CN104450897A - Functional specificity molecular marker for rice high yield allele GY6 - Google Patents

Functional specificity molecular marker for rice high yield allele GY6 Download PDF

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CN104450897A
CN104450897A CN201410681948.8A CN201410681948A CN104450897A CN 104450897 A CN104450897 A CN 104450897A CN 201410681948 A CN201410681948 A CN 201410681948A CN 104450897 A CN104450897 A CN 104450897A
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high yield
rice
molecular marker
gene
functional specificity
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CN104450897B (en
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樊叶杨
庄杰云
朱玉君
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China National Rice Research Institute
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12Q2600/00—Oligonucleotides characterized by their use
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Abstract

The invention provides a functional specificity molecular marker Si2926/HhaI for detecting a rice high yield allele GY6. An upstream primer sequence of the functional specificity molecular marker Si2926/HhaI is shown as SEQ ID No: 1, and a downstream primer sequence of the functional specificity molecular marker Si2926/HhaI is shown as SEQ ID NO: 1; PCR amplification and restriction enzyme digesting are carried out on NDAs extracted in a rice seedling period by utilizing the pair of primers, and whether a material to be detected is carried with the rice high yield allele GY6 or not can be detected, so that the whole detection process is simple in operation and high in accuracy.

Description

The specific Function molecule marker of rice high yield allelotrope GY6
Technical field
The present invention relates to the detection technique field in rice breeding, particularly high yield allelotrope gY61 specific Function molecule marker.
Background technology
Paddy rice is one of most important food crop in the world.Improve rice yield significant to solution Food Security.The single plant yield of paddy rice is determined by three Components: single-strain tassel number, Defined daily doses and thousand seed weight, all belongs to complicated quantitative character, controls by multiple Quantitative Trait Genes (Quantitative trait loci, QTL).In recent years, along with completing of paddy rice genome sequencing, clone's research of rice yield traits QTL achieves fast development, has cloned the QTL that 16 control the rice yield correlated character such as plant type, fringe shape, Defined daily doses, grains per panicle and thousand seed weight.But, relative to the complicacy of yield traits, also quite limited to the understanding of its hereditary basis at present, therefore, in the urgent need to excavating more output gene, be not only and understand its hereditary basis and offer theoretical foundation, and provide genetic resources and technical support for cultivating high-yield variety faster and better.
At present, molecular marker assisted selection (MAS) is applied more and more widely in rice breeding, but most selection is still based on the molecule marker with gene linkage, and the genetic marker of non-genomic itself, particularly functional indicia.Application function mark carries out MAS and can overcome linkage molecule and mark some drawbacks brought: 1) heredity is burdensome, namely goal gene (beneficial gene) and the non-goal gene near it (unfavorable gene) exist chain, while importing goal gene, also bring unfavorable gene into, cause improvement not reach re-set target; 2) label information misleads, and namely marks and still exists, but makes mark and gene isolation owing to recombinating, and causes the disappearance of target gene, causes false-positive generation.
Rice yield gene gY6the output gene (patent No.: the ZL 201210047301.0 that the present inventor clones recently; Denomination of invention: rice yield gene gY6clone and application).In order to accelerate gY6the application process of gene in breeding, is necessary to develop and identifies accurately and effectively gY6the allelic specific Function molecule marker of high yield.
Summary of the invention
The object of this invention is to provide qualification high yield allelotrope gY6specific Function molecule marker Si2926/ hhathe primer of I, described fragment is as shown in sequence table SEQ ID NO:1-2.
Particularly, the nucleotide sequence of described primer pair is as follows:
Upstream primer sequence is 5'-CCGGATCACTAACCTCAGCG-3', as shown in sequence table SEQ ID No:1;
Downstream primer sequence is 5'-GCATCCAAGATATGATCCGTA-3', as shown in sequence table SEQ ID NO.2.
The present invention is achieved through the following technical solutions:
With gY6based on precious Shan 97 type of gene and the allelic Nucleotide of Milyang 46 type and aminoacid sequence, carry out sequence alignment analysis, for the 31st amino acids difference, application Oligo 7.0 software design goes out 1 dCAPS and marks Si2926/ hhai.First apply precious Shan 97 and Milyang 46 carries out pcr amplification and digestion with restriction enzyme, verify its Detection results and polymorphism.Apply again this molecule marker to be derived from precious Shan 97/ Milyang 46, gY6the near isogenic line colony that gene interval exists function difference carries out functional verification, and carries out genotype detection through applying 66 parts of Rice Germplasm Resources further.Show this specific Function molecule marker pair gY6the allelic detecting reliability of high yield is high, can be applied to gY6in the allelic transformation research of high yield.
The present invention has following significantly beneficial effect:
The present invention is by comparing gY6precious Shan 97 type and Milyang 46 type two kinds of allelic Nucleotide and aminoacid sequence, designed and developed 1 specific Function molecule marker based on amino acid difference ectopic sites.Apply this specific Function molecule marker, based on Standard PCR technology, be aided with digestion with restriction enzyme, if the clip size obtained is 135 bp, then rice material to be measured contains high yield allelotrope gY6; The individuality carrying target gene type can not only be screened from breeding population effective and rapidly, and accurately distinguish different rice pest insects gY6allelotype.
The molecule marker of the present invention's exploitation is derived from the polymorphism in gene internal specific Function site, there is not hereditary burden and false-positive problem, by based on to target gene carry out conventional hybridization transformation, pyramiding breeding or transgenosis application, can greatly save time and cost, increase screening accuracy, improve breeding efficiency.
Accompanying drawing explanation
Fig. 1 is precious Shan 97 type and Milyang 46 type gY6allelotrope amino acid alignment result.
Fig. 2 is molecule marker Si2926/ hhathe detected result of I in precious Shan 97 and Milyang 46.Wherein, M:Maker; ZS: precious Shan 97; MY: Milyang 46; Be PCR primer on the left of M, right side is digestion products.
Fig. 3 is molecule marker Si2926/ hhathe detected result of I near isogenic line colony.Wherein, Z: precious Shan 97; M: Milyang 46; 1-15: carry the allelic strain of precious Shan 97 type; 16-30: carry the allelic strain of Milyang 46 type.
Fig. 4 is molecule marker Si2926/ hhathe detected result of I in 66 parts of indica hybrid rice parents.Wherein, 1: II-32B; The excellent 62B of 2:D; 3:V20B; 4: rich B; 5: the fragrant 29A in river; 6: phenanthrene changes B; 7: Feng Yuan A; 8: ridge 46B; 9: golden 23B; 10: Long Tepu B; 11: interior fragrant 2A; 12: interior fragrant 85A; 13: the rich A in sky; 14: the blue or green early B of association; 15: print 32B; 16: excellent IB; 17: Zhenshan 97B; 18: middle 100A; 19: middle 1A; 20: middle 2B; 21: middle 3B; 22: middle 9B; 23: middle Zhejiang A; 24:207; 25:253; 26:926:27:CDR22; 28:R402; 29:To974; 30:ZDZ057; 31: survey 64; 32: No. 1, polyphyly; 33: grace extensive 58; 34: spoke extensive 718; 35: extensively extensive 128; 36: osmanthus 99; 37: Jianghui 151; 38: Lu extensive 17; 39; Milyang 46; 40: Mianhui 501; 41: Mianhui725; 42: bright extensive 63; 43: bright extensive 70; 44: bright extensive 77; 45: bright extensive 86; 46: another name for Sichuan Province extensive 162; 47: another name for Sichuan Province extensive 527; 48: Yanhui 559; 49: Restorer line Yihui1577; 50: Zhenhui 084; 51:CH238; 52:CH59; 53: in extensive 218; 54: in extensive 8006; 55: in extensive 111; 56: in extensive 1176; 57: in extensive 333; 58: in extensive 161; 59: special blue or green; 60:IR24; 61: in extensive 8012; 62: in extensive 8015; 63: in extensive 465; 64: in extensive 7492; 65:R600; 66: extensive 66.
Embodiment
Explain the present invention further below in conjunction with embodiment, but embodiment does not limit in any form to the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Experiment material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
embodiment 1 gY6the exploitation of gene specific molecule marker
1. gY6the qualification in allelotrope amino acid alignment and Specific amino acid site
Sequence alignment program DNASTAR MegAlign module (Lasergene) is utilized to exist to precious Shan 97 and Milyang 46 gY6locus upper amino acid sequence (patent No.: ZL 201210047301.0, SEQ ID No:3 and SEQ ID No:6) carries out sequence alignment analysis.Identify GY6 albumen to there are differences on 6 amino acid sites, be respectively A31V, K105E, N144S, K146R, D147N and A160T(Fig. 1).
2. gY6the design of gene specific molecule marker primer
For the 31st amino acids site in above-mentioned 6 amino acid difference ectopic sites, according to CAPS indicia designs principle, apply online software dCAPS Finder 2.0 (http://helix.wustl.edu/dcaps/dcaps.html) and Oligo 7.0(Molecular Biology Insights) design primer.Its upstream primer sequence is 5'-CCGGATCACTAACCTCAGCG-3', and downstream primer sequence is 5'-GCATCCAAGATATGATCCGTA-3'.
3. DNA Trace bio-element
(1) clip Rice Seedling Leaves 2 ~ 3 cm, is cut into the fragment that 0.5 cm is long, puts into 2.0 mL centrifuge tubes.
(2) add 450 μ l DNA extraction liquid and a steel ball, application organizes beveller to grind.
(3) add 450 μ l chloroform extraction liquid, cover tightly lid, mixing of turning upside down.
(4) 11,000 rpm extremely clear phase-splittings in centrifugal 2 minutes.Aspirate supernatant 400 μ l, proceeds in 1.5 new ml centrifuge tubes, abandons rifle head.
(5) add the dehydrated alcohol of 800 μ l precoolings, cover tightly lid, mixing of turning upside down.Place 30 minutes for-20 DEG C.
(6) 11,000 rpm invest bottom centrifuge tube to precipitation in centrifugal 3 minutes, abandon supernatant liquor.
(7) precipitate 2 times by 70% washing with alcohol, 1.5 ml centrifuge tubes are inverted on paper, seasoning.
(8) 1/10 × TE buffer solution precipitation of 100 μ l is added.
(9) get 2 μ l and carry out pcr amplification.
4. pcr amplification and detection
Amplification reaction system is as follows: 10 × PCR Buffer 2 μ l, dNTPs (2.5 mM each) 1.6 μ l, primer (5 pmol) 2 μ l, Taq enzyme (2.5 U/ μ l, Cwbio) 0.2 μ l, DNA profiling (50 ng/ μ l) 2 μ l, add ddH 2o to 20 μ l.Reaction conditions: 94 DEG C 2 minutes; 94 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 30 seconds, 30 circulation; 72 DEG C 8 minutes; 10 DEG C of preservations.
Get 2 μ l PCR primer and be splined on 2% sepharose, connect electrode, electrophoresis 1 hour under 100V constant voltage, powered-down; Take off gel, GelRed(Biotium) the rear gel imaging of dyeing.
5. PCR primer enzyme is cut and is detected
Endonuclease reaction system is as follows: 10 × Buffer M 2 μ l, restriction enzyme hhai (Takara) 1 μ l, PCR primer 7 μ l, adds ddH 2o to 20 μ l; Reaction conditions: 37 DEG C 3 hours.
Get 10 μ l PCR primer and be splined on 2% sepharose, connect electrode, electrophoresis 1.5 hours under 100V constant voltage, powered-down; Take off gel, GelRed(Biotium) the rear gel imaging of dyeing.
Result shows, and the precious Shan 97 of application Si2926 primer amplification and Milyang 46, its PCR primer size is 155 bp, warp hhaafter I enzyme is cut, precious Shan 97 obtains the fragment of 135 bp, and Milyang 46 remains unchanged (Fig. 2).
embodiment 2 gY6the checking of gene specific molecule marker near isogenic line colony
To be derived from 1 cover near isogenic line colony of precious Shan 97/ Milyang 46 for material, this colony only exists gY6interval separation, containing precious Shan 97 and Milyang 46 two kinds of genotype, compared with the latter, the former examples explain is many, thousand seed weight is large, output is high, right gY6gene specific molecule marker Si2926/ hhai carries out functional verification.The PCR amplification system described in Application Example 1 and reaction conditions, utilize Si2926/ hhai carries out amplification and enzyme is cut.
Electrophoresis result (Fig. 3) shows, Si2926/ hhai presents separation near isogenic line colony, and the strain (No. 1-15) that examples explain is many, thousand seed weight is large, output is high all detects the banding pattern the same with precious Shan 97, shows that they all carry precious Shan 97 type high yield allelotrope; Otherwise all the other strains (No. 16-30) then carry Milyang 46 type allelotrope.This result shows, molecule marker Si2926/ provided by the invention hhai couple gY6the allelic detecting reliability of high yield is high, can conduct gY6the specific Function molecule marker of gene, is applied to gY6in the allelic transformation research of high yield.
embodiment 3 gY6the detection of gene specific molecule marker in Rice Germplasm Resources
Utilize gY6gene function specific molecular marker Si2926/ hhai, detects 66 parts of indica hybrid rice parents.Result shows, 17 parts of parents carry precious Shan 97 type allelotrope, and 49 parts of parents carry Milyang 46 type allelotrope (Fig. 4).
<110> China Paddy Rice Inst
<120> rice high yield allelotrope gY6specific Function molecule marker
<160> 2
<170> PatentIn version 3.3
 
<210> 1
<211> 20
<212> DNA
<213> artificial sequence
<400> 1
ccggatcact aacctcagcg 20
<210> 2
<211> 21
<212> DNA
<213> artificial sequence
<400> 2
gcatccaaga tatgatccgt a 21
 

Claims (1)

1. detect rice high yield allelotrope gY6specific Function molecule marker Si2926/ hhathe primer of I, is characterized in that: its upstream primer sequence is 5'-CCGGATCACTAACCTCAGCG-3', as shown in sequence table SEQ ID No:1; Its downstream primer sequence is 5'-GCATCCAAGATATGATCCGTA-3', as shown in sequence table SEQ ID NO.2.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107760794A (en) * 2017-10-24 2018-03-06 中国水稻研究所 Detect the specific PCR molecular markers of the weak response to temperature allele of qHd1 of rice varieties treasure Shan 97
CN110055348A (en) * 2019-05-07 2019-07-26 华南农业大学 The Functional marker of rice grain shape gene GL3 and its application

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102643829A (en) * 2011-11-25 2012-08-22 中国水稻研究所 Rice yield gene GY6 clone and application thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102643829A (en) * 2011-11-25 2012-08-22 中国水稻研究所 Rice yield gene GY6 clone and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
马文丽主编: "《基因测序实验技术》", 30 November 2012, 化学工业出版社 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107760794A (en) * 2017-10-24 2018-03-06 中国水稻研究所 Detect the specific PCR molecular markers of the weak response to temperature allele of qHd1 of rice varieties treasure Shan 97
CN110055348A (en) * 2019-05-07 2019-07-26 华南农业大学 The Functional marker of rice grain shape gene GL3 and its application

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