Strong-winter winter rape (Brassica campestris) SOD enzyme molecular marker and QTL locus

A Chinese cabbage-type winter rapeseed and molecular marker technology, applied in the fields of molecular biology and genetic breeding, can solve the problem that the research on molecular markers and QTL mapping has not yet been reported, and achieve the advantages of speeding up the breeding process, saving production costs and improving screening efficiency. Effect

Active Publication Date: 2015-11-11
GANSU AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, there have been many studies on SOD enzyme activity in Chinese cabbage-typ

Method used

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  • Strong-winter winter rape (Brassica campestris) SOD enzyme molecular marker and QTL locus
  • Strong-winter winter rape (Brassica campestris) SOD enzyme molecular marker and QTL locus
  • Strong-winter winter rape (Brassica campestris) SOD enzyme molecular marker and QTL locus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Construction of isolated populations of strong winter Chinese cabbage-type winter rapeseed and determination of SOD enzyme activity.

[0033] The specific construction of the isolated population used in this embodiment is as follows:

[0034] (1) In August 2010, multi-generation bagging and self-crossing of the parent rapeseed 'Longyou 7' with strong winter resistance and strong winter resistance and rapeseed 'Longyou 9' with strong winter resistance were used to configure the hybrid combination, harvested in May 2011 F1 generation seeds.

[0035] (2) F1 seeds were sown in August 2011, and bagged and self-crossed in April 2012 to obtain F2 generation seeds.

[0036] The F2 generation seeds were sown in August 2012. At the seedling stage, 103 plants were randomly selected to be listed and marked, and fresh young leaves were collected in early November, brought back to the laboratory in an ice box and stored in a -70°C ultra-low temperature refrigerator. SOD...

Embodiment 2

[0037] Example 2: Extraction of total DNA from leaves of parents and F2 generation segregation populations.

[0038] Total DNA was extracted from the leaves by the CTAB method, and the specific steps were as follows:

[0039] (1) Pick healthy young leaves as materials for extracting rapeseed genomic DNA, wash them with distilled water, and dry them with paper.

[0040] (2) Take about 0.5g of fresh leaves and put them in a mortar, add liquid nitrogen, grind them quickly until they are whitish powdery, and put them into a 2ml centrifuge tube immediately.

[0041] (3) Add 700ul of preheated 2×CTAB extraction buffer to infiltrate the powder and invert the centrifuge tube to fully disperse the powder. Place in a water bath at 65°C for 60 minutes, during which time gently mix 2-3 times.

[0042] (4) Take out the centrifuge tube, cool to room temperature, add 700ul of chloroform / isoamyl alcohol (24 / 1), gently invert to mix, and let stand for 10min.

[0043] (5) Centrifuge at 13000r...

Embodiment 3

[0051] Example 3: Source, synthesis and polymorphism screening of SSR and InDel primers.

[0052] (1) 315 pairs were uniformly selected from the rapeseed microsatellite primer sequences published on www.UKcrop.net and all primers on 10 chromosomes published on BrassicaDatabase website http: / / brassicadb.org / . Among them, there are 51 pairs of SSR primers and 264 pairs of InDel primers. Primers were synthesized by Shanghai Bioengineering Technology Co., Ltd.

[0053] (2) Randomly select 6 strains of DNA from each parent and mix them in equal amounts, and use them as templates for screening primers.

[0054] (3) PCR reaction system

[0055] The PCR reaction system is 10ul:

[0056] MixTaq enzyme 6ul

[0057] Upper primer 0.5ul

[0058] Lower primer 0.5ul

[0059] ddH2O2ul

[0060] Template: 1ul

[0061] (4) PCR amplification program: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 50s, annealing at 55-60°C for 50s (annealing temperature decreased or incre...

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Abstract

The invention discloses a strong-winter winter rape (Brassica campestris) SOD enzyme molecular marker and a QTL locus. A screening process includes following steps: (1) performing hybridization between a multi-generation bagged-selfing parent strong-winter super-cold-winder-resistant rape 'Longyou #7' and strong-cold-winder-resistant rape 'Longyou #9' to construct an F2-generation segregation population; (2) extracting leaf DNA of the two parents and the F2-generation segregation population through the CTAB method; (3) performing polymorphic primer screening to the two parent DNA through SSR and InDel primers; (4) obtaining genotype data through the F2-generation segregation population to construct a genetic linkage map and carry out QTL analysis; and (5) obtaining two QTL loca of SOD enzyme of the strong-winter winter rape (Brassica campestris), which are respectively named Qsod.gsau-6A and Qsod.gsau-7A. Four molecular markers, which are linked with the QTL loca of the SOD enzyme of the strong-winter winter rape (Brassica campestris), are respectively named Ra1-F06, Ra2-D04, 8C0108 and Ol12E03.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and genetic breeding, more specifically relates to the QTL site of the SOD enzyme activity gene of strong winter Chinese cabbage type winter rapeseed, and also relates to the QTL site of the SOD enzyme activity gene of strong winter Chinese cabbage type winter rape. molecular marker. Background technique [0002] Brassica napus has a long history of cultivation in my country and is rich in germplasm resources. It is one of the important oil crops in my country. But the Chinese cabbage-type rapeseed is mainly spring-type, and the germplasm of strong-winter-type rapeseed is lacking, especially the varieties of strong-winter-type rape that are suitable for planting in the northern arid and cold regions. The climate in northern my country is very cold, and winter rapeseed has excellent cold resistance to survive the winter safely. Therefore, breeding of cold-resistant varieties is the key t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 孙万仓杨刚曾秀存刘自刚武军艳方彦李学才孔德晶赵艳宁米超
Owner GANSU AGRI UNIV
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