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SSR (Simples sequence repeats) and InDel (insertion/deletion) molecular marker primer linked with brassica campestris orange head gene Br-or, and application thereof

A br-indel and molecular marker technology, applied in the fields of vegetable breeding and molecular genetics, can solve problems such as difficulty in development, and achieve the effects of speeding up the breeding process, stable amplification and convenient detection.

Inactive Publication Date: 2013-05-15
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

InDel markers are developed based on indel differences between homologous sequences, which is difficult to develop for crops with unknown genome sequences

Method used

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  • SSR (Simples sequence repeats) and InDel (insertion/deletion) molecular marker primer linked with brassica campestris orange head gene Br-or, and application thereof
  • SSR (Simples sequence repeats) and InDel (insertion/deletion) molecular marker primer linked with brassica campestris orange head gene Br-or, and application thereof
  • SSR (Simples sequence repeats) and InDel (insertion/deletion) molecular marker primer linked with brassica campestris orange head gene Br-or, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1: Application of Br-InDel (1) Primer

[0103] (1) Genomic DNA of cabbage was extracted by CTAB method, and the extraction steps were as follows:

[0104] A. Take 0.2g of fresh young leaves with the main veins removed, stuff them into a 1.5ml centrifuge tube, put them into liquid nitrogen for quick freezing, and quickly grind them to powder with a clean plastic pestle washed with 75% alcohol by mass fraction;

[0105] B. Add 700μl of CTAB extract (CTAB: 2%, Tris-HCl (pH8.0): 100mmol / L, EDTA: 20mmol / L, NaCl: 1.4mol / L) preheated at 65°C to the centrifuge tube, Then add 8 μl of β-mercaptoethanol and mix quickly;

[0106] C. Then put the centrifuge tube into a 65°C water bath, shake it every 5 minutes, and keep the water bath for 30 minutes;

[0107] D. Take out the centrifuge tube, add an equal volume of mixture of phenol, chloroform and isoamyl alcohol (phenol: chloroform: isoamyl alcohol = 25:24:1), shake well for 15 minutes, then centrifuge at 12000r / min for 1...

Embodiment 2

[0135] Embodiment 2: the application of Br-SSR6 primer

[0136] (1) Using the CTAB method to extract the genomic DNA of cabbage, the extraction steps are as follows:

[0137]A. Take 0.2g of fresh young leaves with the main veins removed, stuff them into a 1.5mL centrifuge tube, put them into liquid nitrogen for quick freezing, and quickly grind them to powder with a clean plastic pestle cleaned with 75% alcohol;

[0138] B. Add 700 μL of CTAB extract (CTAB: 2%, Tris-HCl (PH8.0): 100 mmol / L, EDTA: 20 mmol / L, NaCl: 1.4 mol / L) preheated at 65 °C to the centrifuge tube, Then add 8 μL β-mercaptoethanol and mix quickly;

[0139] C. Then put the centrifuge tube into a 65°C water bath, shake it every 5 minutes, and keep the water bath for 30 minutes;

[0140] D. Take out the centrifuge tube, add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) mixture, shake well for 15 minutes, then centrifuge at 12000r / min for 10 minutes at room temperature;

[0141] E. Transfer t...

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Abstract

The invention discloses an SSR (simples sequence repeats) and InDel (insertion / deletion) molecular marker primer linked with brassica campestris orange head gene Br-or and an application of the molecular marker primer. The marker primer is obtained by the steps of: based on the genome DNA (deoxyribonucleic acid) of orange brassica campestris and normal white brassica campestris and F2S4 separation group as a template, carrying out polymorphism primer screening to orange head and white head single strain DNA mixed tank in F2S4 by using 480 pairs of SSR and InDel primer pairs, then analyzing single strain of F2S4 group, and screening the molecular marker linked with the brassica campestris orange head gene Br-or to finally obtain fifteen Br-SSR and three Br-InDel molecular markers linked with the Br-or gene, wherein a molecular genetic map of the brassica campestris orange head gene Br-or is created on the ninth link group (A09). According to the link analysis, the genetic distance of two markers most tightly linked with both sides of the Br-or gene are 0.11cM and 0.79cM; and the molecular marker primer has the advantages of being convenient for detection, stable in amplification, and high in repeatability and accuracy, and thus a basis is provided to the molecule assistant selective breeding of brassica campestris orange head character and the breeding process is accelerated.

Description

technical field [0001] The invention belongs to the fields of vegetable breeding and molecular genetics, and specifically relates to the development and application of SSR and InDel molecular marker primers linked with Chinese cabbage orange leafy gene Br-or. Background technique [0002] Chinese cabbage (Brassica campestris L.ssp. pekinensis) belongs to Brassicaceae Brassica Brassica subspecies, is one of the main vegetable crops originated in my country, and is closely related to people's daily life. With the improvement of living standards, people's requirements for the quality of Chinese cabbage are increasing day by day. Therefore, Chinese cabbage quality breeding has been paid more and more attention by domestic and foreign scholars and breeders. [0003] The applicant began to breed orange Chinese cabbage in 1993, and the Chinese cabbage first-generation hybrids "Jinguan 1" and "Jinguan 2" orange Chinese cabbage passed the examination and approval of Shaanxi Province ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 张鲁刚张俊祥马晓彤李会霞张明科惠麦侠
Owner NORTHWEST A & F UNIV
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