CN100562585C - Method of a kind of assistantly screening indica-type rice and japonica rice and primer special thereof - Google Patents
Method of a kind of assistantly screening indica-type rice and japonica rice and primer special thereof Download PDFInfo
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- CN100562585C CN100562585C CNB200810056529XA CN200810056529A CN100562585C CN 100562585 C CN100562585 C CN 100562585C CN B200810056529X A CNB200810056529X A CN B200810056529XA CN 200810056529 A CN200810056529 A CN 200810056529A CN 100562585 C CN100562585 C CN 100562585C
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Abstract
The invention discloses the method for a kind of assistantly screening indica-type rice and japonica rice.The method of assistantly screening indica-type rice provided by the present invention and japonica rice, be to detect in the paddy rice to be measured whether to lack among the AC132003 from 5 ' terminal 122836-124566 position nucleotide sequence, if disappearance, paddy rice then to be measured is candidate's long-grained nonglutinous rice, is candidate japonica rice if do not lack paddy rice then to be measured.The method of assisting sifting paddy rice indica and japonica subspecies of the present invention can be used for identifying in early days long-grained nonglutinous rice and japonica rice variety or local race, understand the genetic variation and genetic differentiation degree of long-grained nonglutinous rice and japonica rice, Xian, the heterotic utilization of japonica rice and cultivation high yield, fine hybridisation rice had important directive function, thereby optimization cross combination, accelerate breeding speed, reduce the breeding cost, have simple to operate, with low cost, the advantage that cycle is short, be suitable for applying, for the evaluation of paddy rice indica and japonica subspecies germplasm provides a kind of system of selection efficiently.
Description
Technical field
The present invention relates to method and the primer special thereof of a kind of assistantly screening indica-type rice and japonica rice.
Background technology
Cultivated rice is one of most important food crop in the world, its 11, in the cultivated rice domestication history in more than 500 year, for adapting to different agroecological environments, cultivated rice has formed abundant genetic diversity and obvious genetic differentiation, two the different ecotypes (Morishima H of long-grained nonglutinous rice (indica) and japonica rice (japonica) have been produced, 0ka HI.Phylogenetic differentiation of cultivated rice.XXII.Numerableevaluation of the Indica-Japonica differentiation.Japanese Journal ofBreeding, 1981,31:402-413; Glaszmann J C.Isozymes and classification ofAsian rice varieties.Theoretical and Applied Genetics, 1987,74:21-30; Wang Z Y, Tanksley S D.Restriction fragment length polymorphism in Oryzasativa L.Genome, 1989,32:1113-1118; Blair M W, Panaud O, McCouch S R.Inter-simple sequence repeat (ISSR) amplification for analysis ofmicrosatellite motif frequency and fingerprinting in rice (Oryza sativa L.) .Theoretical and Applied Genetics, 1999,98 (5): 780-782.)).On many forms and physiological character, exist evident difference (Morishima H between them, Oka H I.Phylogeneticdifferentiation of cultivated rice.Numerical evaluations of theindica-japonica differentiation.Jpn.J.Breed, 1981,31:402-413.WANGXiang-Kun, CHENG Kan-Sheng, HUAN Nai-Wei, LUO Jun, LU Yi-Xuan, LIUGuang-Rong.Study on two important rice types concerning the origin anddifferentiation of Asian cultivated rice.Acta Genetica Sinica, 1987,14 (4): 262-270.).Yet, the Xian of paddy rice-round-grained rice differentiation is an evolutionary process slowly, exists typical long-grained nonglutinous rice or japonica rice in many paddy rice local race, but also exists the Xian round-grained rice to break up unconspicuous osculant, be difficult to identify the Xian round-grained rice differentiation degree of more difficult research paddy rice with traditional method for these types.Therefore, the Marker gene and the molecule marker thereof of development of new are accurately identified long-grained nonglutinous rice and japonica rice, and very necessary with the evolution of its research paddy rice.
Plant cytochrome P450 is that molecular weight is the one erythrocruorin-sulphur ferritin of 40-60KD, similar.It is present in the vegetable cell in conjunction with two kinds of forms with solubility and film, but the reaction of catalysis number of chemical is avoided objectionable impurities infringing party mask the defence plant and played an important role.Also contain rice cytochrome P 450 gene in the paddy rice, as gene LOC_Os11g27730.
Summary of the invention
The method that the purpose of this invention is to provide a kind of assistantly screening indica-type rice and japonica rice.
The method of assistantly screening indica-type rice provided by the present invention and japonica rice, be to detect the nucleotide sequence that whether lacks in the paddy rice to be measured among the AC132003 from 5 ' terminal 122836-124566 position, if disappearance, paddy rice then to be measured is candidate's long-grained nonglutinous rice, is candidate japonica rice if do not lack paddy rice then to be measured.
Described disappearance specifically can detect by pcr amplification.
Wherein, the purpose fragment that increased in detecting of described pcr amplification comprises among the AC132003 in 5 ' terminal 122836-124566 position Nucleotide the fragment of 1000bp at least.
The concrete available agarose gel electrophoresis of the PCR product that obtains in the above-mentioned amplification detects.
During described pcr amplification detected, used primer specifically can be following primer: a sequence wherein was shown in sequence in the sequence table 1, and another sequence is shown in sequence in the sequence table 2.
In the practical application, the method of adoptable a kind of concrete assistantly screening indica-type rice and japonica rice is as follows: the genomic dna with paddy rice to be measured is a template, with nucleotide sequence is respectively that a pair of primer of sequence 1 and sequence 2 carries out pcr amplification in the sequence table, the product that amplification obtains detects with agarose gel electrophoresis, if product is at a band that shows on the agarose between the 1000-2000bp, paddy rice then to be measured is a candidate japonica rice, if product is being shown as a band that does not have between the 1000-2000bp on the sepharose, paddy rice then to be measured is candidate's long-grained nonglutinous rice.
Another object of the present invention provides the primer of a kind of assistantly screening indica-type rice and japonica rice.
The primer of assistantly screening indica-type rice provided by the present invention and japonica rice, can be among the pcr amplification AC132003 in 5 ' terminal 122836-124566 position Nucleotide the used any a pair of nucleotide sequence of 1000bp fragment at least.
Described primer specifically can be following primer: a sequence wherein is shown in sequence in the sequence table 1, and another sequence is shown in sequence in the sequence table 2.
The method of assisting sifting paddy rice indica and japonica subspecies of the present invention can grown early screening long-grained nonglutinous rice and japonica rice variety or local race, understand the genetic variation and genetic differentiation degree of long-grained nonglutinous rice and japonica rice, Xian, the heterotic utilization of japonica rice and cultivation high yield, fine hybridisation rice had important directive function, thereby optimization cross combination, accelerate breeding speed, reduce the breeding cost, have simple to operate, with low cost, the advantage that cycle is short, be suitable for applying, for the evaluation of paddy rice indica and japonica subspecies germplasm provides a kind of system of selection efficiently.
Description of drawings
Fig. 1 is genome sequence comparison result (from TIGR website) and the primer MVRT1D place genome sequence position view that list of gene LOC_Os11g27730 in the warm and fine 93-11 of Japan.
Fig. 2 is a template for the genomic dna with the warm and fine 93-11 of Japan, the result of the pcr amplification product under primer MVRT1D guiding.
Fig. 3 is a template for the genomic dna with 16 rice varieties and local race, the detected result of the pcr amplification product under primer MVRT1D guiding.
Embodiment
Used experimental technique is ordinary method if no special instructions among the following embodiment.
Rice varieties among the following embodiment and local race are all from national germplasm resource bank.
The discovery of embodiment 1, gene LOC_Os11g27730 different expression patterns in long-grained nonglutinous rice and japonica rice
At first the chip data of indica rice at different developmental phases and different tissues position carried out mining analysis, the result shows that gene LOC_Os11g27730 expression amount in long-grained nonglutinous rice is extremely low does not even express, and the expression amount in japonica rice is high.According to former research results, the major cause that causes gene expression amount difference may be the insertion/disappearance (InDel) of base sequence or the regulation and control that are subjected to other gene.
Method with comparative genomics, at first the genome sequence of gene LOC_Os11g27730 in rice variety 93-11 and japonica rice variety Japan is fine carried out nucleotide sequence comparison (Blast) and analyzed, found that there is disappearance (Fig. 1) really in this gene in 93-11.
For further verifying fine difference that whether exists on the genomic level of 93-11 and Japan, according to Japanese fine sequence, there are the zone design primer MVRT1D:MVRT1DF (sequence is shown in sequence in the sequence table 1) and the MVRT1DR (sequence is shown in sequence in the sequence table 2) of genome difference at the two, and are that template is carried out the PCR checking with the genomic dna of the warm and fine 93-11 of Japan respectively.The reaction system of pcr amplification is: oryza sativa genomic dna template 20ng, Taq Plus archaeal dna polymerase 0.5U, 2.0 μ l, 10 * PCR damping fluid (100mM TrisHCl pH9.0,500mM KCl, 15mM Mg
2+, 1%Triton X-100), 100 μ M dNTPs, forward primer 4 μ M, reverse primer 4 μ M are with DEPC water postreaction system to 20 μ l.The PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 30sec subsequently, 58 ℃ of 45sec, 72 ℃ of 1min30sec, totally 35 circulations; 72 ℃ of 10min again.By concentration is that 1%～1.2% agarose gel electrophoresis detects in the amplified production whether the purpose band is arranged.Detected result shows that primer MVRT1D has amplified production in Japan is fine, amplified production is at a band that is shown as on the sepharose between the 1000-2000bp, and in 93-11, there is not amplified production (Fig. 2, swimming lane 1 for fine with Japan be the product that template amplification obtains, swimming lane 2 is for being the product that template amplification obtains with 93-11,3 negative contrasts, Marker is the 1Kb DNA Ladder of Pu Boxin bio tech ltd, production code member: PBZ0304-1).
The feasibility checking of embodiment 2, usefulness primer MVRT1D assisting sifting paddy rice indica and japonica subspecies
Rice varieties and local race are all come national germplasm resource bank: 93-11, and thin numb toothed oak paddy is purple glutinous, Nepa (deep water rice), and osmanthus is towards No. 2, Aus6538 (Bamalaia), IR24, special blue or green, Japan is fine, short macrospecies, texture of wood (bare hull japonica rice), glutinous japonica rice, rich more, autumn light, No. 7, ruddiness and Yun Feng.
At first utilize respectively each 8 kinds and local race of random choose indica and japonica subspecies of morphological index and pertinent literature retrieval, as table 1:
The indica and japonica subspecies qualification result of table 1,16 rice varieties and local race
| The kind name | Affiliated subspecies | The place of production |
| 93-11 | Xian | China Zhejiang |
| Thin numb toothed oak paddy | Xian | Chinese yunnan |
| Purple glutinous | Xian | Chinese yunnan |
| Nepa (deep water rice, deep water rice) | Xian | Bangladesh |
| Osmanthus is towards No. 2 | Xian | GuangZhou, China |
| Aus6538(Bamalaia) | Xian | Burma |
| IR24 | Xian | World paddy rice institute |
| Special blue or green | Xian | GuangZhou, China |
| Japan is fine | Round-grained rice | Japan |
| Short macrospecies | Round-grained rice | China Anhui |
| Texture of wood (bare hull japonica rice) | Round-grained rice | Chinese yunnan |
| Glutinous japonica rice | Round-grained rice | Chinese yunnan |
| Rich more | Round-grained rice | Japan |
| Qiu Guang | Round-grained rice | Japan |
| Ruddiness | Round-grained rice | Japan |
| No. 7, cloud peak | Round-grained rice | Korea S |
Extract the genomic dna of above-mentioned different rice varieties and local race respectively, under the guiding of primer MVRT1D, carry out pcr amplification.Reaction system is as follows: oryza sativa genomic dna template 20ng, Taq Plus archaeal dna polymerase 0.5U, 2.0 μ l, 10 * PCR damping fluid (100mM TrisHCl pH9.0,500mM KCl, 15mM Mg
2+, 1%TritonX-100), 100 μ M dNTPs, forward primer 4 μ M, reverse primer 4 μ M are with DEPC water postreaction system to 20 μ l.The PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 30sec subsequently, 58 ℃ of 45sec, 72 ℃ of 1min30sec, totally 35 circulations; 72 ℃ of 10min again.Reaction finishes, and utilizing concentration is that 1% agarose gel electrophoresis detects the PCR product.3 repetitions are established in experiment.
The unanimity as a result of three repeated experiments, detected result as shown in Figure 3 (swimming lane 1-8 respectively rice variety and local race 93-11, thin numb toothed oak paddy, purple glutinous, Nepa (deep water rice, deep water rice), osmanthus towards No. 2, Aus6538 (Bamalaia), IR24 and special blue or green amplified production electrophoresis result; 9-16 is respectively the electrophoresis result of No. 7, fine, the short macrospecies of 8 japonica rice varieties and local race Japan, texture of wood (bare hull japonica rice), glutinous japonica rice, rich more, autumn light, ruddiness and Yun Feng).The result shows, primer MVRT1D is in 7 rice varieties and local race, all do not amplify banding pattern, identical with the amplification of 93-11, and in 7 japonica rice varieties and local race, all can amplify the fine identical banding pattern with Japan, amplified production is at a band that is shown as on the sepharose between the 1000-2000bp.Above result shows that InDel primer MVRT1D identifies that the accuracy of long-grained nonglutinous rice and japonica rice has all reached 100%.Therefore, MVRT1D can be used for paddy rice indica and japonica subspecies assisting sifting mark.
Sequence table
<160>2
<210>1
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
cctcatgttcctccgacttg 20
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
tgccgctttgcttatcttct 20
Claims (8)
1, the method for a kind of assistantly screening indica-type rice and japonica rice, be to detect the nucleotide sequence that whether lacks in the paddy rice to be measured among the AC132003 from 5 ' terminal 122836-124566 position, if disappearance, paddy rice then to be measured is candidate's long-grained nonglutinous rice, is candidate japonica rice if do not lack paddy rice then to be measured.
2, method according to claim 1 is characterized in that: described disappearance detects by pcr amplification.
3, method according to claim 2 is characterized in that: the purpose fragment that described pcr amplification is increased in detecting comprises among the AC132003 in 5 ' terminal 122836-124566 position Nucleotide the fragment of 1000bp at least.
4, method according to claim 3 is characterized in that: described pcr amplification detects with agarose gel electrophoresis in detecting.
5, according to claim 2,3 or 4 described methods, it is characterized in that: the primer sequence of using in the amplification described in described pcr amplification detects is shown in sequence in the sequence table 1 and sequence 2.
6, method according to claim 5, it is characterized in that: during described pcr amplification detects, the product that amplification obtains detects with agarose gel electrophoresis, if product is at a band that is shown as on the sepharose between the 1000-2000bp, paddy rice then to be measured is a candidate japonica rice, if product is being shown as a band that does not have between the 1000-2000bp on the sepharose, paddy rice then to be measured is candidate's long-grained nonglutinous rice.
7, the primer of assistantly screening indica-type rice and japonica rice, be among the pcr amplification AC132003 in 5 ' terminal 122836-124566 position Nucleotide the used a pair of nucleotide sequence of 1000bp fragment at least.
8, primer according to claim 7 is characterized in that: a sequence in the described primer is shown in sequence in the sequence table 1, and another sequence is shown in sequence in the sequence table 2.
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| CN108251552B (en) * | 2018-03-16 | 2020-11-03 | 中国科学院植物研究所 | Phosphoglycoside-oleate mutase gene fragment for increasing rice yield 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 |
| CN116058276A (en) * | 2022-07-19 | 2023-05-05 | 湖北大学 | Method for preparing strong-dominance indica-japonica intersubular hybrid rice by utilizing tetraploid rice restored diploid and application thereof |
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Non-Patent Citations (6)
| Title |
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| . GenBank登录号AC132003. 2004 |
| . GenBank登录号AC132003. 2004 * |
| Development of genome-wide DNA polymorphismdatabasefor map-based cloning of rice genes.. Shen Y J 等人.Plant Physiology,Vol.135 . 2004 |
| Development of genome-wide DNA polymorphismdatabasefor map-based cloning of rice genes.. Shen Y J 等人.Plant Physiology,Vol.135 . 2004 * |
| 籼稻93-11和粳稻日本晴DNA插入缺失差异片段揭示的水稻籼-粳分化. 蔡星星等.复旦学报（自然科学版），第45卷第3期. 2006 |
| 籼稻93-11和粳稻日本晴DNA插入缺失差异片段揭示的水稻籼-粳分化. 蔡星星等.复旦学报（自然科学版），第45卷第3期. 2006 * |
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