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PCR marker related to cabbage dominant nuclear gene male sterility and application thereof

A male sterility and nuclear gene technology, applied in the field of PCR markers, can solve the problems of poor versatility of molecular markers, and achieve the effects of shortening the breeding cycle, high stability, and economical genotyping

Active Publication Date: 2019-07-09
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing molecular markers have poor versatility, so it is necessary to develop efficient and versatile markers

Method used

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  • PCR marker related to cabbage dominant nuclear gene male sterility and application thereof
  • PCR marker related to cabbage dominant nuclear gene male sterility and application thereof
  • PCR marker related to cabbage dominant nuclear gene male sterility and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Development of PCR markers for male sterility-assisted screening of dominant nuclear genes in cabbage

[0043] The inventors compared and analyzed the resequencing results of the normal inbred line "01-20", the heterozygous sterile material "DGMS01-20", and the homozygous sterile material "h-DGMS01-20", and detected positions C09:29,141,514 There is a 1bp deletion mutation, which was confirmed by first-generation sequencing. Position C09:29,141,514 indicates the physical position 29,141,514 on the chromosome of Brassica oleracea C09, which is determined based on the whole genome sequence of the cabbage inbred line "02-12", the whole genome of the cabbage inbred line "02-12" The sequence is described in the Bolbase database (reference genome website http: / / www.ocri-genomics.org / bolbase / index.html).

[0044] In order to verify the correlation between this variation and the male sterility of cabbage, the 50 bp sequence (SEQID NO.1) of its upstream and downstrea...

Embodiment 2

[0049] Example 2. Correlation verification between PCR markers developed by the present invention and cabbage male sterility

[0050] Step 1. Extract Genomic DNA

[0051] Genomic DNA from cabbage leaves of normal inbred lines, heterozygous sterile lines and homozygous sterile lines were extracted by CTAB (Hexadecyl trimethyl ammonium Bromide) method.

[0052] Step 2, using the primer set K9-6 to carry out genotyping detection on the cabbage material

[0053] BGI was entrusted to synthesize the three-segment primer of K9-6, and the amplification of the primer in cabbage was identified by KASP technology. When synthesizing primers, add a FAM tag sequence 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID NO.5) to the 5' end of the forward primer K9-6-X to obtain a forward primer F1; A HEX tag sequence 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID NO.6) was added to the 5' end of the primer K9-6-Y to obtain the forward primer F2.

[0054] Forward primer F1:

[0055] GAAGGTGACCAAGTTCATGCTCATGTAAGTGCAG...

Embodiment 3

[0067] Embodiment 3. Kit and its preparation

[0068] Forward primer F1, forward primer F2 and reverse primer in Example 2 were artificially synthesized, divided into centrifuge tubes, or prepared as KASP Primer mix and loaded into a kit. Put the reagent KASP Master mix for PCR into the kit.

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Abstract

The invention belongs to the technical field of molecular biological breeding, and relates to a PCR marker related to cabbage dominant nuclear gene male sterilityand application thereof. A method foridentifying or assisting in identifying the cabbage dominant nuclear gene male sterility is provided, it is detected that the genotype of to-be-detected cabbage C09 chromosome 29, 141, 514 positions is A / A or A / - or - / -, and the fertility of cabbage is determined according to the genotype of to-be-detected cabbage; the to-be-detected cabbage of the A / A genotype is normal; the to-be-detected cabbage of the A / - genotype is the heterozygous sterile; to-be-detected cabbage of the A / - genotype is homozygous sterile. By means of the PCR marker and a primer, the male sterility of the cabbage is assisted to be identified; the PCR marker has the advantages of being easy to operate, high in specificity and good in stability; the coincidence rate between identification results and field phenotypes reaches100%, early breeding of cabbage varieties can be achieved, and the PCR marker has great application prospects.

Description

technical field [0001] The invention relates to the technical field of molecular biological breeding, in particular to a PCR marker related to the male sterility of the cabbage dominant nuclear gene and its application. Background technique [0002] Cabbage belongs to the genus Brassica of the family Cruciferae (Cruciferae), and contains many varieties, among which varieties such as head cabbage, broccoli, cauliflower, and kohlrabi are important vegetable crops. Cabbage has strong adaptability and stress resistance, and has high nutritional and health value, so it is widely cultivated in China and even all over the world. Because cabbage has strong heterosis, most of the fine varieties of cabbage at home and abroad are first-generation hybrids. At present, domestic and foreign cabbage hybrids are mostly prepared by male sterile lines. [0003] In the spring of 1979, academician Fang Zhiyuan discovered the male sterile strain 79-399-3 in the natural population of head cabbag...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6858
CPCC12Q1/6858C12Q1/6895C12Q2600/13C12Q2600/156C12Q2531/113C12Q2537/161C12Q2563/107
Inventor 吕红豪韩风庆袁凯文方智远杨丽梅庄木张扬勇王勇
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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