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Molecular markers and qtl sites of pod enzymes in strong winter Chinese cabbage type winter rapeseed

A cabbage-type winter rapeseed, molecular marker technology, applied in the field of molecular biology and genetic breeding, can solve the problems that the research on molecular markers and QTL positioning has not been reported yet, and achieve the goals of accelerating the breeding process, improving screening efficiency, and saving production costs Effect

Active Publication Date: 2018-08-31
GANSU AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, there have been many studies on POD enzyme activity in Chinese cabbage-type winter rapeseed, but there have been no reports on its molecular markers and QTL mapping.

Method used

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  • Molecular markers and qtl sites of pod enzymes in strong winter Chinese cabbage type winter rapeseed
  • Molecular markers and qtl sites of pod enzymes in strong winter Chinese cabbage type winter rapeseed
  • Molecular markers and qtl sites of pod enzymes in strong winter Chinese cabbage type winter rapeseed

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Construction of strong winter cabbage type winter rapeseed population and determination of POD enzyme activity.

[0027] The specific structure of the segregated population used in this embodiment is as follows:

[0028] (1) In August 2010, the hybrid combination of strong winter super-resistant winter rapeseed'Longyou 7'and strong winter rapeseed'Longyou 9', which were self-bred with multi-generation bagging, was harvested in May 2011 F1 generation seeds.

[0029] (2) F1 seeds were sown in August 2011 and self-bred in bagging in April 2012 to obtain F2 seeds.

[0030] F2 generation seeds were sown in August 2012. At the seedling stage, 103 plants were randomly selected for listing, and fresh young leaves were collected in early November, brought back to the laboratory in an ice box and stored in an ultra-low temperature refrigerator at -70°C. The guaiacol colorimetric method was used to determine the POD enzyme activity. The results showed that the POD enzyme act...

Embodiment 2

[0031] Example 2: Extraction of total DNA from leaves of parental and F2 generation separated populations.

[0032] The CTAB method is used to extract total DNA from leaves, and the specific steps are as follows:

[0033] (1) Pick healthy young leaves as materials for extracting rape genomic DNA, wash them with distilled water, and blot them dry with paper.

[0034] (2) Take about 0.5g of fresh leaves and place them in a mortar, add liquid nitrogen, quickly grind them to whitish powder, and immediately put them into a 2ml centrifuge tube.

[0035] (3) Add 700ul of preheated 2×CTAB extraction buffer solution to soak the powder and invert the centrifuge tube to fully disperse the powder. In the 65°C water bath for 60 minutes, gently mix 2-3 times.

[0036] (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 10 min.

[0037] (5) Centrifuge at 13000rpm for 10min.

[0038] (6) Take 600ul of the supe...

Embodiment 3

[0045] Example 3: SSR and InDel primer source, synthesis and polymorphism screening.

[0046] (1) From the rape microsatellite primer sequence published on www.UK crop.net and all the primers on the 10 chromosomes published on the Brassica Database website http: / / brassicadb.org / , 315 pairs were uniformly selected. Among them, 51 pairs of SSR primers and 264 pairs of InDel primers. The primers were synthesized by Shanghai Bioengineering Technology Co., Ltd.

[0047] (2) Randomly select 6 strains of DNA from each parent and mix them in equal amounts as the template for screening primers.

[0048] (3) PCR reaction system

[0049] The PCR reaction system is 10ul:

[0050] Mix Taq enzyme 6ul

[0051] Upper primer 0.5ul

[0052] Lower primer 0.5ul

[0053] ddH2O 2ul

[0054] Template: 1ul

[0055] (4) PCR amplification program: 94°C pre-denaturation 4min; 94°C denaturation 50s, 55-60°C renaturation 50s (annealing temperature decrease or increase 0.5°C for each cycle), 72°C extension 50s, 35 cycl...

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Abstract

The invention discloses molecular markers and a QTL site of POD enzyme of strong winter Brassia campestris L. Screening comprises the following steps: (1) constructing an F2-generation segregation population through hybridization of parents obtained through multi-generation bagged self-crossing, i.e., strong winter superstrong-cold-resistance winter rape Longyou No. 7 and strong-cold-resistance winter rape Longyou No. 9; (2) extracting leaf DNAs of the two parents and the F2-generation segregation population by using a CTAB method; (3) carrying out polymorphism primer screening on the DNAs of the two parents by using SSR and InDel primer pairs; (4) constructing a genetic linkage map according to obtained genotype data of the F2-generation segregation population and carrying out QTL analysis; and (5) naming the obtained one QTL site of the POD enzyme of strong winter Brassia campestris L. as Qpod. gsau-10A and acquiring the two molecular marks linked with the QTL site of the POD enzyme of strong winter Brassia campestris L., i.e., O112E03 and BrID90115.

Description

Technical field [0001] The invention belongs to the technical field of molecular biology and genetic breeding, and specifically relates to a strong winter cabbage type winter rape POD enzyme activity gene QTL locus, and also relates to a strong winter cabbage type winter rape POD enzyme activity gene QTL locus linkage Molecular markers. Background technique [0002] Cabbage-type rape has a long history of cultivation in my country and has abundant germplasm resources. It is one of the important oil crops in my country. However, the cabbage type rape is mainly spring, and the strong winter cabbage type winter rapeseed is lacking in germplasm, especially the strong winter rape variety that is suitable for planting in northern arid and cold regions. The climate in northern my country is severely cold, and winter rape has excellent cold resistance to survive the winter safely. Therefore, the selection of cold-resistant varieties is the key to the development of winter rape in northe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
Inventor 孙万仓杨刚刘自刚曾秀存武军艳方彦李学才孔德晶刘林波侯献飞
Owner GANSU AGRI UNIV
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