CN104450897B - The specific Function molecular marker of rice high yield allele GY6 - Google Patents
The specific Function molecular marker of rice high yield allele GY6 Download PDFInfo
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Abstract
The invention provides detection rice high yield alleleGY61 specific Function molecular marker Si2926/HhaI, its forward primer sequence as shown in SEQ ID No:1, downstream primer sequence is as shown in SEQ ID NO.2, utilize this DNA that rice seedling is extracted by primer to carry out PCR amplification and digestion with restriction enzyme, can detect whether detected materials carries rice high yield alleleGY6, whole detection process operation is simple and accuracy is high.
Description
Technical field
1 function that the present invention relates to the detection technique field in rice breeding, particularly high yield allele GY6 is special
Property molecular marker.
Background technology
Oryza sativa L. is one of the most most important cereal crops.Improve rice yield, to solving Food Security, there is weight
Want meaning.The single plant yield of Oryza sativa L. is determined by three Components: single-strain tassel number, Defined daily doses and mass of 1000 kernel, belongs to multiple
Miscellaneous quantitative trait, is controlled by multiple Quantitative Trait Genes (Quantitative trait loci, QTL).In recent years, along with
Completing of Oryza sativa L. genome sequencing, clone's research of rice yield traits QTL achieves fast development, has cloned 16 controls
The QTL of the rice yield correlated traits such as plant type processed, fringe shape, Defined daily doses, grains per panicle and mass of 1000 kernel.But, relative to
The complexity of yield traits, the understanding to its hereditary basis is the most fairly limited at present, therefore, in the urgent need to excavating more yield
Gene, the most only understands that its hereditary basis offers theoretical foundation, and provides base for faster and better cultivating high-yield variety
Because of resource and technical support.
At present, molecular marker assisted selection (MAS) is applied in rice breeding more and more widely, but most
Select to be still based on the molecular marker with gene linkage, and the genetic marker of non-genomic itself, particularly functional indicia.Application
Functional indicia carries out some drawbacks that MAS can overcome linkage molecule labelling to be brought: 1) heredity burden, i.e. genes of interest
(beneficial gene) non-genes of interest (unfavorable gene) neighbouring with it exists chain, also brings into while importing genes of interest
Unfavorable gene, causes improvement not reach target；2) label information misleads, i.e. labelling yet suffers from, but owing to restructuring makes mark
Note and Gene segregation, cause the disappearance of target gene, cause false-positive generation.
Rice yield gene GY6 is the yield gene (patent No.: the ZL that the present inventor clones recently
201210047301.0；Denomination of invention: the clone of rice yield gene GY6 and application thereof).In order to accelerate GY6 gene in breeding
In application process, it is necessary to develop accurately and effectively qualification GY6 high yield allelic specific Function molecular marker.
Summary of the invention
It is an object of the invention to provide the specific Function molecular marker Si2926/Hha I identifying high yield allele GY6
Primer, described fragment is as shown in sequence table SEQ ID NO:1-2.
Specifically, the nucleotide sequence of described primer pair is as follows:
Forward 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:
Based on precious Shan 97 type of GY6 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 labelling Si2926/
Hha I.First the precious Shan 97 of application and Milyang 46 carry out PCR amplification and digestion with restriction enzyme, verify its Detection results and polymorphic
Property.Reapply this molecular marker to being derived from precious Shan 97/ Milyang 46, there is the near isogene of function difference in GY6 gene interval
It is that colony carries out functional verification, and carries out genotype detection through 66 parts of Rice Germplasm Resources of application further.Show that this function is special
Opposite molecule labelling detection allelic to GY6 high yield reliability is high, can apply to the allelic transformation of GY6 high yield and grinds
In studying carefully.
The present invention has a beneficial effect will be apparent below:
The present invention by comparing GY6 treasure's Shan 97 type and two kinds of allelic nucleotide of Milyang 46 type and aminoacid sequence,
1 specific Function molecular marker has been designed and developed based on amino acid difference ectopic sites.Apply this specific Function molecular marker, with
It is aided with digestion with restriction enzyme based on Standard PCR technology, if the clip size obtained is 135 bp, Oryza sativa L. material the most to be measured
Material is containing high yield allele GY6；The individuality carrying target gene type can not only be screened from breeding population effective and rapidly,
And the GY6 allelotype of different rice pest insects can be accurately distinguished.
The molecular marker of present invention exploitation is derived from the polymorphism in gene internal specific Function site, there is not heredity burden
With false-positive problem, target gene carried out conventional hybridization transformation, pyramiding breeding or transgenic application by based on, permissible
It is greatly saved time and cost, increases screening accuracy, improve breeding efficiency.
Accompanying drawing explanation
Fig. 1 is precious Shan 97 type and Milyang 46 type GY6 allele amino acid alignment result.
Fig. 2 is molecular marker Si2926/Hha I testing result in precious Shan 97 and Milyang 46.Wherein, M:Maker；
ZS: precious Shan 97；MY: Milyang 46；Being PCR primer on the left of M, right side is digestion products.
Fig. 3 is molecular marker Si2926/Hha I testing result in NIL colony.Wherein, Z: precious Shan 97；
M: Milyang 46；1-15: carry the precious Shan 97 allelic strain of type；16-30: carry the allelic strain of Milyang 46 type.
Fig. 4 is molecular marker Si2926/Hha I testing result in 66 parts of indica hybrid rice parents.Wherein, 1: II-
32B；The excellent 62B of 2:D；3:V20B；4: rich B；5: CHUANXIANG 29A；6: phenanthrene changes B；7: Feng Yuan A；8: ridge 46B；9: gold 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: in
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: polyphyly 1；33: grace extensive 58；34: spoke extensive 718；35: the most 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.
Detailed description of the invention
The present invention is explained further below in conjunction with embodiment, but the present invention is not done any type of limit by embodiment
Fixed.Experimental technique in following embodiment, if no special instructions, is conventional method.Experiment material used in following embodiment
Material, reagent etc., if no special instructions, the most commercially obtain.
The exploitation of embodiment 1 GY6 gene specific molecular marker
1. GY6 allele amino acid alignment and the qualification in Specific amino acid site
Utilize sequence alignment program DNASTAR MegAlign module (Lasergene) to precious Shan 97 and Milyang 46 at GY6
Gene locus upper amino acid sequence (patent No.: ZL 201210047301.0, SEQ ID No:3 and SEQ ID No:6) carries out sequence
Row comparison is analyzed.Identify GY6 albumen to there are differences on 6 amino acid sites, respectively A31V, K105E, N144S,
K146R, D147N and A160T(Fig. 1).
2. the design of GY6 gene specific molecular marker primer
For the 31st amino acids site in above-mentioned 6 amino acid difference ectopic sites, according to CAPS indicia designs principle, should
With online software dCAPS Finder 2.0 (http://helix.wustl.edu/dcaps/dcaps.html) and Oligo
7.0(Molecular Biology Insights) design primer.Its forward primer sequence is 5'-
CCGGATCACTAACCTCAGCG-3', 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 of 0.5 cm length, puts in 2.0 mL centrifuge tubes.
(2) adding 450 μ l DNA extraction liquid and steel balls, application tissue beveller is ground.
(3) add 450 μ l chloroform extracts, cover tightly lid, mixing of turning upside down.
(4) 11,000 rpm are centrifuged 2 minutes to clear split-phase.Aspirate supernatant 400 μ l, proceeds to 1.5 new ml and is centrifuged
Guan Zhong, abandons rifle head.
(5) add the dehydrated alcohol of 800 μ l pre-coolings, cover tightly lid, mixing of turning upside down.Place 30 minutes for-20 DEG C.
(6) 11,000 rpm are centrifuged 3 minutes and invest bottom centrifuge tube to precipitation, abandon supernatant.
(7) precipitate 2 times by 70% washing with alcohol, 1.5 ml centrifuge tubes are inverted on paper, natural drying.
(8) 1/10 × TE buffer solution precipitation of 100 μ l is added.
(9) take 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 ddH2O to 20 μ
l.Reaction condition: 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 guarantors
Deposit.
Take 2 μ l PCR primer and be splined on 2% agarose gel, connect electrode, electrophoresis 1 hour under 100V constant voltage,
Close power supply；Take off gel, GelRed(Biotium) dyeing after gel imaging.
5. PCR primer enzyme action and detection
Endonuclease reaction system is as follows: 10 × Buffer M 2 μ l, restricted enzyme Hha I (Takara) 1 μ l, PCR
Product 7 μ l, adds ddH2O to 20 μ l；Reaction condition: 37 DEG C 3 hours.
Taking 10 μ l PCR primer and be splined on 2% agarose gel, connect electrode, under 100V constant voltage, electrophoresis 1.5 is little
Time, close power supply；Take off gel, GelRed(Biotium) dyeing after gel imaging.
Result shows, application Si2926 primer amplification treasure's Shan 97 and Milyang 46, its PCR primer size is 155 bp, warp
After Hha I enzyme action, precious Shan 97 obtains the fragment of 135 bp, and Milyang 46 keeps constant (Fig. 2).
The checking in NIL colony of the embodiment 2 GY6 gene specific molecular marker
Be derived from precious Shan 97/ Milyang 46 1 set NIL colony as material, this colony only separates in GY6 interval,
Containing precious Shan 97 and two kinds of genotype of Milyang 46, compared with the latter, the former is real, and grain number is many, mass of 1000 kernel is big, yield is high, to GY6 gene
Specific molecular marker Si2926/Hha I carries out functional verification.PCR amplification system described in Application Example 1 and reaction bar
Part, utilizes Si2926/Hha I to carry out expanding and enzyme action.
Electrophoresis result (Fig. 3) shows, Si2926/Hha I presents separation in NIL colony, and real grain number is many, thousand
Grain strain (No. 1-15) great, that yield is high all detects the banding pattern as precious Shan 97, shows that they all carry precious Shan 97 type
High yield allele；Otherwise, remaining strain (No. 16-30) then carries Milyang 46 type allele.This result shows, the present invention carries
Molecular marker Si2926/Hha I detection allelic to the GY6 high yield reliability of confession is high, can be as the function of GY6 gene
Specific molecular marker, is applied in GY6 high yield allelic transformation research.
The detection in Rice Germplasm Resources of the embodiment 3 GY6 gene specific molecular marker
Utilize GY6 gene function specific molecular marker Si2926/Hha I, 66 parts of indica hybrid rice parents are examined
Survey.Result shows, 17 parts of parents carry precious Shan 97 type allele, and 49 parts of parents carry Milyang 46 type allele (Fig. 4).
<110>China Paddy Rice Inst
<120>the specific Function molecular marker of rice high yield allele GY6
<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. the primer of the specific Function molecular marker Si2926/Hha I of detection rice high yield allele GY6, its feature exists
In: its forward 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.
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| 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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