CN104877994B - Strong winter habit Chinese cabbage type winter rape malonaldehyde MDA molecular labeling and QTL positioning - Google Patents
Strong winter habit Chinese cabbage type winter rape malonaldehyde MDA molecular labeling and QTL positioning Download PDFInfo
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Abstract
The invention discloses strong winter habit Chinese cabbage type winter rape malonaldehyde MDA molecular labeling and QTL site, screening step includes: (1) using mostly for superpower cold-resistant winter rape ' Gansu Province oil 7 of the strong winter habit of parent of bagging selfing ' and strong cold-resistant winter rape ' Gansu Province oil 9 ' hybridize building F2 for segregating population；(2) two parents and F2 are extracted for the leaf DNA of segregating population using CTAB method；(3) polymorphism primer screening is carried out using two parent DNA of SSR and InDel primer pair；(5) genotype data is obtained by F2 segregating population and constructs genetic linkage map and qtl analysis；(6) QTL site for obtaining the last 2 winter habit Chinese cabbage type winter rape malonaldehyde MDA, is respectively designated as Qmda.gsau-1A and Qmda.gsau-8A；4 chain molecular labelings are respectively as follows: BrID101211, OI12F11 (R1), BrID10839 and Ra2-E12 with the QTL site of the last 2 winter habit Chinese cabbage type winter rape malonaldehyde MDA.
Description
Technical field
The invention belongs to molecular biology and Biotechnology in Genetic Breeding fields, and in particular to a kind of strong winter habit Chinese cabbage type winter rape
Malonaldehyde MDA gene QTL site also relates to chain with strong winter habit Chinese cabbage type winter rape malonaldehyde MDA gene QTL site
Molecular labeling.
Background technique
Turnip type rape is long in China's cultivation history, and it is important one of the oil crops in China that germ plasm resource is abundant.But
Based on spring habit, winter habit Chinese cabbage type winter rape germplasm lacks turnip type rape, is particularly suitable for the strong winter habit of North Dry Han Qu plantation
Winter rape variety lacks.Northern China weather severe cold, winter rape have excellent winter resistance ability safe overwintering, therefore cold-resistant product
Kind breeding is the key that northern winter rape development.It is time-consuming and laborious using conventional breeding methods breeding cold-resistant material, and efficiency of selection
It is lower, if can carry out in seedling stage superseded by molecular mark, not only saves and production cost but also greatly improve
Efficiency of selection.
Malonaldehyde MDA is one of most important product of Membrane-Lipid Peroxidation in Plants, therefore its generation can aggravate the damage of film
Malonaldehyde MDA content is a common counter in plant cold resistance physiological Study.About malonaldehyde MDA in plant variation in vivo
Study a lot of, but there is not been reported for the research positioned to its molecular labeling and QTL.This research is positioned by QTL, it is intended to be filtered out
The QTL site and molecular labeling of strong winter habit Chinese cabbage type winter rape malonaldehyde MDA, is used for strong winter habit Chinese cabbage type winter rape malonaldehyde
The clone of MDA gene and the molecular marker assisted selection of winter rape cold-tolerance breeding.
Summary of the invention
The technical problem to be solved by the present invention is in the prior art the shortcomings that and provide 2 strong winter habit Chinese cabbage type
The QTL site of winter rape malonaldehyde MDA, the effect value in 2 sites is relatively high, to strong winter habit Chinese cabbage type winter rape malonaldehyde
The regulation of MDA content plays an important role, and can be used as positional cloning and molecular marker assisted selection.
The present invention also provides 4 molecule marks chain with the QTL site of strong winter habit Chinese cabbage type winter rape oil malonaldehyde MDA
The genetic distance of note, these labels and QTL site is relatively close and is SSR and the InDel label of based on PCR technology, thus reliable
And it is easy to use, convenience will be provided to strong winter habit Chinese cabbage type winter rape cold-tolerance breeding.
Technical problem to solve of the invention adopts the following technical scheme that
Strong winter habit Chinese cabbage type winter rape malonaldehyde MDA molecular labeling and QTL site, screening includes following steps:
(1) segregating population is constructed: using mostly for superpower cold-resistant winter rape ' Gansu Province oil 7 of the strong winter habit of parent of bagging selfing ' and
Strong cold-resistant winter rape ' Gansu Province oil 9 ' configuration cross combination, first familiar generation selfing generate F2 for segregating population.The reality that the present invention uses
Material is tested to be provided by genetics breeding of rape seminar, Gansu Agriculture University.
(2) rape leaf DNA is extracted: extracting parent ' Gansu Province oil 7 using CTAB method ' and ' Gansu Province oil 9 ' and F2 generation separation
The leaf DNA of group.
(3) Primer Source and synthesis: synthesizing 315 pairs of primers by Shanghai biotechnology Co., Ltd, Primer Source in
The rape micro-satellite primers sequence and Brassica Database website http that www.UK crop.net is delivered: //
All primers on 10 chromosomes announced on brassicadb.org/, wherein SSR primer 51 is right, InDel primer 2 64
It is right.
(4) polymorphism primer screens: carrying out PCR amplification using the two parent DNA of primer pair of synthesis, filters out with polymorphic
The primer of property, the process are related to PCR amplification, polyacrylamide gel electrophoresis, silver staining, photograph.
(5) polymorphism primer detects in F2 generation: the polymorphism primer filtered out being detected in F2 generation, obtains gene
Type data, the process and (4) step are essentially identical.
(6) genetic linkage map building and QTL positioning: according to F2 for genotype data, QTL IciMapping V3.3 is utilized
Software carries out genetic linkage map building and QTL positioning.
Obtain 2 QTL sites of strong winter habit Chinese cabbage type winter rape malonaldehyde MDA, be respectively designated as: Qmda.gsau-1A and
Qsmda.gsau-8A, 4 chain molecular labelings with the QTL site of winter habit Chinese cabbage type winter rape malonaldehyde MDA 2 strong, point
Be not: the sequence difference of BrID101211, OI12F11 (R1), BrID10839 and Ra2-E12,4 molecular labelings are as follows:
BrID101211-F:TCTCTCCCACAGAAGAAAAA
BrID101211-R:GGAGTTTGAGACTTCGATGA
OI12F11 (R1)-F:AAGGACTCATCGTGCAATCC
OI12F11 (R1)-R: GTGTCAGTGGCTACAGAGAC
BrID10839-F:AGGAACAATACCCATTTGTG
BrID10839-R:GGTGTTTGTGTGTTCGGTA
Ra2-E12-F:TGTCAGTGTGTCCACTTCGC
Ra2-E12-R:AAGAGAAACCCAATAAAGTAGAACC.
The site Qmda.gsau-1A is located on 1A chromosome, chain with molecular labeling BrID101211 and OI12F11 (R1),
Interpretable 28.9663% phenotypic variation, additive effect 0.8136, dominant effect 15.8908.
Qsmda.gsau-8A is located on 8A chromosome, chain with molecular labeling BrID10839 and Ra2-E12, can be explained
11.1783% phenotypic variation, additive effect are -2.1194, dominant effect -3.1315.
Beneficial effects of the present invention: the present invention positions for the first time obtains winter habit Chinese cabbage type winter rape malonaldehyde MDAQTL 2 strong
Site, and 4 molecular labelings chain with the QTL site of strong winter habit Chinese cabbage type winter rape oil malonaldehyde MDA are given, it solves respectively
Release 28.9663% and 11.1783% phenotypic variation.In conventional cold-tolerance breeding, test material need to judge that it is anti-by overwintering
Cold power, it is time-consuming and laborious.By detecting the QTL site of strong winter habit Chinese cabbage type winter rape malonaldehyde MDA, in seedling stage
Screening material substantially increases screening efficiency, saves production cost, accelerates breeding process.Strong winter habit Chinese cabbage type winter rape simultaneously
The acquisition of the QTL site of malonaldehyde MDA can further clone the base that control synthesizes strong winter habit Chinese cabbage type winter rape malonaldehyde MDA
Cause, the exploration for strong winter habit Chinese cabbage type winter rape cold-resistant mechanism provide technical support.
Detailed description of the invention
Fig. 1 is superpower cold-resistant winter rape ' Gansu Province oil 7 of the strong winter habit of the present invention ' and strong cold-resistant winter rape ' Gansu Province oil 9 ' hybridization F2
For group's malonaldehyde MDA content distribution figure；
Fig. 2 is that the present invention utilizes two parental line selection polymorphism primers；
Fig. 3 is testing result of the polymorphism primer Ol13C12a of the present invention in F2 generation；
Fig. 4 is position and LOD curve synoptic diagram of the site Qmda.gsau-1A of the present invention in 2A chromosome；
Fig. 5 is position and LOD curve synoptic diagram of the site Qmda.gsau-8A of the present invention in 8A chromosome.
Specific embodiment
The principles and features of the present invention are described below, and the given examples are served only to explain the present invention, is not intended to limit
Determine the scope of the present invention.
Embodiment 1: the building of strong winter habit Chinese cabbage type winter rape segregating population and malonaldehyde MDA assay.
Segregating population used in the present embodiment specifically constructs as follows:
In August, (1) 2010 is using mostly for superpower cold-resistant winter rape ' Gansu Province oil 7 ' He Qiangkang of the strong winter habit of parent of bagging selfing
Severe winter rape ' Gansu Province oil 9 ' configuration cross combination, harvests F1 generation seed in May, 2011.
In August, (2) 2011 sows F1 seed, and bagging in April, 2012 selfing obtains F2 for seed.
The sowing of in August, 2012 F2 is for seed.103 plants of listing marks are randomly selected to seedling stage, the fresh children of acquisition at the beginning of 11 months
Leaf takes back laboratory with ice chest and is stored in -70 DEG C of ultra low temperature freezers.Using thiobarbituricacidα- (TBA) determination of color third
Dialdehyde (MDA) content.The result shows that: F2 is in normal distribution for malonaldehyde (MDA) content, and range of variation is very wide, it was demonstrated that the third two
Aldehyde (MDA) character belongs to quantitative character (Fig. 1).
Embodiment 2: the extraction of parent and F2 for segregating population blade total DNA.
Blade total DNA is extracted using CTAB method, the specific steps are as follows:
(1) healthy spire is picked as the material for extracting rape genomic DNA, is cleaned with distilled water, is blotted with paper.
(2) it takes fresh blade 0.5g or so to be placed in mortar, liquid nitrogen is added, is ground to rapidly after whitish powder shape immediately
It is packed into 2ml centrifuge tube.
(3) warmed-up 2 × CTAB Extraction buffer 700ul is added to be impregnated with powder and reverse centrifuge tube, keeps powder abundant
It scatters, in 65 DEG C of water-bath 60min, soft mixing 2-3 times therebetween.
(4) centrifuge tube is taken out, is cooled to room temperature, chloroform/isoamyl alcohol (24/1) of 700ul is added, it is light and slow to be mixed by inversion, it is quiet
Set 10 min.
(5) 13000rpm is centrifuged 10min.
(6) it takes supernatant 600ul to be transferred in new centrifuge tube, isometric chloroform/isoamyl alcohol (24/1) is added, it is light and slow reverse
It mixes, is placed at room temperature for 10min.
(7) 13000rpm is centrifuged 10min, and supernatant 400ul is taken to be transferred in new centrifuge tube, adds the isopropyl being pre-chilled in equal volume
Alcohol is stored in -20 DEG C, stands 20 min.
(8) 13000rpm is centrifuged 10min, and centrifuge tube is inverted on blotting paper, supernatant is drained, with the second of 600ul 70%
It is alcohol washing precipitating 2 times, micro- dry at room temperature.300ul deionized water dissolving precipitating is added.
(9) 1ul RNase(10mg/ml is added) 37 DEG C of 60 min of water-bath are placed in, each centrifuge tube adds 300ul deionization
Water, is added isometric chloroform/isoamyl alcohol extraction 2 times.
(10) it draws supernatant 400ul and is transferred to new centrifuge tube, be added the 3mol/LNaAC solution of supernatant volume 1/10,2.5 times
The pre-cooling dehydrated alcohol of volume precipitates DNA, is placed in -20 DEG C, and 20 min or so, 12000rpm are centrifuged 10min, abandon supernatant.
(11) it is precipitated 2 times with the ethanol washing of 600ul 70%, aeration-drying or natural drying.
(12) 30ul TE buffer solution DNA is added, 4 DEG C save backup.(- 20 °C of long-term preservations)
Embodiment 3:SSR and InDel Primer Source, synthesis and polymorphism screening.
(1) the rape micro-satellite primers sequence delivered from www.UK crop.net and the website Brassica Database
315 pairs are uniformly chosen in all primers on 10 chromosomes announced on http://brassicadb.org/.Wherein SSR draws
Object 51 is right, and InDel primer 2 64 is right.Primer is synthesized by Shanghai biotechnology Co., Ltd.
(2) 6 plants of DNA mixed in equal amounts are respectively randomly selected from parent, the template as screening primer.
(3) PCR reaction system
PCR reaction system is 10ul:
Mix Taq enzyme 6ul
Upper primer 0.5ul
Lower primer 0.5ul
ddH2O 2ul
Template: 1ul
(4) PCR amplification program: 94 DEG C of initial denaturation 4min；94 DEG C of denaturation 50s, 55-60 DEG C of each circulation of renaturation 50s(are moved back
Fiery temperature reduces or increases 0.5 DEG C), 72 DEG C of extension 50s, 35 circulations；72 DEG C of extension 10min；Take out 4 DEG C of PCR product guarantors
It deposits.
(5) polyacrylamide gel electrophoresis
Pcr amplification product electrophoresis, concrete operations on 8% polyacrylamide gel are as follows:
The a abundant cleaning glass plate of ddH2O, sample comb and rubber sleeve, are placed on bracket and dry.
After b dries, two pieces of glass plate alignment are anchored to together, are put into rubber sleeve, and sealed with 1% agarose gel, it is quiet
Set 30min.
The glass plate that two pairs are sealed carefully is attached on electrophoresis tank by c.
The acrylamide gel solution of d configuration 50ml: by 30% acrylamide 13.3ml, distilled water 31ml, 10 × TBE
5ml, 10% ammonium persulfate 10.7ml and TEMED 3.5ul are rapidly added in 100ml small beaker, are stirred evenly with glass bar.
Between the above-mentioned acrylamide gel solution prepared is slowly poured into two pieces of glass plates along glass notch board top by e, two pairs
After glass plate glue fills, it is inserted into comb, stands 1h or so.
A little 1 × tbe buffer liquid is added with liquid-transfering gun around comb by f, gently extracts comb, and 1 is filled in electrophoresis tank
× TBE electrode buffer.
PCR product 2-3ul is added in each loading wells of g.Power supply is connected, in voltage 200V, electric current 100mA electrophoresis 1h or so.
H closes power supply, recycles electrode buffer, removes glass plate, pries open glass plate and takes out gel, rinses number with ddH2O
Secondary preparation silver staining.
(6) silver staining process
A is fixed: pouring into 300ml fixer in the glue box equipped with gel, the jog 8min on decolorization swinging table, recycling is fixed
Liquid.
B dyeing: 300ml dyeing liquor being added into glue box, and the jog 10-15min on decolorization swinging table recycles dyeing liquor, uses
DdH2O is rinsed twice.
C development: 300ml developing solution being added into glue box, and jog is clear to band on decolorization swinging table, pours out developing solution,
It is rinsed twice with ddH2O.
D photograph: gel is placed on film illuminator and is taken a picture.
Preparation of reagents needed for during silver staining:
Fixer: 100ml dehydrated alcohol, 5ml glacial acetic acid are settled to 1L with ddH2O.
Dyeing liquor: 2g AgNO3,100ml dehydrated alcohol, 5ml glacial acetic acid are settled to 1L with ddH2O.
Developer solution: 1.5ml formaldehyde (matching while using) is added in 300ml 3%NaOH solution.
(7) polymorphism primer screens
According to electrophoresis result, filters out 39 pairs and expand repetition stabilization between " Gansu Province oil 7 " and " Gansu Province oil 9 ", band is clear
It is clear, the primer with polymorphism.
Embodiment 4: distribution, genetic linkage map building and qtl analysis of the polymorphism primer in F2 generation.
(1) 39 pairs of polymorphism primers for obtaining embodiment 3 carry out PCR expansion to the DNA of 103 single plants of F2 group respectively
Increase, silver staining photograph and banding pattern interpretation after polyacrylate hydrogel electrophoresis, banding pattern identical as parent " Gansu Province oil 7 " is denoted as " A ", with parent
" Gansu Province oil 9 " identical banding pattern is denoted as " B ", and heterozygote is denoted as " H ", and the banding pattern of missing is denoted as "-".
(2) banding pattern is counted as a result, calculating the genetic distance between primer using QTL IciMapping V3.3 software, and
Construct genetic linkage map and qtl analysis.The 39 pairs of primers filtered out are respectively distributed on 10 chromosomes of turnip type rape.To third
Dialdehyde (MDA) content carries out qtl analysis, detects the QTL of 2 control malonaldehyde (MDA) contents altogether, is located at chromosome
On 1A and 8A, in which:
QTL(Fig. 4 on 1A chromosome) be named as Qmda.gsau-1A, be located between positioning area label BrID101211 ~
Between OI12F11 (R1).Qmda.gsau-1A interpretable phenotypic variation is 28.9663%.
QTL(Fig. 5 on 8A chromosome) be named as Qmda.gsau-8A, be located between positioning area BrID10839 ~
Between Ra2-E12.Qmda.gsau-8A interpretable phenotypic variation is 11.1783%.
The chain molecular labeling of the QTL site of table 1 and strong winter habit Chinese cabbage type winter rape malonaldehyde MDA
Table the last 2 winter habit Chinese cabbage type winter rape malonaldehyde MDA activity QTL site essential information
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Any modification, equivalent replacement, improvement and so within principle, should all be included in the protection scope of the present invention.
Claims (1)
- The last 1. winter habit Chinese cabbage type winter rape malonaldehyde MDA molecular labeling, it is characterised in that the amplimer of the molecular labeling is such as Under:BrID101211-F:TCTCTCCCACAGAAGAAAAABrID101211-R:GGAGTTTGAGACTTCGATGA.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102286492A (en) * | 2011-08-17 | 2011-12-21 | 中国农业科学院油料作物研究所 | Major gene locus for thousand-grain weight trait of rape and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286492A (en) * | 2011-08-17 | 2011-12-21 | 中国农业科学院油料作物研究所 | Major gene locus for thousand-grain weight trait of rape and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 不同生态区冬前低温下白菜型冬油菜不同抗寒品种(系)的比较;刘自刚等;《作物学报》;20141231;第40卷(第2期);表2 |
| 白菜型冬油菜抗寒性与生理生化特性关系;蒲媛媛等;《分子植物育种》;20101231;第8卷(第2期);摘要 |
| 白菜型油菜抗寒生理生化特性动态研究;张东昱等;《甘肃农业大学学报》;20110630;第46卷(第3期);摘要部分 |
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