CN110904267A - Method for identifying rice variety by using 1-3 molecular markers - Google Patents
Method for identifying rice variety by using 1-3 molecular markers Download PDFInfo
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- CN110904267A CN110904267A CN201911386868.9A CN201911386868A CN110904267A CN 110904267 A CN110904267 A CN 110904267A CN 201911386868 A CN201911386868 A CN 201911386868A CN 110904267 A CN110904267 A CN 110904267A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a method for identifying rice varieties by using 1-3 molecular markers, which comprises the following steps: establishing a standard substance combination containing a plurality of rice varieties by taking known rice varieties as standard substances, and screening SSR markers capable of specifically identifying corresponding rice standard substances aiming at least one rice variety in the standard substance combination; aiming at a certain standard substance, performing PCR amplification and gel electrophoresis analysis on all rice in the standard substance combination by using an SSR marker specifically identified by the standard substance to obtain an electrophoresis pattern, numbering the standard substance according to pattern band information by using a 'variety name + SSR marker + band position', and thus forming numbering information of a plurality of standard substances; according to the suspected variety of the rice variety to be identified, the SSR markers of the corresponding variety are correspondingly selected from the standard substance combination for PCR amplification and gel electrophoresis analysis, and the authenticity of the rice variety to be detected is judged according to the map. The method has high practical application value and can save the medicine and labor cost.
Description
Technical Field
The invention relates to identification of variety resources, in particular to a method for identifying rice varieties by using 1-3 molecular markers.
Background
The rice is the first large grain crop in China, more than half of people use the rice as staple food, the variety is more due to factors such as variety characteristics, ecological conditions of planting areas, farming systems, farmer preferences and the like in production, and scientific workers, farmers, consumers and crop managers hope to have a simple, quick and effective detection method for variety authenticity and purity identification and identification of rice brands in markets.
Disclosure of Invention
The invention provides a method for identifying rice varieties by using 1-3 molecular markers, which is simple to operate and can be used for identifying the authenticity of conventional varieties.
The invention provides a method for identifying rice varieties by using 1-3 molecular markers, which comprises the following steps:
(1) the method comprises the steps of establishing a standard substance combination containing a plurality of rice varieties by taking known rice varieties as standard substances, and screening SSR markers capable of specifically identifying the corresponding rice standard substances aiming at least one rice variety in the standard substance combination, wherein one rice variety corresponds to 1-3 SSR markers;
(2) aiming at a certain standard substance, performing PCR amplification and gel electrophoresis analysis on all rice in the standard substance combination by using an SSR marker specifically identified by the standard substance to obtain an electrophoresis pattern, numbering the standard substance according to pattern band information by using a 'variety name + SSR marker + band position', and thus forming numbering information of a plurality of standard substances;
(3) according to the suspected variety of the rice variety to be identified, the SSR markers of the corresponding variety are correspondingly selected from the standard substance combination for PCR amplification and gel electrophoresis analysis, and the authenticity of the rice variety to be detected is judged according to the map.
And (3) performing PCR amplification and gel electrophoresis analysis on the rice variety to be identified and the corresponding real rice variety in the standard product combination at the same time, wherein if the two strips are consistent, the rice variety to be identified is real, and otherwise, the rice variety is a fake variety. Because the screened SSR markers have uniqueness and specificity, only real rice varieties can appear in specific positions, and other varieties cannot have specific bands.
Or, in the step (3), performing PCR amplification and gel electrophoresis analysis on the rice variety to be identified, and according to the position of the strip in the map, if the position of the strip is consistent with the serial number position of the corresponding variety, determining that the rice variety to be identified is true, otherwise, determining that the rice variety is a fake variety. All the strips of the variety are made into Marker-shaped strips, and then the strip combination corresponding to the detected variety has uniqueness, so that the detection can be realized.
The strip positions, i.e., the upper and lower positions in the electropherogram, are numbered sequentially upward, e.g., the lowermost strip position is numbered 1.
The standard rice product comprises 88 of Zhejiang japonica rice, 100 of Zhejiang japonica rice and 96 of Zhejiang japonica rice. For example, the rice standard may be 65 rice varieties as shown in table 1.
The SSR for identifying Zhejiang japonica 88 is marked as RM5420, the SSR for identifying Zhejiang japonica 100 is marked as RM1364+ RM3414, and the SSR for identifying Zhejiang japonica 96 is marked as RM6404+ RM6327+ RM 1287.
In particular, the method comprises the following steps of,
forward primer for RM 5420: 5'-CCTGATCTCAACACACACGC-3' the flow of the air in the air conditioner,
reverse primer for RM 5420: 5'-GAAGTCTTGTTGCGCGTATG-3', respectively;
forward primer for RM 1364: 5'-AAGAAATTCAAAACACATGA-3' the flow of the air in the air conditioner,
reverse primer for RM 1364: 5'-AAAACATCTACTTTGATCCA-3', respectively;
forward primer of RM 3414: 5'-TAGGGCAATTGTGCAAGTGG-3' the flow of the air in the air conditioner,
reverse primer for RM 3414: 5'-TTGGGAATTGGGTAGGACAG-3', respectively;
forward primer for RM 6404: 5'-GGGATGATGGATCGGGAG-3' the flow of the air in the air conditioner,
reverse primer for RM 6404: 5'-CTACCAGCCTTGTTTCCTCG-3', respectively;
forward primer for RM 6327: 5'-CAGCCTAGGGCGTCATAGAC-3' the flow of the air in the air conditioner,
reverse primer for RM 6327: 5'-GATTGGGTGATGGATAGCAC-3', respectively;
forward primer of RM 1287: 5'-GGAAGCATCATGCAATAGCC-3' the flow of the air in the air conditioner,
reverse primer for RM 1287: 5'-GGCCGTAGTTTTGCTACTGC-3' are provided.
Has the advantages that:
the SSR markers are simple to operate, high in efficiency and good in repeatability, and for the authenticity of a certain variety, the method can utilize 1-3 SSR markers for successful identification.
The variety is numbered by the 'variety name-SSR mark-strip position' to form an information base, so that the mark can be used, and the strip can identify the variety at the position simply and clearly. .
The variety is identified by using 1-3 marks, the practical application value is high, the variety can be accurately identified by using 1-3 marks, the medicine and labor cost can be saved in the process of actually identifying the variety, and the product can be used as a computer operation interface, is clear and more visual to agricultural science and technology workers, consumers and farmers.
Drawings
FIG. 1 is a graph showing the detection result of marker RM 5420;
FIG. 2 is a graph showing the detection result of the marker RM 1364;
FIG. 3 is a graph showing the detection result of marker RM 3414;
FIG. 4 is a graph showing the detection result of marker RM 6404;
FIG. 5 is a graph showing the detection result of marker RM 6327;
FIG. 6 is a graph showing the detection result of marker RM 1287.
Detailed Description
The invention will be further elucidated with reference to the following specific examples.
Example 1
1. Material source and labeling:
65 parts of rice cultivars were used as test materials (Table 1). Bongjing 15, Jiahe 236, Jiahe 268, Zhongjing No. 6, Zhongjing 12, Zhongjing No. 2, Zhongjing 15 and Zhongjing 16 are purchased from Jiaxing green agricultural seeds Co., Ltd; zhengjing tea 16, hong jing 25, Jia 16, Hanzhen, Hanxiang, Zhe jing 165 are purchased from Olympic Co., Ltd of Huzhou family; zhendao 521, Zhendao 522, Zhendao 523, Changjing 11, Suxiu 867, Chunjiang 151 and Chunjiang 153 were purchased from Shaoxing Shunhada Semiji.
TABLE 1
The SSRs employed in this example are labeled RM5420, RM1364, RM3414, RM6404, RM6327, RM1287, where:
RM5420 has forward and reverse primers, the nucleotide sequence of the forward primer from 5 'end to 3' end is CCTGATCTCAACACACACGC, and the nucleotide sequence of the reverse primer from 5 'end to 3' end is GAAGTCTTGTTGCGCGTATG.
RM1364 has forward and reverse primers, the nucleotide sequence of the forward primer from 5 'end to 3' end is AAGAAATTCAAAACACATGA, and the nucleotide sequence of the reverse primer from 5 'end to 3' end is AAAACATCTACTTTGATCCA.
RM3414 has forward and reverse primers, the nucleotide sequence of the forward primer from 5 'end to 3' end is TAGGGCAATTGTGCAAGTGG, and the nucleotide sequence of the reverse primer from 5 'end to 3' end is TTGGGAATTGGGTAGGACAG.
RM6404 has forward and reverse primers, the nucleotide sequence of the forward primer from 5 'end to 3' end is GGGATGATGGATCGGGAG, and the nucleotide sequence of the reverse primer from 5 'end to 3' end is CTACCAGCCTTGTTTCCTCG.
RM6327 has forward and reverse primers, the nucleotide sequence of the forward primer from 5 'end to 3' end is CAGCCTAGGGCGTCATAGAC, and the nucleotide sequence of the reverse primer from 5 'end to 3' end is GATTGGGTGATGGATAGCAC.
RM1287 has forward and reverse primers, wherein the nucleotide sequence of the forward primer from 5 'end to 3' end is GGAAGCATCATGCAATAGCC, and the nucleotide sequence of the reverse primer from 5 'end to 3' end is GGCCGTAGTTTTGCTACTGC.
2. Extraction of DNA by CTAB method:
1) fresh rice leaves of 2-3cm in length are taken and put into a 2ml centrifuge tube, and a steel ball is put into each tube, and the rice leaves are fully ground by using a grinding oscillator.
2) Adding 500ml CTAB extractive solution, and placing in 65 deg.C water bath for 30-60 min.
3) The tube was removed and cooled for 10min, and 500. mu.L of chloroform was added and shaken.
4) Placing into a centrifuge, rotating at 12000r/min for 7min, and sucking 500 μ L of supernatant and 1.5ml test tube.
5) Then 500. mu.L of ice isopropanol was added to a 1.5ml centrifuge tube, centrifuged at 12,000r/min for 7min, the supernatant was discarded, and 800. mu.L of 75% ethanol was added to wash the precipitate.
6) The mixture was centrifuged at 12,000r/min for 5min, the supernatant was discarded, and the precipitate was dried.
7) Add 96. mu.L of ddH2And O, storing at the temperature of minus 20 ℃.
3. And (3) PCR amplification:
PCR amplification System: 1. mu.L of DNA, 1. mu.L of forward and reverse primers, 1. mu.L of dNTPs, 1. mu.L of CRbuffer, 0.6. mu.L of DNA polymerase, and 4.4. mu.L of ddH2O。
And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 3 min; 35 cycles of 94 ℃ for 35s, 50-60 ℃ for 35s, 72 ℃ for 1 min; extending for 6min at 72 ℃; 4 ℃ forever.
4. Polyacrylamide gel:
performing electrophoresis by using 8% non-denatured polyacrylamide gel, wherein the voltage is 150V, taking out gel after the electrophoresis is finished, washing with clear water for 2 times, placing the gel in silver staining solution (0.1% silver nitrate solution) for gentle shaking for 20min, taking out clear water after the electrophoresis is finished, washing for 2 times, placing developing solution (10g of sodium hydroxide and 5ml of formaldehyde are dissolved in 1L of water), gentle shaking until clear bands appear, taking out gel, washing with clear water, taking a picture, and manually reading the bands.
As a result, as shown in FIGS. 1-6, RM5420 can identify Zhejiang japonica 88 from 65 varieties, RM1364+ RM3414 can identify Zhejiang japonica 100 from 65 varieties, and RM6404+ RM6327+ RM1287 can identify Zhejiang japonica 96 from 65 varieties.
The variety is numbered, the product variety name, the SSR mark and the strip position are numbered, the strip position at the lowest end is numbered as 1, and the product variety is numbered sequentially upwards. Thus, Zhe jing 88 is numbered: zhe Jing 88-RM5420-2, Zhe Jing 100 number: zhe Jing 100-RM1364-3-RM3414-2, Zhe Jing 96: zhe round-grained nonglutinous acid 96-RM6404-3-RM6327-2-RM 1287-1.
5. And (3) authenticity identification of the variety:
for the variety identification, the following steps were used: extracting DNA from the standard variety and the variety to be detected according to the method, performing PCR and PAGE gel electrophoresis by using the markers, and judging whether the band of the sample to be detected is on the same line with the band of the standard variety, wherein if the band is on the same line, the variety to be detected is true.
Specific case 1: the authenticity of seeds (hereinafter referred to as sample A) claiming Zhe Jing 88 on the market is identified.
Method (1): taking a sample A, taking a real standard Zhe japonica 88 seed as a reference, extracting DNA of the two seeds, performing PCR amplification by adopting the RM5420 marker, performing electrophoresis on an amplification product by using 8% non-denatured polyacrylamide gel, and observing whether the two strips are consistent, wherein if the two strips are consistent, the sample A is the real Zhe japonica 88, and if the two strips are not consistent, the sample A is a dummy seed.
Method (2): extracting DNA of the sample A, carrying out PCR amplification by adopting the RM5420 marker, carrying out electrophoresis on an amplification product by using 8% non-denatured polyacrylamide gel, and observing whether the position of a strip is the same as the number position of Zhejiang japonica 88, wherein if the position of the strip is the same as the number position of the Zhejiang japonica 88, the sample A is real Zhejiang japonica 88, and if the position of the strip is different, the sample A is a pseudovariety.
The variety is identified by utilizing 1-3 marks, the practical application value is high, the variety can be accurately identified by utilizing 1-3 marks, in the process of actually identifying the variety, the medicine and labor cost can be saved, the result can be observed on a computer operation interface, the method is clear and more visual for agricultural science and technology workers, consumers and farmers.
The number is carried out by adopting the 'variety name + SSR mark + strip position', so that which mark can be used when the number is used can be simply and clearly seen, and which variety can be identified at which position by the strip.
Sequence listing
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University of chekiang
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Claims (6)
1. A method for identifying rice varieties by using 1-3 molecular markers is characterized by comprising the following steps:
(1) the method comprises the steps of establishing a standard substance combination containing a plurality of rice varieties by taking known rice varieties as standard substances, and screening SSR markers capable of specifically identifying the corresponding rice standard substances aiming at least one rice variety in the standard substance combination, wherein one rice variety corresponds to 1-3 SSR markers;
(2) aiming at a certain standard substance, performing PCR amplification and gel electrophoresis analysis on all rice in the standard substance combination by using an SSR marker specifically identified by the standard substance to obtain an electrophoresis pattern, numbering the standard substance according to pattern band information by using a 'variety name + SSR marker + band position', and thus forming numbering information of a plurality of standard substances;
(3) according to the suspected variety of the rice variety to be identified, the SSR markers of the corresponding variety are correspondingly selected from the standard substance combination for PCR amplification and gel electrophoresis analysis, and the authenticity of the rice variety to be detected is judged according to the map.
2. The method for identifying rice varieties by using 1-3 molecular markers according to claim 1, wherein in the step (3), the rice variety to be identified and the corresponding real rice variety in the standard combination are subjected to PCR amplification and gel electrophoresis analysis simultaneously, if the bands of the two are consistent, the rice variety to be identified is real, otherwise, the rice variety is a fake variety.
3. The method for identifying rice varieties by using 1-3 molecular markers according to claim 1, wherein in the step (3), only the rice varieties to be identified are subjected to PCR amplification and gel electrophoresis analysis, and according to the positions of the bands in the map, if the positions of the bands are consistent with the numbering positions of the corresponding varieties, the rice varieties to be identified are true, otherwise, the rice varieties are false.
4. The method as claimed in claim 1, wherein the rice standard comprises Zhe jing 88, Zhe jing 100, and Zhe jing 96.
5. The method for identifying rice varieties by using 1-3 molecular markers as claimed in claim 2, wherein the SSR marker for identifying Zhejing 88 is RM5420, the SSR marker for identifying Zhejing 100 is RM1364+ RM3414, and the SSR marker for identifying Zhejing 96 is RM6404+ RM6327+ RM 1287.
6. The method for identifying rice cultivars using 1-3 molecular markers according to claim 2,
forward primer for RM 5420: 5'-CCTGATCTCAACACACACGC-3' the flow of the air in the air conditioner,
reverse primer for RM 5420: 5'-GAAGTCTTGTTGCGCGTATG-3', respectively;
forward primer for RM 1364: 5' -AAGAAATTCAAAACACATGA of the reaction mixture, wherein,
reverse primer for RM 1364: 5' -AAAACATCTACTTTGATCCA;
forward primer of RM 3414: 5' -TAGGGCAATTGTGCAAGTGG of the reaction mixture, wherein,
reverse primer for RM 3414: TTGGGAATTGGGTAGGACAG, respectively;
forward primer for RM 6404: 5' -GGGATGATGGATCGGGAG of the reaction mixture, wherein,
reverse primer for RM 6404: CTACCAGCCTTGTTTCCTCG, respectively;
forward primer for RM 6327: 5' -CAGCCTAGGGCGTCATAGAC of the reaction mixture, wherein,
reverse primer for RM 6327: GATTGGGTGATGGATAGCAC, respectively;
forward primer of RM 1287: 5' -GGAAGCATCATGCAATAGCC of the reaction mixture, wherein,
reverse primer for RM 1287: GGCCGTAGTTTTGCTACTGC are provided.
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