CN101408528A - Method for identifying Y excellent series hybrid rice germ plasm - Google Patents
Method for identifying Y excellent series hybrid rice germ plasm Download PDFInfo
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- CN101408528A CN101408528A CNA2007100358774A CN200710035877A CN101408528A CN 101408528 A CN101408528 A CN 101408528A CN A2007100358774 A CNA2007100358774 A CN A2007100358774A CN 200710035877 A CN200710035877 A CN 200710035877A CN 101408528 A CN101408528 A CN 101408528A
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Abstract
The invention relates to a method for identifying the germ plasm of Y-superior serial hybrid rice, comprising the following steps: (1) a primer which can be used for identifying the germ plasm of Y-superior serial hybrid rice is selected; (2) the PCR augmentation on rice samples to be identified is carried out by the obtained primer; (3) the electrophoresis detection for a DNA special marker is carried out; (4) the molecule id card number which is corresponding to the rice samples to be identified is manufactured according to the obtained DNA special marker scattergram, when the first digit of the number is '1', only one digit '1' can appear from the second digit to the fifth digit, and the rice samples to be indentified is Y-superior serial hybrid rice, otherwise the identifying conclusion is contrary. By making use of the method, the varieties and the parent-strain germ plasm of the Y-superior serial hybrid rice can be indentified. The invention has the advantages of short detecting time, large quantity, high efficiency and accurate seed purity identification, thereby the risk of the large-area production of the hybrid rice can be greatly reduced.
Description
Technical field
The present invention relates to a kind of authentication method of Y excellent series hybrid rice germ plasm, belong to plant variety authenticate technology field.
Background technology
Effectively guaranteeing hybrid rice seeds purity, is the important step that hybrid rice is applied, and sets up the technical system that a cover fast, accurately, is efficiently identified hybrid rice germ, is the key that addresses this problem.Field planting is identified and is considered to accurate, the most reliable method in the hybrid rice seeds purity authentication method at present.But field planting grows and is subject to the influence of external environment qualification cycle, has bigger limitation.At this problem, seed seed identification of morphology has successively been proposed, esterase isozyme identifies that methods such as DNA (abbreviation of English deoxyribonucleic acid, Chinese translation are DNA (deoxyribonucleic acid)) molecular markers for identification are used to identify seed purity of hybrid rice.But preceding two kinds of methods are limited in inter-variety variance, add esterase isozyme and have tissue specificity, and to the strict demand of having drawn materials, so these two kinds of methods all can't widespread use.The dna molecular marker evaluation rule is because of the genetic diversity of its reflection bion at dna level, have accurately and reliably, rich polymorphism, draw materials conveniently, be not subjected to advantages such as season and tissue specificity restriction, operative technique are simple and show one's talent, become the main object of present seed purity of hybrid rice authentication method research.Plant variety is removed labeled species with a plurality of primers in identifying, forms special dna fragmentation, and the genetic marker system of combining composition through the band line of electrophoresis formation is called dna fingerprinting, is the strongest molecular labeling system of present marked capacity.Since nineteen ninety-five, China scientific research institutions at different levels and scholar are used for crop varieties at dna fingerprinting and identify and seed purity that especially a large amount of research has been carried out in purity of hybrid evaluation aspect.Sum up present achievement in research, SSR (the abbreviation of English simple sequence repeat, the Chinese translation is a simple repeated sequence) molecular marking technique because of simple, fast, good reproducibility, quantity of information be relatively large, have characteristics such as codominance, can identify germplasm and resolution kind sibship accurately and efficiently.Along with development with SSR primer development technology is finished in rice genome order-checking, make paddy rice SSR labelling technique break through the high limitation of primer development cost, more be convenient to exchange because of the SSR primer, plurality of advantages such as applied widely, and become the first-selected molecular marking technique of setting up dna fingerprinting.
In recent years, the two-line hybrid rice research and development is rapid, particularly the seed selection of eurytopicity photo-thermo-sensitive genetic male sterile line Y58S is high yield, high-quality, how anti-two-line hybrid rice breeding of new variety has been established important foundation, a collection of is the Y major clique series hybrid rice new varieties of maternal preparation with Y58S owing to have high yield, high-quality, many plurality of advantages such as anti-, obtains large tracts of land rapidly and promote on producing.But the two-line hybrid rice production of hybrid seeds is subjected to the regulation and control of ecological factors such as light, temperature because of its maternal fertility, and there are some risks all the time in seed production purity, and the hybrid rice seed that obtains after the large tracts of land production requirement production of hybrid seeds can carry out purity detecting fast and accurately; Simultaneously, the protection of the kind of Y major clique row new varieties power also needs molecular labeling that new technical support is provided.Therefore, press for and set up a kind of molecular labeling and make up for the fast detecting of seed purity or the evaluation of Y major clique row kind germplasm technical support is provided.
Summary of the invention
The technical problem to be solved in the present invention is, deficiency at the prior art existence, a kind of method of carrying out Y major clique series hybrid rice kind Germplasm Identification with the dna molecular level is proposed, utilize this method can identify Y major clique series hybrid rice and parent's germplasm thereof, and can detect the germplasm of a large amount of seeds at short notice, thereby reach the purpose of identification of seed purity, reduce the risk that hybrid rice seeds is produced.
Technical solution of the present invention is that the method that described Y excellent series hybrid rice germ plasm is identified is following step:
(1), filter out the primer that can be used for the evaluation of Y excellent series hybrid rice germ plasm in the following manner:
1., in Gramene database (Cornell Univ USA builds), find 12 chromosomes of paddy rice, from every chromosome, select out 5-10 SSR primer at random.
2., 1. select out the primer that can be used for identifying the Y excellent series hybrid rice germ plasm the gained SSR primer from step, thisly can be used for identifying that the primer of Y excellent series hybrid rice germ plasm is the 5th chromosomal RM (primer numbering prefix) 164, the dna sequence dna of described RM164 is:
Forward sequence TCTTGCCCGTCACTGCAGATATCC,
Reverse sequence GCAGCCCTAATGCTACAATTCTTC,
Or the 7th chromosomal RM18, the dna sequence dna of described RM18 is:
Forward sequence TTCCCTCTCATGAGCTCCAT,
Reverse sequence GAGTGCCTGGCGCTGTAC,
Or the 10th chromosomal RM258, the dna sequence dna of described RM258 is:
Forward sequence TGCTGTATGTAGCTCGCACC,
Reverse sequence TGGCCTTTAAAGCTGTCGC,
Or the combination of above-mentioned primer.
(2), the primer that uses above-mentioned steps (1) gained to can be used for the evaluation of Y excellent series hybrid rice germ plasm carries out PCR (abbreviation of English Polymerase chain reaction, Chinese translation are the reaction of polymerase connection formula) amplification to sending mirror paddy rice sample:
1., utilize the PCR system to carry out pcr amplification, the composition of used PCR system is:
Concentration is the dna profiling 1 μ l of 25ng/ μ l
PH value is 8.3 10 * PCR damping fluid, 2 μ l
Concentration is the dNTPs 1 μ l of 2.5mM
Concentration is the Taq enzyme 0.2 μ l of 5U/ μ l
Concentration is the forward aligning primer 0.5 μ l of 10pM
Concentration is the negative sense aligning primer 0.5 μ l of 10pM
Deionization sterilized water 14.8l
2., the PCR system of utilizing above-mentioned steps 1. to provide is implemented pcr amplification according to following program:
A.94 under ℃ temperature above-mentioned PCR system is carried out the DNA degenerative treatments of 4 fens clock times;
B.94 under ℃ temperature to step a gained through the PCR system of DNA degenerative treatments carry out 45 second the DNA degenerative treatments;
C.52-59 the PCR system that under ℃ temperature above-mentioned steps b is provided is carried out renaturation processing in 45 seconds;
D.72 the PCR system of under ℃ temperature step c gained being handled through renaturation is carried out the synthetic extension processing of 45 DNA in second;
E. repeating step b～steps d in regular turn repeats 35～40 times;
F.72 under ℃ temperature step e gained is carried out 5 minutes synthetic extensions of DNA through the PCR system of 35～40 step b～steps d re-treatments and handle, get the DNA specific mark.Pcr amplification finishes.
(3), step (2) gained DNA specific mark is carried out electrophoresis detection:
1., using polyacrylamide content is that 6% gel carries out electrophoretic separation to step (2) gained DNA specific mark and handles 1.5～2 hours processing times;
2., to step 1. the DNA specific mark handled through electrophoretic separation of gained carry out silver and dye color development treatment and get DNA specific mark distribution plan.Electrophoresis detection finishes.
(4), make and send the corresponding molecule ID (identity number) card No. of paddy rice sample of reflecting according to step (3) gained DNA specific mark distribution plan.Concrete method for making is, obtain the position reference numbers " 1 " of the DNA specific mark demonstration of amplification in the DNA specific mark distribution plan, obtain the position reference numbers " 0 " that the DNA specific mark of amplification shows, form thus and send the corresponding molecule ID (identity number) card No. of paddy rice sample of reflecting.When first figure place of molecule ID (identity number) card No. was " 1 ", as long as second occur one digit number to the five-digit number and be " 1 ", can making this, to send mirror paddy rice sample be the expert's conclusion of Y major clique series hybrid rice, otherwise expert's conclusion is opposite.For example excellent No. 7 and the excellent No. 8 molecule ID (identity number) card No. of Y of excellent No. 1 of the Y among the Y major clique series hybrid rice, Y is respectively 10010011001100,10010011001000 and 10100110001001 in regular turn.
First figure place of the above-mentioned Y major clique series hybrid rice molecule ID (identity number) card No. that this step (4) is obtained is that 1 correspondence markings is carried out dna sequencing, obtain the sequence tagged site of excellent No. 1 of the Y (claim again Y two excellent No. 1) of one of Y major clique series hybrid rice, its sequence is as follows:
TCTTGCCCGTCACTGCAGATATCCGACACACACGGACATAAAAATATAGCCAAATCATATAGGAATTCAAAACACAAAGATTTAAAAGATACTGTGTTTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTGTGTGTGTGAGAGAGGAGCATCAGAGAGAGGCGGCGTTGGAGTAAACAAAAGGGGAAAAGAAGAATTGTAGCTTTTTTTTTTGAAACAGAAGAATTGTAGCATTAGGGCTGC，
The dna sequence dna of this sequence tagged site can be used as the replenishing of Y two excellent No. 1 Germplasm Identification of one of Y major clique series hybrid rice.
The invention has the beneficial effects as follows, utilize this method can identify Y major clique series hybrid rice kind and parent's germplasm thereof.Detection time is short, quantity is big, efficient is high, and seed purity is identified accurately, thereby can be significantly reduced the risk that the hybrid rice large tracts of land is produced.
Embodiment:
Embodiment 1:
(1), filter out the primer that can be used for the evaluation of Y excellent series hybrid rice germ plasm in the following manner:
1., in the Gramene database, find 12 chromosomes of paddy rice, from every chromosome, select out 5 SSR primers at random.
2., 1. find out the 5th chromosomal RM164 the gained SSR primer, the dna sequence dna of this RM164 is from step:
Forward sequence TCTTGCCCGTCACTGCAGATATCC,
Reverse sequence GCAGCCCTAATGCTACAATTCTTC.
(2), use above-mentioned steps (1) gained primer to carry out pcr amplification to sending mirror paddy rice sample 07A1 and paddy rice sample 07A2:
1., utilize the PCR system to carry out pcr amplification, the composition of used PCR system is:
Concentration is the dna profiling 1 μ l of 25ng/ μ l
PH value is 8.3 10 * PCR damping fluid, 2 μ l
Concentration is the dNTPs 1 μ l of 2.5mM
Concentration is the Taq enzyme 0.2 μ l of 5U/l
Concentration is the forward aligning primer 0.5 μ l of 10pM
Concentration is the negative sense aligning primer 0.5 μ l of 10pM
Deionization sterilized water 14.8 μ l
2., the PCR system of utilizing above-mentioned steps 1. to provide is implemented pcr amplification according to following program:
A.94 under ℃ temperature above-mentioned PCR system is carried out the DNA degenerative treatments of 4 fens clock times;
B.94 under ℃ temperature to step a gained through the PCR system of DNA degenerative treatments carry out 45 second the DNA degenerative treatments;
C.52 the PCR system that under ℃ temperature above-mentioned steps b is provided is carried out renaturation processing in 45 seconds;
D.72 the PCR system of under ℃ temperature step c gained being handled through renaturation is carried out the synthetic extension processing of 45 DNA in second;
E. repeating step b～steps d in regular turn repeats 35 times;
F.72 under ℃ temperature step e gained is carried out 5 minutes synthetic extensions of DNA through the PCR system of 35 step b～steps d re-treatments and handle, get the DNA specific mark.Pcr amplification finishes.
(3), step (2) gained DNA specific mark is carried out electrophoresis detection:
1., using polyacrylamide content is that 6% gel carries out electrophoretic separation to step (2) gained DNA specific mark and handles 1.5 hours processing times;
2., to step 1. the DNA specific mark handled through electrophoretic separation of gained carry out silver and dye color development treatment and get DNA specific mark distribution plan.Electrophoresis detection finishes.
(4), make and send reflect paddy rice sample 07A1 and the corresponding molecule ID (identity number) card No. of paddy rice sample 07A2 according to step (2) gained DNA specific mark distribution plan.Obtain the position reference numbers " 1 " that the DNA specific mark of amplification shows in the DNA specific mark distribution plan, obtain the position reference numbers " 0 " that the DNA specific mark of amplification shows, form thus and send the corresponding molecule ID (identity number) card No. of paddy rice sample of reflecting.Qualification result shows that the preceding five-digit number of the molecule ID (identity number) card No. of paddy rice sample 07A1 is 10010, and to send the germplasm of the paddy rice sample 07A1 that reflects be the expert's conclusion of Y major clique series hybrid rice so can draw; The preceding five-digit number of the molecule ID (identity number) card No. of paddy rice sample 07A2 is 00011, is non-Y major clique series hybrid rice kind so send mirror paddy rice sample 07A2.
Embodiment 2:
(1), filter out the primer that can be used for the evaluation of Y excellent series hybrid rice germ plasm in the following manner:
1., in the Gramene database, find 12 chromosomes of paddy rice, from every chromosome, select out 10 SSR primers at random.
2., 1. find out primer the 5th chromosomal RM164, the 7th chromosomal RM18 and the 10th chromosomal RM258 that can be used for identifying the Y excellent series hybrid rice germ plasm the gained SSR primer from step, wherein,
The dna sequence dna of the 5th chromosomal RM164 is:
Forward sequence TCTTGCCCGTCACTGCAGATATCC,
Reverse sequence GCAGCCCTAATGCTACAATTCTTC,
The dna sequence dna of the 7th chromosomal RM18 is:
Forward sequence TTCCCTCTCATGAGCTCCAT,
Reverse sequence GAGTGCCTGGCGCTGTAC,
The dna sequence dna of the 10th chromosomal RM258 is:
Forward sequence TGCTGTATGTAGCTCGCACC,
Reverse sequence TGGCCTTTAAAGCTGTCGC.
(2), use above-mentioned steps (1) gained primer to carry out pcr amplification to sending mirror paddy rice sample 07B1 and paddy rice sample 07B2:
1., utilize the PCR system to carry out pcr amplification, the composition of used PCR system is:
Concentration is the dna profiling 1 μ l of 25ng/ μ l
PH value is 8.3 10 * PCR damping fluid, 2 μ l
Concentration is the dNTPs 1 μ l of 2.5mM
Concentration is the Taq enzyme 0.2l of 5U/ μ l
Concentration is the forward aligning primer 0.5 μ l of 10pM
Concentration is the negative sense aligning primer 0.5 μ l of 10pM
Deionization sterilized water 14.8 μ l
2., the PCR system of utilizing above-mentioned steps 1. to provide is implemented pcr amplification according to following program:
A.94 under ℃ temperature above-mentioned PCR system is carried out the DNA degenerative treatments of 4 fens clock times;
B.94 under ℃ temperature to step a gained through the PCR system of DNA degenerative treatments carry out 45 second the DNA degenerative treatments;
C.59 the PCR system that under ℃ temperature above-mentioned steps b is provided is carried out renaturation processing in 45 seconds;
D.72 the PCR system of under ℃ temperature step c gained being handled through renaturation is carried out the synthetic extension processing of 45 DNA in second;
E. repeating step b～steps d in regular turn repeats 40 times;
F.72 under ℃ temperature step e gained is carried out 5 minutes synthetic extensions of DNA through the PCR system of 40 step b～steps d re-treatments and handle, get the DNA specific mark, pcr amplification finishes.
(3), step (2) gained DNA specific mark is carried out electrophoresis detection:
1., using polyacrylamide content is that 6% gel carries out electrophoretic separation to step (2) gained DNA specific mark and handles 2 hours processing times;
2., to step 1. the DNA specific mark handled through electrophoretic separation of gained carry out silver and dye color development treatment and get DNA specific mark distribution plan, electrophoresis detection finishes.
(4), make and send reflect paddy rice sample 07B1 and the corresponding molecule ID (identity number) card No. of paddy rice 07B2 according to step (2) gained DNA specific mark distribution plan.Obtain the position reference numbers " 1 " that the DNA specific mark of amplification shows in the DNA specific mark distribution plan, obtain the position reference numbers " 0 " that the DNA specific mark of amplification shows, form thus and send the corresponding molecule ID (identity number) card No. of paddy rice sample of reflecting.Qualification result shows that sending the molecule ID (identity number) card No. of mirror paddy rice sample 07B1 is 10100110001001, is the expert's conclusion of Y major clique series hybrid rice so can draw paddy rice sample 07B1.This molecule ID (identity number) card No. 10100110001001 and above-mentioned Y major clique series hybrid rice molecule ID (identity number) card No. contrast can further be drawn this, and to send mirror paddy rice sample 07B1 be excellent No. 8 expert's conclusion of Y of one of Y major clique series hybrid rice; Sending the molecule ID (identity number) card No. of mirror paddy rice sample 07B2 is 10010011001000, also is the expert's conclusion of Y major clique series hybrid rice so can draw paddy rice sample 07B2.This molecule ID (identity number) card No. 10010011001000 and above-mentioned Y major clique series hybrid rice molecule ID (identity number) card No. contrast can further be drawn this, and to send mirror paddy rice sample 07B2 be excellent No. 7 expert's conclusion of Y of one of Y major clique series hybrid rice.
Claims (1)
1, a kind of authentication method of Y excellent series hybrid rice germ plasm, this method are following step:
(1), filter out the primer that can be used for the evaluation of Y excellent series hybrid rice germ plasm in the following manner:
1., in the Gramene database, find 12 chromosomes of paddy rice, from every chromosome, select out 5-10 SSR primer at random,
2., 1. select out the primer that can be used for identifying the Y excellent series hybrid rice germ plasm the gained SSR primer, thisly can be used for identifying that the primer of Y excellent series hybrid rice germ plasm is the 5th chromosomal RM164, the dna sequence dna of described RM164 is from step:
Forward sequence TCTTGCCCGTCACTGCAGATATCC,
Reverse sequence GCAGCCCTAATGCTACAATTCTTC,
Or the 7th chromosomal RM18, the dna sequence dna of described RM18 is:
Forward sequence TTCCCTCTCATGAGCTCCAT,
Reverse sequence GAGTGCCTGGCGCTGTAC,
Or the 10th chromosomal RM258, the dna sequence dna of described RM258 is:
Forward sequence TGCTGTATGTAGCTCGCACC,
Reverse sequence TGGCCTTTAAAGCTGTCGC,
Or the combination of above-mentioned primer,
(2), the primer that uses above-mentioned steps (1) gained to can be used for the evaluation of Y excellent series hybrid rice germ plasm carries out pcr amplification to sending mirror paddy rice sample:
1., utilize the PCR system to carry out pcr amplification, the composition of used PCR system is:
Concentration is the dna profiling 1 μ l of 25ng/ μ l
PH value is 8.3 10 * PCR damping fluid, 2 μ l
Concentration is the dNTPs 1 μ l of 2.5mM
Concentration is the Taq enzyme 0.2 μ l of 5U/ μ l
Concentration is the forward aligning primer 0.5 μ l of 10pM
Concentration is the negative sense aligning primer 0.5 μ l of 10pM
Deionization sterilized water 14.8 μ l
2., the PCR system of utilizing above-mentioned steps 1. to provide is implemented pcr amplification according to following program:
A.94 under ℃ temperature above-mentioned PCR system is carried out the DNA degenerative treatments of 4 fens clock times;
B.94 under ℃ temperature to step a gained through the PCR system of DNA degenerative treatments carry out 45 second the DNA degenerative treatments;
C.52-59 the PCR system that under ℃ temperature above-mentioned steps b is provided is carried out renaturation processing in 45 seconds;
D.72 the PCR system of under ℃ temperature step c gained being handled through renaturation is carried out the synthetic extension processing of 45 DNA in second;
E. repeating step b～steps d in regular turn repeats 35～40 times;
F.72 under ℃ temperature step e gained is carried out 5 minutes synthetic extensions of DNA through the PCR system of 35～40 step b～steps d re-treatments and handle, get the DNA specific mark, pcr amplification finishes,
(3), step (2) gained DNA specific mark is carried out electrophoresis detection:
1., using polyacrylamide content is that 6% gel carries out electrophoretic separation to step (2) gained DNA specific mark and handles 1.5～2 hours processing times;
2., to step 1. the DNA specific mark handled through electrophoretic separation of gained carry out silver and dye color development treatment and get DNA specific mark distribution plan, electrophoresis detection finishes,
(4), make and send the corresponding molecule ID (identity number) card No. of paddy rice sample of reflecting according to step (3) gained DNA specific mark distribution plan, concrete method for making is, obtain the position reference numbers " 1 " of the DNA specific mark demonstration of amplification in the DNA specific mark distribution plan, obtain the position reference numbers " 0 " of the DNA specific mark demonstration of amplification, form thus and send mirror paddy rice sample corresponding molecule ID (identity number) card No., when first figure place of molecule ID (identity number) card No. is " 1 ", be " 1 " as long as second occurs one digit number to the five-digit number, can making this, to send mirror paddy rice sample be the expert's conclusion of Y major clique series hybrid rice, otherwise expert's conclusion is opposite.
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Cited By (4)
| 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 |
| CN103194539A (en) * | 2013-04-09 | 2013-07-10 | 中国农业科学院麻类研究所 | Method for identifying ramie variety by simple sequence repeat (SSR) molecular marker |
| CN105734123A (en) * | 2016-02-02 | 2016-07-06 | 陈日胜 | PCR identification method for sea rice chromosome based on sea rice DNA variation mark and relevant PCR primer and kit |
| CN111507444A (en) * | 2020-04-20 | 2020-08-07 | 甘肃烽火台数据信息技术有限责任公司 | Method and equipment for obtaining germplasm and medicinal material traceability data based on SSR (simple sequence repeat) |
-
2007
- 2007-10-10 CN CNA2007100358774A patent/CN101408528A/en active Pending
Cited By (6)
| 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 |
| CN103194539A (en) * | 2013-04-09 | 2013-07-10 | 中国农业科学院麻类研究所 | Method for identifying ramie variety by simple sequence repeat (SSR) molecular marker |
| CN103194539B (en) * | 2013-04-09 | 2014-07-09 | 中国农业科学院麻类研究所 | Method for identifying ramie variety by simple sequence repeat (SSR) molecular marker |
| CN105734123A (en) * | 2016-02-02 | 2016-07-06 | 陈日胜 | PCR identification method for sea rice chromosome based on sea rice DNA variation mark and relevant PCR primer and kit |
| CN105734123B (en) * | 2016-02-02 | 2019-07-05 | 陈日胜 | The PCR identification method and related PCR primer and kit of extra large rice crops chromosome based on extra large rice crops DNA variation label |
| CN111507444A (en) * | 2020-04-20 | 2020-08-07 | 甘肃烽火台数据信息技术有限责任公司 | Method and equipment for obtaining germplasm and medicinal material traceability data based on SSR (simple sequence repeat) |
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Application publication date: 20090415 |