant of the male parent, female parent, and F2 generation was extracted by the CTAB method to form 4 mixed pools, and the DNA of these 4 mixed pools was resequenced, and the original reads obtained by sequencing (paired-end sequence ) for quality assessment and filtering to obtain clear reads, which were compared with the reference genome sequence, and based on the comparison results, the GATK software package was...

Embodiment 2

[0044] Super-BSA technology based on whole genome resequencing

[0045] (1) Genomic DNA was extracted from 5 male and 5 female plants, 50 sterile and 50 fertile plants in the F2 population by CTAB method, and mixed in equal amounts to form 5 mixed pools.

[0046] (2) Technical route

[0047] Extract genomic DNA, use Covaris for random fragmentation, electrophoresis to recover DNA fragments of the required length (0.2-5Kb), add adapters, perform cluster preparation (Solexa) or E-PCR (SOLiD), and finally use Paired-End (Solexa ) or Mate-Pair (SOLiD) method to resequence the insert. The result of resequencing is to scan and detect gene sequence differences and structural variations related to infertility traits at the genome-wide level, so as to realize genetic evolution analysis and prediction of important trait candidate genes.

Embodiment 3

[0049] Application of SNP markers related to male sterility traits

[0050] The primer pair Chr3-320626[T / G]-1-2 and Chr3-320626[T / G]-3-2 were used to amplify and analyze the genomic DNA of the double-affinity F2 generation respectively, and the PCR reaction system was as follows:

[0051] 1×PCR buffer, 1.35mMMgCl2, 0.08mMdNTPs, 1.0UTaq DNA polymerase (both purchased from MBIFermentas, Lithuania company), 100ngDNA, forward and reverse primers 0.45μM each, ddH2O supplemented to a final volume of 20μl. The thermal cycle parameters are: 95°C for 3min; 95°C for 30sec, renaturation temperature for 58°C for 30sec, 72°C for 1min, 35 cycles; 72°C for 5min, 1 cycle; 4°C storage, the reaction is completed on the PTC-ALD1244 PCR instrument . Both materials were performed in duplicate. The amplified product was detected by 1.5% agarose gel electrophoresis on a horizontal electrophoresis tank, using 1×TAE buffer (0.04MTris-acetate, 0.001MEDTA, pH8.0), voltage 8V / cm, electrophoresis for 3...

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Abstract

The invention discloses a cucumber male sterility gene related SNP (Single Nucleotide Polymorphism) marking method and application. The marking method comprises the steps of taking a YL-5 male sterile line as a female parent to hybridize with a distant related variety D37-1, so as to obtain a hybrid F1, performing selfing by the hybrid F1 to obtain an F2 population, and two phenotypes of male sterility and male fertility exist in an F2 segregation population, then utilizing a method of combination of re-sequencing and BSA, screening an SNP locus unrelated with the male sterility, and developing a male sterility gene related molecular marker. The marking method disclosed by the invention has the advantages of short period, low cost and high efficiency. Meanwhile, an important development approach is provided for molecular markers for molecular breeding, phyletic evolution and germplasm resource identification.

Description

technical field [0001] The invention relates to the technical field of molecular genetic breeding, in particular to the marker and application of SNP related to cucumber male sterility gene. Background technique [0002] Cucumber (Cucumissativus L) is one of the most important vegetable crops in my country. In the key project of "Key Technology Integration and Industrialization of Male Sterile Breeding of Main Vegetable Crops and Industrialization of Improved Seeds" carried out by the "Eleventh Five-Year" National Science and Technology Support Program of the Ministry of Science and Technology, cucumber was Listed as an important research project. Male sterility genes have been found in many crops, but in the literature reports at home and abroad, there are very few reports about cucumber male sterility genes, let alone the development of any markers related to cucumber male sterility genes. [0003] In the spring of 2012, we first discovered a male sterile mutant of cucumbe...

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 韩毅科杜胜利王仙云高尚魏爱民陈正武刘楠赵峰月
Owner TIANJIN RES INST OF VEGETABLE
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