CN107630103A - A kind of CAPS molecule labelling methods for identifying rice varieties and application - Google Patents
A kind of CAPS molecule labelling methods for identifying rice varieties and application Download PDFInfo
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Abstract
The invention discloses a kind of identification rice long-grained nonglutinous rice and the CAPS molecule labelling methods of japonica rice variety and application.Methods described comprises the following steps：S1. rice sample genomic dna is extracted；S2. performing PCR amplification is entered with primer pair step S1 genomic DNAs described in claim 3；S3. step S2 PCR primer is passed throughHhaI digestion rear electrophoresis, obtain CAPS marks；S4. electrophoresis result judges：The rice sample is long-grained nonglutinous rice if 440bp electrophoretic band is obtained, if obtaining 220bp electrophoretic band, the rice varieties are japonica rice.The CAPS molecule labelling methods specificity of the present invention is high, and the germ plasm resource that rice cultivar conveniently accurately can be carried out to long-grained nonglutinous rice and japonica rice is distinguished.Meanwhile molecular labeling provided by the invention is in actual applications, PCR combination digestions are only needed, and cost is low, flux is high, specific height, is highly suitable for production practices.
Description
Technical field
The invention belongs to plant biotechnology field.More particularly, to a kind of CAPS molecule marks for identifying rice varieties
Note method and application.
Background technology
Rice Cropping kind is mainly Asian Cultivated Rice, and is divided into long-grained nonglutinous rice according to a series of phenotypic differences
（OryzasalivasubspIndica）And japonica rice（OryzasalivasubspJapanica）, China is planted according to different geographical
Condition and production requirement, Developing Cultivate thousands of kinds of long-grained nonglutinous rices and japonica rice variety.Ensure stable high yield, the seed of rice varieties is pure
Degree is one of factor of most critical.Rice varieties purity is traditionally identified, in different bearings such as Seedling Stage, heading stage, stage of wax ripeness
Phase is carried out.The character of identification mainly has three aspects, i.e. tree characteristics, panicled characters and grain character, but should in actual production
Use is influenceed greatly by the experience of assessor.And as modern breeding develops, the phenotypic character of part kind and classical Xian
Rice japonica rice differs greatly, for example, part japonica rice grain be not it is wealthy and short, it is thicker, oval or oval but be partial to paddy
Grain is long and narrow etc..Therefore, in production practices, it is necessary to find quick accurate identification long-grained nonglutinous rice, the method for japonica rice difference.For rice
Hereditary difference of the hereditary information DNA sequence dna between long-grained nonglutinous rice and japonica rice carries out the method that molecular markers for identification is efficiently and accurately.SNP
（single nucleotide polymorphism）Mark and the CAPS developed according to SNP marker are marked（cleaved
amplified polymorphic sequence）It is more abundant can to obtain that other molecular marking techniques can not show for technology
Polymorphism（RafalskiA.Applications of single nucleotide polymorphisms in
Cropgenetics.CurrOpin Plant Biol, 2002,5 (2) 94~100）.
The content of the invention
The technical problems to be solved by the invention are to overcome to lack existing for traditional rice long-grained nonglutinous rice and japonica rice variety authentication technique
Fall into and deficiency, there is provided it is a kind of can quick, precise Identification rice whether be long-grained nonglutinous rice or japonica rice variety CAPS molecule labelling methods.
Molecular labeling provided by the invention in actual applications, only needs PCR combination digestions, and cost is low, flux is high, high plus specificity,
It is highly suitable in production practices.
It is an object of the invention to provide a kind of specific molecular marker V14SNP1 of rice V14 genes.
It is a further object of the present invention to provide a kind of primer for detecting above-mentioned molecular labeling.
Another object of the present invention is to provide a kind of CAPS molecule labelling methods for identifying rice long-grained nonglutinous rice and japonica rice variety.
The above-mentioned purpose of the present invention is to give realization by the following technical programs：
A kind of specific molecular marker V14SNP1 of rice V14 genes, its nucleotide sequence in japonica rice such as SEQ ID NO：
Shown in 1, the sequence such as SEQ ID NO in long-grained nonglutinous rice：Shown in 2.
The specific molecular marker V14SNP1 of the present invention, it is in japonica rice variety and common wild-rice, long-grained nonglutinous rice and Nivara
All highly conserved in wild rice, there is a G ← → C SNP positions in sequences of the V14SNP1 in japonica rice and long-grained nonglutinous rice at 222bp
Point difference, the present invention compared for the V14SNP1 of numerous rice varieties, it was demonstrated that it is highly conserved in rice japonica rice and long-grained nonglutinous rice, table
It is bright to differentiate rice varieties using specific molecular marker V14SNP1.
Therefore, SEQ ID NO：1 and SEQ ID NO：Specific molecular marker V14SNP1 shown in 2 is in identification Indica
Application in rice and japonica rice variety is also in the scope of the present invention.
A kind of primer sets for being used to detect above-mentioned molecular labeling V14SNP1, the sequence such as SEQ ID NO of the primer：3、
SEQ ID NO：Shown in 4；
Sense primer：5’-TGAGAAACTTACCATTGGC-3’（ SEQ ID NO：3）；
Anti-sense primer：5’-AAGCATCCTCTTCATATCCTTG-3’（ SEQ ID NO：4）.
SEQ ID NO：3 and SEQ ID NO：Primer sets shown in 4 can be used as CAPS labeled primers to be used to identify rice varieties,
Therefore, application of the primer sets in identification rice long-grained nonglutinous rice and japonica rice variety is also in the scope of the present invention.
A kind of CAPS molecule labelling methods for identifying rice long-grained nonglutinous rice and japonica rice variety, comprise the following steps：
S1. rice sample genomic dna is extracted；
S2. with above-mentioned primer pair SEQ ID NO：3 and SEQ ID NO：4 pairs of step S1 genomic DNAs enter performing PCR amplification；
S3. step S2 PCR primer is passed throughHhaI digestion rear electrophoresis, obtain CAPS marks；
S4. electrophoresis result judges：The rice sample is long-grained nonglutinous rice if 440bp electrophoretic band is obtained, if obtaining 220bp electrophoresis
Band, then the rice varieties are japonica rice.
Preferably, the PCR reaction systems are 10 × PCR Buffer, 10 μ L, 10mmol/L dNTPs 0.2 μ L, 0.1 μ
Mol/L PCR primers each 1 μ L, High fidelity PCR polymerase 5U, the μ g of DNA profiling 0.2, deionized water are supplemented to 100 μ L.
Preferably, the PCR response procedures are 95 DEG C of pre-degeneration 2min；95 DEG C of 15sec, 52 DEG C of 20sec, 72 DEG C
30sec, totally 30 circulations；72 DEG C of extension 5min.
The molecular labeling V14SNP1 long-grained nonglutinous rices type of the present invention is compared with japonica rice type sequence, a G ← → C SNP site be present
Difference；Japonica rice is in the restrictive restriction endonuclease of site Beside neighbour's sequenceHhaI digestion recognition sites：GCGC.And long-grained nonglutinous rice is in the enzyme
Cutting the SNP mutation GCGG of recognition site, cause can not be by restriction enzymeHhaI digestions identify.Therefore existHhaI digestions are known
After other above PCR fragment, japonica rice fragment is digested as the DNA fragmentation of 220bp sizes, and long-grained nonglutinous rice still keeps the DNA of 440bp sizes
Fragment.
Above-mentioned molecular labeling V14SNP1 can also be used to Testing and appraisal parental rice homozygosity, therefore, the molecular labeling
Applications of the V14SNP1 in rice long-grained nonglutinous rice and the detection of japonica rice parental purity is also in the scope of the present invention.
Meanwhile application of the above-mentioned CAPS molecule labelling methods in rice long-grained nonglutinous rice and the detection of japonica rice parental purity is also in this hair
In bright protection domain.
Compared with prior art, the invention has the advantages that：
The invention provides a kind of CAPS molecule labelling methods for being used to identify rice long-grained nonglutinous rice and japonica rice variety.The CAPS molecules
Labeling method specificity is high, and the germ plasm resource that rice cultivar conveniently accurately can be carried out to long-grained nonglutinous rice and japonica rice is distinguished.Together
When, molecular labeling provided by the invention in actual applications, only needs PCR combination digestions, and cost is low, flux is high, specific height,
It is highly suitable for production practices.
Brief description of the drawings
Fig. 1 for be CAPS labeled primers PCR amplification andHhaI digestion result figures.Wherein, rice variety 1~7 is respectively
93-11, short-foot Nan Te, Guanglu ai 4, precious Shan 97, bright extensive 63, Huang Sizhan, Huang Huazhan；Japonica rice variety 1~7 is respectively Japan
Fine, 65 in platform, middle to spend 11, land reclamation and cultivation 58, cloud round-grained rice 23, Hubei Province is late No. 3, Shanxi rice No. 1.The pcr amplification product of above kind is carried outHhaI digestions result is that long-grained nonglutinous rice and Nivara wild rices fragment can not be digested, and japonica rice variety and common wild-rice fragment quilt
Digestion is 220bp DNA fragmentation.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
Limit in any form.Unless stated otherwise, the reagent of the invention used, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following examples agents useful for same and material are purchased in market.
Embodiment 1 differentiates the molecular labeling and detection primer of rice
The present inventor's early-stage Study finds that, there is a kind of molecular labeling V14-SNP1 in rice, its sequence in japonica rice is such as
SEQ ID NO：Shown in 1, the sequence such as SEQ ID NO in long-grained nonglutinous rice：It is 440bp shown in 2, and at 222bp, there is one
Individual G ← → C SNP site difference, thus it is speculated that go to identify rice long-grained nonglutinous rice and japonica rice variety using above-mentioned molecular labeling.Analysis hair
It is existing, because japonica rice is in the restrictive restriction endonuclease of SNP site site Beside neighbour's sequenceHhaI digestion recognition sites：GCGC, and Xian
Rice is caused in the SNP mutation GCGG of the digestion recognition site can not be by restriction enzymeHhaI digestions identify, therefore can pass through
CAPS molecule labelling methods remove Testing and appraisal rice long-grained nonglutinous rice and japonica rice variety, when withHhaMolecular labeling V14-SNP1 is known in I digestions
Afterwards, japonica rice variety can be digested as the DNA fragmentation of 220bp sizes, and long-grained nonglutinous rice still keeps the DNA fragmentation of 440bp sizes, thus come
Identify the long-grained nonglutinous rice and japonica rice variety of rice.
Meanwhile by comparing sequences of the molecular labeling V14-SNP1 in japonica rice and long-grained nonglutinous rice, devise following pcr amplification primer
Thing is as CAPS labeled primers：
Sense primer：5’-TGAGAAACTTACCATTGGC-3’（ SEQ ID NO：3）；
Anti-sense primer：5’-AAGCATCCTCTTCATATCCTTG-3’（ SEQ ID NO：4）.
PCR reaction systems are the μ L of 10 × PCR Buffer, 10 μ L, 10mmol/L dNTPs 0.2, and 0.1 μm of ol/L PCR draws
Thing each 1 μ L, High fidelity PCR polymerase 5U, the μ g of DNA profiling 0.2, deionized water are supplemented to 100 μ L.
PCR response procedures are 95 DEG C of pre-degeneration 2min；95 DEG C of 15sec, 52 DEG C of 20sec, 72 DEG C of 30sec, totally 30 are followed
Ring；72 DEG C of extension 5min.
Embodiment 2 identifies the CAPS molecule labelling methods of rice long-grained nonglutinous rice and japonica rice variety
The experiment material that the present invention is used to identify the CAPS molecule labelling methods of rice long-grained nonglutinous rice and japonica rice variety is including common wild
Rice and Nivara wild rices；Rice variety is respectively 93-11, short-foot Nan Te, Guanglu ai 4, precious Shan 97, and bright extensive 63, Huang Sizhan,
Huang Huazhan；Japonica rice variety is respectively Nipponbare, middle to spend 11 65 in platform, land reclamation and cultivation 58, cloud round-grained rice 23, Hubei Province evening No. 3, Shanxi rice No. 1.Tool
Body step is as follows：
1st, rice sample genomic dna extracts
（1）Rice leaf about 0.5g is taken, blade is shredded, is put into the mortar of precooling, liquid nitrogen is added, by blade grind into powder
Afterwards, it is put into 2 mL centrifuge tubes, adds 800 μ L 2 × CTAB extraction buffers, mixes, 65 DEG C of water-bath 30min；
（2）Add isometric chloroform, isoamyl alcohol and absolute ethyl alcohol（76：4：20）, 10 min are vibrated, are fully mixed；
（3）12min is centrifuged under conditions of 12000 revs/min, supernatant is transferred in another 1.5mL centrifuge tube, is added
Its 0.6 times of volume isopropanol or two volumes absolute ethyl alcohol, mix, 12000 revs/min of centrifugation 2min, remove supernatant, use 0.5ml
70% ethanol rinsing twice, is air-dried, is dissolved in 100-200 μ L 0.5 × TE buffer solutions；
（4）1 μ L are taken to be used for follow-up pcr amplification reaction.
2nd, pcr amplification reaction
Above-mentioned rice sample genomic dna is expanded with the CAPS labeled primers of embodiment 1；
The pcr amplification reaction system is：10 × PCR Buffer, 10 μ L, 10mmol/L dNTPs 0.2 μ L, 0.1 μm of ol/L
PCR primer each 1 μ L, High fidelity PCR polymerase 5U, the μ g of DNA profiling 0.2, deionized water are supplemented to 100 μ L.
The pcr amplification reaction is carried out on DNA cloning instrument, and PCR response procedures are 95 DEG C of pre-degeneration 2min；95℃
15sec, 52 DEG C of 20sec, 72 DEG C of 30sec, totally 30 circulations；72 DEG C of extension 5min, are saved backup with after 4 DEG C；
3rd, digestion
The pcr amplification product of above-mentioned rice sample is usedHhaI digestions, agarose of the pcr amplification product 1.2% after digestion
Electrophoresis 1.5h (5V/cm) on gel, ethidium bromide (EtBr) dyeing, is observed and photographed to record with gel imaging system.
4th, result and analysis
16 different rice material genomic DNAs are entered with performing PCR amplification, can amplify gem-pure single band, and 8
Individual stripe size is identical, through 3 repetitions, as a result stable and consistent（As shown in Figure 1A）.By positive and negative sequencing, 16 PCR productions
Thing length is 440bp；Also, PCR amplifications primer used is all correctly measured at both ends.Wherein, japonica rice variety and general
Common wild rice sequence is shown in sequence such as SEQ ID NO：Shown in 1, long-grained nonglutinous rice and Nivara wild rices sequence are such as SEQ ID NO：2 institutes
Show.
HhaAfter I digestions identify the PCR fragment of above-mentioned rice varieties, japonica rice fragment is digested as the DNA of 220bp sizes
Fragment, and long-grained nonglutinous rice still keeps the DNA fragmentation of 440bp sizes（As shown in Figure 1B）, therefore the CAPS molecule labelling methods of the present invention
It can be used to identify rice long-grained nonglutinous rice and japonica rice variety.
Sequence table
<110>Agricultural University Of South China
<120>A kind of CAPS molecule labelling methods for identifying rice varieties and application
<130> 1713457ZBSH042
<141> 2017-10-13
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 440
<212> DNA
<213>Rice (Oryza sativa)
<400> 1
gagaaactta ccattggcaa tattctacat ttccagcatt ctaatatatt ctcttactct 60
tttcaggatg atctatcagg tttggagtat cctggtgtac tttattcaaa caatcctcgt 120
gctccaatca agaaaccagg tatgaaaggc tgtgaaaatc cctgatatcc agtggttact 180
ttgtattaat actttattct ccaggccggg aaaaaccagc gctgaaacaa aactgggaag 240
gaagacaacc taaaacacga gacagatgtg acacttcaaa aaaagtcgat gctctgcatg 300
ccaagagtaa agctagcaga tcaactggtc ttgtggacat agataatgaa gtagaggtag 360
actactaagg ttgattcagt gtgtccatgt tcttgtttat gttcaatatt taacgtatgc 420
aggataaatg attgatttat 440
<210> 2
<211> 440
<212> DNA
<213>Rice (Oryza sativa)
<400> 2
gagaaactta ccattggcaa tattctacat ttccagcatt ctaatatatt ctcttactct 60
tttcaggatg atctatcagg tttggagtat cctggtgtac tttattcaaa caatcctcgt 120
gctccaatca agaaaccagg tatgaaaggc tgtgaaaatc cctgatatcc agtggttact 180
ttgtattaat actttattct ccaggccggg aaaaaccagc gctgaaacaa aactgggaag 240
gaagacaacc taaaacacga gacagatgtg acacttcaaa aaaagtcgat gctctgcatg 300
ccaagagtaa agctagcaga tcaactggtc ttgtggacat agataatgaa gtagaggtag 360
actactaagg ttgattcagt gtgtccatgt tcttgtttat gttcaatatt taacgtatgc 420
aggataaatg attgatttat 440
<210> 3
<211> 19
<212> DNA
<213>Rice (Oryza sativa)
<400> 3
tgagaaactt accattggc 19
<210> 4
<211> 22
<212> DNA
<213>Rice (Oryza sativa)
<400> 4
aagcatcctc ttcatatcct tg 22
Claims (9)
1. a kind of specific molecular marker V14SNP1 of rice V14 genes, it is characterised in that the V14SNP1 is in long-grained nonglutinous rice
Nucleotide sequence such as SEQ ID NO：Shown in 1, the sequence such as SEQ ID NO in japonica rice：Shown in 2.
2. applications of the specific molecular marker V14SNP1 in identification rice long-grained nonglutinous rice and japonica rice variety described in claim 1.
3. a kind of primer sets that the 1 molecular labeling V14SNP1 is required for test right, it is characterised in that the primer
Sequence such as SEQ ID NO：3, SEQ ID NO：Shown in 4.
4. application of the primer sets described in claim 3 in identification rice long-grained nonglutinous rice and japonica rice variety.
5. a kind of CAPS molecule labelling methods for identifying rice long-grained nonglutinous rice and japonica rice variety, it is characterised in that comprise the following steps：
S1. rice sample genomic dna is extracted；
S2. performing PCR amplification is entered with primer pair step S1 genomic DNAs described in claim 3；
S3. step S2 PCR primer is passed throughHhaI digestion rear electrophoresis, obtain CAPS marks；
S4. electrophoresis result judges：The rice sample is long-grained nonglutinous rice if 440bp electrophoretic band is obtained, if obtaining 220bp electrophoresis
Band, then the rice varieties are japonica rice.
6. according to the method for claim 5, it is characterised in that the PCR reaction systems are the μ of 10 × PCR Buffer 10
L, 10mmol/L dNTPs 0.2 μ L, 0.1 μm of ol/L PCR primer each 1 μ L, High fidelity PCR polymerase 5U, the μ of DNA profiling 0.2
G, deionized water are supplemented to 100 μ L.
7. according to the method for claim 5, it is characterised in that the PCR response procedures are 95 DEG C of pre-degeneration 2min；95℃
15sec, 52 DEG C of 20sec, 72 DEG C of 30sec, totally 30 circulations；72 DEG C of extension 5min.
8. applications of the molecular labeling V14SNP1 described in claim 1 in rice long-grained nonglutinous rice and the detection of japonica rice parental purity.
9. any one of the claim 5~7 CAPS molecule labelling methods are in rice long-grained nonglutinous rice and the detection of japonica rice parental purity
Using.
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| CN109735646A (en) * | 2019-01-07 | 2019-05-10 | 华南农业大学 | A kind of CAPS molecular labeling and method and its application for identifying rice varieties |
| CN110066886A (en) * | 2019-05-28 | 2019-07-30 | 广州瑞科基因科技有限公司 | A kind of reagent, method and application for identifying rice varieties |
| CN111485030A (en) * | 2019-01-25 | 2020-08-04 | 华南农业大学 | Application of rice transcription factor BTF3 in identification of japonica rice and indica rice and method for identifying japonica rice and indica rice |
| CN111485031A (en) * | 2019-01-25 | 2020-08-04 | 华南农业大学 | Rice molecular marker DOF8 and application thereof, and method for identifying japonica rice and indica rice by using rice molecular marker DOF8 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109735646A (en) * | 2019-01-07 | 2019-05-10 | 华南农业大学 | A kind of CAPS molecular labeling and method and its application for identifying rice varieties |
| CN109735646B (en) * | 2019-01-07 | 2022-04-08 | 华南农业大学 | CAPS molecular marker and method for identifying rice variety and application thereof |
| CN111485030A (en) * | 2019-01-25 | 2020-08-04 | 华南农业大学 | Application of rice transcription factor BTF3 in identification of japonica rice and indica rice and method for identifying japonica rice and indica rice |
| CN111485031A (en) * | 2019-01-25 | 2020-08-04 | 华南农业大学 | Rice molecular marker DOF8 and application thereof, and method for identifying japonica rice and indica rice by using rice molecular marker DOF8 |
| CN110066886A (en) * | 2019-05-28 | 2019-07-30 | 广州瑞科基因科技有限公司 | A kind of reagent, method and application for identifying rice varieties |
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