CN103146831B - Method for identifying rice and dry rice - Google Patents
Method for identifying rice and dry rice Download PDFInfo
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- CN103146831B CN103146831B CN201310087005.8A CN201310087005A CN103146831B CN 103146831 B CN103146831 B CN 103146831B CN 201310087005 A CN201310087005 A CN 201310087005A CN 103146831 B CN103146831 B CN 103146831B
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Abstract
The invention belongs to the field of utilization of biological discrimination and germplasm resources, and specifically relates to a method for rapidly and efficiently identifying rice and dry rice ecotype in places with cultivated rice. The invention utilizes five rice microsatellite (SSR) points with great difference in the rice and dry rice ecotype to design a molecular method for rapidly and efficiently identifying rice and dry rice ecotype. The method comprises the steps of sample DNA extraction, three-primer system PCR amplification and electrophoresis detection of amplification product, different genetypes are assigned according to results of a great deal of experiments, the integration judgement values of five points are calculated comprehensively, and the water and dying ecotype of cultivated rice is judged through a judging value. The method provided by the invention has the advantages that the accuracy rating is high, the adaptability is wide, the method is rapid and convenient, and the method has extensive application prospects on aspects of cultivated rice germplasm collection, judgment, research and utilization.
Description
Technical field
The present invention relates to a kind of qualification cultivated rice paddy rice or the ecotypic method of dryland rice, refer more particularly to one and utilize SSR mark fast, efficiently to identify the ecotypic molecular method of cultivated rice local variety paddy rice or dryland rice.
Technical background
Cultivated rice is one of most important food crop in the world, and the population over half for the whole world provides grain.Cultivated rice is produced the water consuming and is accounted for the over half of whole agricultural waters, and China has and exceedes 60% paddy field and can be subject to drought in various degree, and therefore drought and water shortage becomes one of major issue threatening China's grain security.Under severe like this situation, saving water, resisting drought rice (water-saving and drought-resistant rice, WDR) research and development just become direction important in rice breeding work, and it can contribute for the Sustainable development of Rice Production and the construction of harmonious ecology society.The core concept of saving water, resisting drought rice, exactly ubiquitous drought resistance in dryland rice is imported in the paddy rice of high yield, cultivate that output is high under normal condition, quality good and can under slight or medium drought condition, still can keep the cultivated rice kind of high yield and quality.Therefore collecting, identify, preserve and utilize paddy rice, Upland Rice Germplasm Resources is development and cultivation saving water, resisting drought rice and the crucial and guarantee that maintains China's grain-production Sustainable development.
Paddy rice (irrigated rice) and dryland rice (upland rice) be Asian Cultivated Rice (
oryza sativa) in two kinds of most important ecotypes, account for respectively 55% and 12% of rice cultivar germplasm area.Wherein paddy rice is adapted to have the Agro-habitats of irrigating with storing facilities of water, has the characteristics such as high-quality, high yield, disease and insect resistance; And dryland rice is often planted in middle-and-low-yielding fields, and lack and irrigate and water storage condition, therefore have the characteristics such as drought-enduring, water saving, nutrient efficient.But paddy rice and dryland rice are difficult to distinguish in phenotype, habitat simple classification in germplasm collection in the past and preservation work during often according to collection of resources, therefore easily cause rice cultivar germplasm resource water, land is ecotypic obscures, and has caused great puzzlement to utilization and the research of germ plasm resource.Therefore no matter at the collection and use of rice cultivar germplasm resource, and in the research and development of saving water, resisting drought rice, all need badly a kind of reliable and fast method local paddy rice and the dryland rice ecotype of planting of cultivated rice identified.
Although paddy rice, dryland rice do not have fairly obvious phenotypic difference, but due to paddy rice and dryland rice for a long time in different Agro-habitats and and the selective action of mankind's differentiation under, rice germplasm and dryland rice germplasm will inevitably produce the differentiation of certain level in heredity, and this differentiation embodies a concentrated reflection of on the gene relevant to drought stress.
Summary of the invention
The object of this invention is to provide a kind of reliably and distinguish fast the method for paddy rice and Upland Rice Germplasm Resources in qualification cultivated rice, its method adopting is to utilize simple repeated sequence (SSR) molecule marker that is positioned at drought-induced gene inside to distinguish fast the molecular assay method of paddy rice, dryland rice.Specifically utilize the specificity difference segment of the DNA sequence dna in the remarkable genetic variation and genetic differentiation of multiple generations site in paddy rice and dryland rice colony, design of amplification primers combination and derivative thereof, inclined to one side paddy rice type allelotype and dryland rice type allelotype are carried out respectively to assignment, and determine paddy rice and the ecotypic method of dryland rice according to these multidigit point qualification value sums.
In order to realize above-mentioned technical purpose, the invention provides a kind of qualification cultivated rice paddy rice or dryland rice germplasm ecotype method, its step comprising has: extract DNA sample from tissue or the organ of rice plant, DNA sample is carried out to polymerase chain (PCR) amplification, PCR product is carried out to electrophoresis detection, utilize analysis software to read PCR product clip size, determine the genotype of PCR product, pcr gene type is carried out to assignment, calculate comprehensive qualification value, according to the size of comprehensive qualification value, determine the ecotype of paddy rice or dryland rice.
Preferential embodiment of the present invention is that the tissue of described rice plant or organ include seed, seedling, strain, stem, the leaf of paddy rice.
Preferential embodiment of the present invention is that described polymerase chain (PCR) amplification system is TP-PCR system.
Preferential embodiment of the present invention is, in described TP-PCR system, every group of primer comprises three sequences, and described three sequences comprise a pair of SSR Auele Specific Primer and a M13 universal primer.
Preferential embodiment of the present invention is that a described M13 universal primer 5 ' is held fluorescent mark.
Preferential embodiment of the present invention is that a primer 5 ' end that in described a pair of SSR Auele Specific Primer, annealing temperature is higher connects one section of M13 sequence.
Preferential embodiment of the present invention is that described M13 sequence is CACGACGTTGTAAAACGAC.
Preferential embodiment of the present invention is, described genotype is 5 pairs of length oligonucleotide that is 13-40bp and derivative sequence thereof
SSR primer, described 5 pairs of SSR primer genetic information are as shown in table 1
Preferential embodiment of the present invention is that the reaction conditions (20ul system) of described polymerase chain (PCR) amplification system is
1) 10 × Taq damping fluid (containing 25mM MgCl2), 2 μ l
2) 10mM dNTP Mixture 0.4μl
3) 20 μ M primer 1 0.2 μ l
4) primer 2 (containing M13 sequence) 0.04 μ l after 20 μ M
5) 20 μ M fluorescent mark M13 primer 0.16 μ l
6) Taq DNA Polymerase 1Unit
7) DNA profiling 20-30ng
8) ddH2O (distilled water) complements to 20 μ l
Preferential embodiment of the present invention is that the response procedures (20ul system) of described polymerase chain (PCR) amplification system is:
1) 94 DEG C of denaturations 5 minutes;
2) 94 DEG C of sex change 40 seconds;
3) annealing temperature: 55-57.5 DEG C, annealing time: 30 seconds;
4) 72 DEG C are extended 40 seconds;
5) circulation 2), 3), 4) step, totally 34 circulations
6) 72 DEG C are extended 10 minutes.
Preferential embodiment of the present invention is that described electrophoresis detection is fluorescent capillary electrophoresis tube.
Preferential embodiment of the present invention is that described electrophoresis detection is that polyacrylamide gel electrophoresis detects.
Preferential embodiment of the present invention is that the information of the variant genotypic paddy rice in described 5 SSR sites, dryland rice qualification value is as shown in table 2:
Referring to table 2, determining the different Identity of allele values in each site, its be by the research and analysis to a large amount of (>500 part) experiment materials and according to the different allelotypes in 5 SSR sites the empirical value of the distribution frequency in paddy rice, dryland rice colony, obtain the qualification value of each site different genotype, the degree of this genotype deflection paddy rice of unit point qualification value representation or dryland rice, the value of qualification that its concrete meaning is genotype A is 0.1, and the sample that genotype is AA is that the likelihood ratio paddy rice of dryland rice is high by 10%.
The not homoallelic assignment of SSR mark of listing according to table 2, then calculates the comprehensive qualification value in 5 sites, and according to qualification value, the ecotype of germplasm is judged, the qualification accuracy rate of different range qualification value is as shown in table 3.Wherein, comprehensively the value of qualification is 5 SSR site qualification value sums.What table 3 showed is that different comprehensive qualifications are worth corresponding water, the land ecotype and put letter rate
What table 3 showed is the ecotypic letter rate of putting of judging paddy rice, dryland rice according to comprehensive qualification value
* accuracy rate represents, in the time differentiating that a certain kind is paddy rice or dryland rice, and the probability of this accuracy of judgement.
According to the inspection to from more than the 500 part of material in the whole world, there is the local qualification value of planting of 19.5% cultivated rice in-0.4 ~ 0.09 scope, utilize this technological method cannot judge its water, dryland rice attribute.
Utilization of the present invention produces the special primer of 5 SSR site designs of genetic variation and genetic differentiation in paddy rice, dryland rice colony, by to these 5 SSR sites not isoallele carry out assignment operation, and utilize the comprehensive qualification in 5 sites to be worth to judge water, the land attribute of cultivated rice.
Technique effect of the present invention is: authentication method is easy, and effect quickness and high efficiency can be identified more than 80% local kind of cultivated rice, Average Accuracy exceedes 85%, no matter for the categorised collection of germplasm, or the development and utilization aspect of dryland rice germplasm, good promotion and application prospect had.
Brief description of the drawings
Fig. 1, identity process figure of the present invention
Fig. 2, the genotype of utilizing GeneScaner V1.0 software to read the PCR product clip size in E1177 site (utilize the PCR product in the E1177 site of freeware GeneScaner V1.0 to two samples to read (parameter is selected: Size Standard GS500LIZ_3130, Sizing Default-NPP).In figure, X-coordinate represents the size (unit: base) of PCR product, and ordinate zou represents the power of PCR product signal; The large I of PCR product directly reads in software.Accompanying drawing comprises upper and lower two samples, and the PCR product size of first sample is 158 bp, and the PCR product size of second sample is 156 bp, corresponds respectively to different genotype).
Embodiment
embodiment 1:
Utilize paddy rice, the dryland rice ecotype of 30 parts of domestic rice cultivar germplasm resources of 5 pairs of SSR molecular markers for identification.Specific experiment material is in table 4.
30 parts of paddy rice from China different areas of table 4., the local material of planting of dryland rice.
Extract DNA sample from any organ or tissue (comprising seed, seedling, strain, stem, leaf etc.) of rice plant, and sample DNA is carried out to polymerase chain amplification, the polymerase chain amplification system that the present invention uses is conventional TP-PCR system, and the PCR instrument that any company produces and any biological reagent company's production and synthetic reagent all can use and achieve the goal.PCR reaction conditions and program following (20 μ l system) in the present invention:
PCR reaction conditions:
1) 10 × Taq damping fluid (containing 25mM MgCl2), 2 μ l
2) 10mM dNTP Mixture 0.4μl
3) primer 0.2 μ l before 20 μ M
4) primer (containing M13 sequence) 0.04 μ l after 20 μ M
5) 20 μ M fluorescent mark M13 primer 0.16 μ l
6) Taq DNA Polymerase 1Unit
7) DNA profiling 20-30ng
8) ddH2O (distilled water) complements to 20 μ l
PCR response procedures:
1) 94 DEG C of denaturations 5 minutes;
2) 94 DEG C of sex change 40 seconds;
3) annealing temperature: 55-57.5 DEG C, annealing time: 30 seconds;
4) 72 DEG C are extended 40 seconds;
5) circulation 2), 3), 4) step, totally 34 circulations
6) 72 DEG C are extended 10 minutes.
Electrophoresis detection
What the present invention adopted is that fluorescent capillary electrophoresis tube or polyacrylamide gel electrophoresis detect PCR product, and (parameter is selected: Size Standard GS500LIZ_3130 to utilize freeware GeneScaner V1.0 to read its clip size, Sizing Default-NPP), determine genotype, then the each genotype assignment providing according to table 2 and table 3, the ecotype of calculating and identification experiment material, qualification result can be with reference to shown in table 5.
Table 5. utilizes 5 pairs of SSR marks to 30 parts of paddy rice from China different areas, the local qualification result of planting material of dryland rice.
embodiment 2:
Utilize paddy rice, the dryland rice ecotype of the international rice cultivar germplasm resource of 5 couples of SSR molecular markers for identification 20Fen.Specific experiment material is in table 6.
20 parts of paddy rice from different areas, the world of table 6., the local material of planting of dryland rice.
Extract DNA sample from any organ or tissue (comprising seed, seedling, strain, stem, leaf etc.) of rice plant, and sample DNA is carried out to polymerase chain amplification.The polymerase chain amplification system that the present invention uses is conventional TP-PCR system, and the PCR instrument that any company produces and any biological reagent company's production and synthetic reagent all can use and achieve the goal.PCR reaction conditions and program following (20 μ l system):
PCR reaction conditions
1) 10 × Taq damping fluid (containing 25mM MgCl2), 2 μ l
2) 10mM dNTP Mixture 0.4μl
3) 20 μ M primer 1 0.2 μ l
4) primer 2 (containing M13 sequence) 0.04 μ l after 20 μ M
5) 20 μ M fluorescent mark M13 primer 0.16 μ l
6) Taq DNA Polymerase 1Unit
7) DNA profiling 20-30ng
8) ddH2O (distilled water) complements to 20 μ l
PCR response procedures:
1) 94 DEG C of denaturations 5 minutes;
2) 94 DEG C of sex change 40 seconds;
3) annealing temperature: 55-57.5 DEG C, annealing time: 30 seconds;
4) 72 DEG C are extended 40 seconds;
5) circulation 2), 3), 4) step, totally 34 circulations
6) 72 DEG C are extended 10 minutes.
Electrophoresis detection
What the present invention adopted is fluorescent capillary electrophoresis tube.PCR reaction product is carried out kapillary fluorescence electrophoresis and utilized freeware GeneScaner V1.0 to read (the parameter selection: Size Standard GS500LIZ_3130 of its clip size, Sizing Default-NPP), determine genotype, then the each genotype assignment providing according to table 2 and table 3, calculates and the ecotype of identification experiment material.Concrete qualification result is as shown in table 7.
Table 7. utilizes 5 pairs of SSR marks to 30 parts of paddy rice from China different areas, the local qualification result of planting material of dryland rice.
Claims (5)
1. identify the method for paddy rice and dryland rice for one kind, comprise the steps: to extract DNA sample from tissue or the organ of rice plant, DNA sample is carried out to polymerase chain amplification, PCR product is carried out to electrophoresis detection, utilize analysis software to read PCR product clip size, determine the genotype of PCR product, pcr gene type is carried out to assignment, calculate comprehensive qualification value, according to the size of comprehensive qualification value, determine the ecotype of paddy rice or dryland rice, it is characterized in that, utilize oligonucleotide that 5 pairs of length are 13-40bp and the SSR primer of derivative sequence thereof, described 5 pairs of SSR primers are as shown in table 1:
The variant genotypic paddy rice in described 5 SSR sites, dryland rice qualification are worth as shown in table 2:
The size of described comprehensive qualification value also determines that the ecotypic foundation of paddy rice or dryland rice is as shown in table 3:
Described polymerase chain amplification system is TP-PCR system, in described TP-PCR system, every group of primer comprises three sequences, described three sequences comprise a pair of SSR Auele Specific Primer and a M13 universal primer, a described M13 universal primer 5 ' end fluorescent mark, a primer 5 ' the end that in described a pair of SSR Auele Specific Primer, annealing temperature is higher connects one section of M13 sequence, and described M13 sequence is CACGACGTTGTAAAACGAC.
2. the method for qualification paddy rice according to claim 1 and dryland rice, is characterized in that the reaction conditions of described polymerase chain amplification system:
20ul system:
3. the method for qualification paddy rice according to claim 8 and dryland rice, is characterized in that the response procedures of described polymerase chain amplification system:
20ul system:
1) 94 DEG C of denaturations 5 minutes;
2) 94 DEG C of sex change 40 seconds;
3) annealing temperature: 55-57.5 DEG C, annealing time: 30 seconds;
4) 72 DEG C are extended 40 seconds;
5) circulation 2), 3), 4) step, totally 34 circulations
6) 72 DEG C are extended 10 minutes.
4. the method for qualification paddy rice according to claim 1 and dryland rice, is characterized in that, described electrophoresis detection is fluorescent capillary electrophoresis tube.
5. the method for qualification paddy rice according to claim 1 and dryland rice, is characterized in that, described electrophoresis detection is that polyacrylamide gel electrophoresis detects.
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| CN104561363A (en) * | 2015-02-04 | 2015-04-29 | 安徽省农业科学院水稻研究所 | Primer of snoRNA genetic molecular markers for screening drought-resistant rice varieties |
| CN105506141B (en) * | 2016-01-22 | 2019-04-16 | 江西省农业科学院水稻研究所 | Primer combination and its application for the analysis of long-grained nonglutinous rice improved variety genetic integrity |
| CN105543377B (en) * | 2016-01-22 | 2019-04-16 | 江西省农业科学院水稻研究所 | Primer combination and its application for the analysis of common wild-rice genetic integrity |
| CN107058508B (en) * | 2017-02-27 | 2020-04-17 | 浙江理工大学 | Salvia miltiorrhiza germplasm resource identification method |
| CN111172241B (en) * | 2019-12-10 | 2022-11-22 | 吉林省农业科学院 | Sheep four-base repeated microsatellite marker and screening method, primer set and application thereof |
Citations (1)
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| CN102220428A (en) * | 2011-05-11 | 2011-10-19 | 合肥丰乐种业股份有限公司 | Method for inspecting purity of two-line hybrid rice seeds by utilizing SSR (Simple Sequence Repeat) method |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220428A (en) * | 2011-05-11 | 2011-10-19 | 合肥丰乐种业股份有限公司 | Method for inspecting purity of two-line hybrid rice seeds by utilizing SSR (Simple Sequence Repeat) method |
Non-Patent Citations (6)
| Title |
|---|
| Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.);O. Panaud. X.Chen. S. R. McCouch;《Mol Gen Genet》;19961231;第597-607页 * |
| O. Panaud. X.Chen. S. R. McCouch.Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.).《Mol Gen Genet》.1996,第597-607页. |
| 利用SSR 标记分析部分粳稻品种的遗传多样性;宣云等;《核农学报》;20070331;第21卷(第3期);第217～220页 * |
| 利用水稻功能基因SSR标记鉴定水稻种质资源;赵勇等;《中国农业科学》;20020430;第35卷(第4期);第349-350页 * |
| 宣云等.利用SSR 标记分析部分粳稻品种的遗传多样性.《核农学报》.2007,第21卷(第3期),第217～220页. |
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