CN104946745A - Method for identifying tea tree variety by using DNA bar code - Google Patents
Method for identifying tea tree variety by using DNA bar code Download PDFInfo
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Abstract
The present invention relates to a tea tree variety identifying method, specifically to a method for identifying the tea tree variety by using DNA bar code. The method comprises: respectively extracting DNA from a plurality of tea tree varieties; screening SSR primers; respectively preparing PCR reagents from the plurality of the extracted NDA, and carrying out reaction amplification; carrying out gel electrophoresis on each PCR reaction product, staining, and shooting and recording by using a gel imager; and combining the letters corresponding to the variety conversion on the SSR primers to form the DNA bar code. According to the present invention, the molecular marker technology is utilized, the method for identifying the tea tree variety by using the DNA bar code is established through the technical combination of genome DNA extraction, labeling screening, PCR amplification, product analysis, stripe data reading, letter bar code establishing and the like, and compared with the traditional morphology identifying method, the method of the present invention has the following characteristics that: the method is not affected by the environmental conditions, the method is simple, rapid and accurate, and the accurate identifying can be performed on the tea variety authenticity.
Description
Technical field
The present invention relates to the discrimination method of tea tree breed.
Background technology
How China's at present seed selection 114 national breedings and 154 provincial breedings, carry out precise Identification to these kinds significant to breeder's Right protection and new variety popularization etc.New tea cultivar DUS (abbreviation of specificity, consistence, stability) Testing Guideline is first new variety of plant DUS Testing Guideline that China works out for UPOV (UPOV), and its formulation is that China's Resources of Tea Plant evaluation and protection provide technical foundation and authorize foundation.Along with molecular marking technique development, UPOV includes DNA marker qualification in variety of crops DUS content measurement.DNA molecular marker technology not only has the reliability of stability, the identifiability of inter-variety variance, minimum product intraspecific variablity and experimental result, and there is the advantages such as easy, rapid, very be applicable to cultivar identification, overcome the shortcoming that the traditional form Marker Identification cycle is long, error is large, Traits change is little.The marking method of constructed dna fingerprint database is defined as SSR and SNP by International Plant variety right alliance (UPOV) in BMT Testing Guideline draft.And SSR marker because primer development is cheap, simple to operate, be easy to promote, be verified as the mark system of the most widespread use of Plant new variety protection by UPOV biological chemistry and molecular engineering working group.Though UPOV includes the qualification of crop SSR marker in variety of crops DUS content measurement, clear and definite regulation be there is no to the testing process of Different Crop and concrete grammar.
Summary of the invention
For dependence tea tree Agronomic trait carries out length qualification cycle to tea tree breed at present, error is large, identification traits difference little kind difficulty is large, to problems such as assessor's skill requirement enrich, the invention provides a kind of method simply, fast, accurately utilizing DNA bar code discriminating tea tree breed.
The technical scheme that the present invention solves the problems of the technologies described above employing is: a kind of method utilizing DNA bar code to differentiate tea tree breed, comprises the following steps:
(1) DNA extraction: the DNA extracting several tea tree breed respectively;
(2) primer screening: clear, stable and reproducible several to SSR primer of screening band;
(3) pcr amplification: the SSR primer that each DNA in the DNA of said extracted screens with often pair is respectively mixed and is made into PCR reaction reagent and increases;
(4) the PCR primer analysis of SSR marker: each PCR reaction product is carried out gel electrophoresis poststaining, then adopts gel imaging instrument Taking Pictures recording;
(5) structure of molecular barcode: by the electrophorogram of each kind on often pair of SSR primer of above-mentioned record clearly band be designated as " 1 ", same position is counted " 0 " without band or the weak band not easily differentiated, sets up raw data base; Then 1,0 data in each kind site on often pair of SSR primer are converted to letter; Again several letter to grade change corresponding on SSR primer is combined, form the DNA bar code of each kind;
(6) differentiate: with the true and false of above-mentioned DNA bar code for standard discriminating tea tree breed.
As preferably, RNA isolation kit is adopted to extract DNA.
As preferably, filter out
F:CTCCGATTACTTTCTTCC
R:GCAGGTTAGCGGTGGTTA；
F:TAGGGTTTTAGTTTCAG
R:AACATCCTTGCCTCGTC；
F:GTGAAGTTAGTTGTTACTCTTTTTTGG
R:AGGGGAAGTGAGGAGGCAT；
F:ATGCTTCAGGGAGTGACCAT
R:ATTTATGCCAAACTACCAACAG
Four pairs of SSR primers.
As preferably, each PCR reaction reagent is number 6.65 parts of ddH by volume
2the Mg of the DNA of a kind of the said extracted of O, 1.0 parts of 10ng/ μ L, 1.0 parts of 10 × Buffer, 0.7 part of 25mM
2+, 0.2 part of dNTP of 10mM, primers and the 0.25 part of 2U/ μ L of each 0.1 part 10 μMs Taq polymerase mixing be made into and form.
As preferably, pcr amplification condition is 94 DEG C of sex change 3min, then 94 DEG C of sex change 30s, 53 DEG C of annealing 30s, and 72 DEG C extend 50s, and totally 30 circulations, complete with after cocycle, and 72 DEG C of extension 10min, finally by PCR primer cool to room temperature or 4 DEG C.
As preferably, above-mentioned pcr amplification repeats once, compares twice amplification, if two times result is inconsistent, pcr amplification repeats once again, is as the criterion there to be the consistent result of twice amplification.
As preferably, PCR primer is mixed with 6 × loading buffer, adopt the polyacrylamide gel electrophoresis of 10%, carry out cma staining after electrophoresis, until there is clear band to occur.
As preferably, the step of cma staining is: 1. fix 5 ~ 8min with 10% ethanol and 0.5% acetic acid; 2. again 5 ~ 8min is fixed with 10% ethanol and 0.5% acetic acid; 3. 0.2%AgNO is used
3dyeing 10 ~ 15min; 4. the distilled water flushing several seconds is used; 5. with 1.5%NaOH and the colour developing of 0.5% formaldehyde.
As preferably, the rule that 1,0 data are converted to letter is: 1. have 2 sites, and that only has first site by the size in site is encoded to A; That only has second site is encoded to B; What two sites had is encoded to C; What two sites did not all have is encoded to N; 2. have 3 sites, that only has 1 site is encoded to A, B, C by size successively; Only have 2 sites, be encoded to D, E, F successively by Sites Combination; What three sites had is encoded to G; If three sites are not all encoded to N; 3. have 4 sites, that only has 1 site is encoded to A, B, C, D by size successively; Only have 2 sites then to encode letter from E, be encoded to E, F, G, H, I, J successively by Sites Combination; That only has 3 sites is encoded to K, L by Sites Combination successively from K; What 4 sites had is encoded to M, and what 4 sites did not all have is encoded to N.
As can be known from the above technical solutions, the present invention utilizes molecular marking technique, read by extracting genome DNA, label screening, pcr amplification, product analysis, strip data and the technical combinations such as foundation of alphabet strip shape code, establish a kind of method that DNA bar code differentiates tea tree breed, the method is compared with traditional form authentication method, not by environmental influence, method is simple, quick, accurate, can carry out precise Identification to the verity of tea tree breed.
Accompanying drawing explanation
Fig. 1 is the amplification figure of primer 2 34.
Fig. 2 is the amplification figure of primer 685.
Fig. 3 is the amplification figure of primer A58.
Fig. 4 is the amplification figure of primer A142.
Embodiment
The present invention is introduced in detail below in conjunction with accompanying drawing:
The present invention's sweet oak leaf is neat, green No. 2 of Hunan ripple, Fuding white tea, jade green and sharp wave yellow No. 13 totally 5 kinds make test sample.First, the step that DNA extraction method provides according to test kit is carried out, and last dissolving DNA is to 100ul.Nanodrop detects DNA concentration, and the concentration redistilled water of DNA is diluted to the concentration of 10-20ng/ul.
Then, 4 pairs of SSR primers that screening band is clear, stable and reproducible, concrete primer information is as following table 1.
The primer information that table 1 screens
Then, according to the agent prescription of table 2, first mixed by the 6 kinds of reagent of other except DNA, during mixing, Taq polymerase finally adds, and is then distributed into 5 parts and adds in 20ulPCR test tube respectively, finally in each pipe examination, adds 1ulDNA respectively.
| Sequence number | Reagent | Add front concentration | Volume (ul) |
| 1 | ddH 2O | - | 6.65 |
| 2 | DNA | 10ng/μL | 1.0 |
| 3 | 10×Buffer | - | 1.0 |
| 4 | Mg 2+ | 25mM | 0.7 |
| 5 | dNTP | 10mM | 0.2 |
| 6 | primers | 10μM | Each 0.1 |
| 7 | Taq polymerase | 2U/μL | 0.25 |
Table 2 10ulPCR reaction system agent prescription
Above-mentioned test tube is placed in PCR instrument expand, extender is: 94 DEG C of sex change 3min, 94 DEG C of sex change 30s, 53 DEG C of annealing 30s, 72 DEG C extend 50s, totally 30 circulations, complete with after cocycle, 72 DEG C extend 10min, finally by PCR primer cool to room temperature or 4 DEG C.Analyze after PCR instrument is taken out.The analysis of product is polyacrylamide gel electrophoresis: the PCR primer mixing 1ul 6 × loading buffer pipetting 4ul is used for 10% polyacrylamide gel electrophoresis, and carry out cma staining after electrophoresis, electrophoretic voltage is set as 150V, and the time is 180min; The step of cma staining is (100ml system): 1. fix 5 ~ 8min with 10% ethanol+500 μ L acetic acid; 2. again 5 ~ 8min is fixed with 10% ethanol+500 μ L acetic acid; 3. 0.2%AgNO is used
3dyeing 10 ~ 15min; 4. the distilled water flushing several seconds is used; 5. with the colour developing of 1.5%NaOH+500ml formaldehyde, until there is clear band to occur.Each step has been shaken on shaking table above, and the gel after colour developing adopts imager Taking Pictures recording.
Then, utilize manual method to read tape, by electrophorogram clearly band be designated as " 1 ", same position is counted " 0 " without band or the weak band not easily differentiated, sets up raw data base, and then 1,0 data in site in often pair of SSR primer are converted to alphabetical bar code.Coding rule is: if 1. there are 2 sites, then by the size in site, that only has first site is encoded to A, and that only has second site is encoded to B, and what two sites had is encoded to C, if two sites are not all encoded to N, as table 3; If 2. there are 3 sites, that only has 1 site is encoded to A, B, C by size successively, has 2 sites, is encoded to D, E, F successively by Sites Combination, and what three sites had is encoded to G, if three sites are not all encoded to N, as table 4; If 3. there are 4 sites, that only has 1 site is encoded to A, B, C, D by size successively, 2 sites are had then to encode letter from E, E, F, G, H, I, J is encoded to successively by Sites Combination, what have 3 sites is encoded to K, L by Sites Combination successively from K, 4 are encoded to M, and 4 sites are not all encoded to N, as table 5.
| Site situation | Only has the first site | Only has the second site | Two sites are all used | Two sites all do not have |
| Bar code | A | B | C | N |
Table 3 has the bar code of 2 locis to change rule
Table 4 has the bar code of 3 locis to change rule
Table 5 has the bar code of 4 locis to change rule
Primer 2 34,685, the electrophorogram of A58 and A142 amplification is shown in Fig. 1, Fig. 2, Fig. 3 and Fig. 4, the order of sample in the drawings successively: 1 is that sweet oak leaf is neat, and 2 is green No. 2 of Hunan ripple, 3 is Fuding white tea, 4 is jade green, and 5 is yellow No. 13 of sharp wave, and M is 100bp DNA ladder.According to the object clip size (table 1) of each primer, 4 primer original band data logging are in table 6, the position size 1. representing site 1 wherein in Fig. 1, Fig. 2, Fig. 3 and Fig. 4,2. the position size in site 2 is represented, 3. represent the position size in site 3,4. represent the position size in site 4.
The alphabetical bar code changed according to the transformation rule of aforementioned table 3, table 4, table 5 is in table 7, and the DNA bar code of 5 kinds that these 4 combination of primers are formed is in table 8.With the barcode in table 8 for standard, above 4 pairs of combination of primers can be adopted to increase to tea tree breed to be identified, and according to the bar code transformation rule provided, amplification is converted into bar code, if the bar code of tea tree breed to be identified is identical with the bar code of standard variety, then this kind is standard variety; If bar code is different, then it not standard variety.
1,0 data of table 64 pairs of primers amplification in 5 kinds
| Primer | Sweet oak leaf is neat | Green No. 2 of Hunan ripple | Fuding white tea | Jade green | Yellow No. 13 of sharp wave |
| 234 | B | C | C | D | L |
| 685 | F | G | B | D | E |
| A58 | D | E | B | D | C |
| A142 | G | D | G | D | G |
The alphabetical bar code that 1,0 data that table 74 pairs of primers increase in 5 kinds are converted to
| Primer | Sweet oak leaf is neat | Green No. 2 of Hunan ripple | Fuding white tea | Jade green | Yellow No. 13 of sharp wave |
| DNA bar code | BFDG | CGED | CBBG | DDDD | LECG |
The DNA alphabet strip shape code of table 85 kinds
Be described in detail the technical scheme that the present invention's example provides above, the explanation of above embodiment is only applicable to the principle helping to understand the embodiment of the present invention; Meanwhile, for one of ordinary skill in the art, according to the embodiment of the present invention, embodiment and range of application all will change, and in sum, this description should not be construed as limitation of the present invention.
Claims (9)
1. utilize DNA bar code to differentiate a method for tea tree breed, comprise the following steps:
(1) DNA extraction: the DNA extracting several tea tree breed respectively;
(2) primer screening: clear, stable and reproducible several to SSR primer of screening band;
(3) pcr amplification: the SSR primer that each DNA in the DNA of said extracted screens with often pair is respectively mixed and is made into PCR reaction reagent and increases;
(4) the PCR primer analysis of SSR marker: each PCR reaction product is carried out gel electrophoresis poststaining, then adopts gel imaging instrument Taking Pictures recording;
(5) structure of molecular barcode: by the electrophorogram of each kind on often pair of SSR primer of above-mentioned record clearly band be designated as " 1 ", same position is counted " 0 " without band or the weak band not easily differentiated, sets up raw data base; Then 1,0 data in each kind site on often pair of SSR primer are converted to letter; Again several letter to grade change corresponding on SSR primer is combined, form the DNA bar code of each kind;
(6) differentiate: with the true and false of above-mentioned DNA bar code for standard discriminating tea tree breed.
2. method according to claim 1, is characterized in that: adopt RNA isolation kit to extract DNA.
3. method according to claim 2, is characterized in that: filter out
F:CTCCGATTACTTTCTTCC
R:GCAGGTTAGCGGTGGTTA；
F:TAGGGTTTTAGTTTCAG
R:AACATCCTTGCCTCGTC；
F:GTGAAGTTAGTTGTTACTCTTTTTTGG
R:AGGGGAAGTGAGGAGGCAT；
F:ATGCTTCAGGGAGTGACCAT
R:ATTTATGCCAAACTACCAACAG
Four pairs of SSR primers.
4. method according to claim 3, is characterized in that: each PCR reaction reagent is number 6.65 parts of ddH by volume
2the Mg of a DNA of the said extracted of O, 1.0 parts of 10ng/ μ L, 1.0 parts of 10 × Buffer, 0.7 part of 25 mM
2+, the dNTP of 0.2 part of 10 mM, primers and the 0.25 part of 2U/ μ L of each 0.1 part 10 μMs Taq polymerase mixing be made into and form.
5. method according to claim 4, it is characterized in that: pcr amplification condition is 94 DEG C of sex change 3 min, then 94 DEG C of sex change 30 s, 53 DEG C of annealing 30 s, 72 DEG C extend 50 s, and totally 30 circulations, complete with after cocycle, 72 DEG C extend 10 min, finally by PCR primer cool to room temperature or 4 DEG C.
6. method according to claim 5, it is characterized in that: above-mentioned pcr amplification repeats once, compare twice amplification, if two times result is inconsistent, pcr amplification repeats once again, is as the criterion there to be the consistent result of twice amplification.
7. method according to claim 6, is characterized in that: PCR reaction product mixed with 6 × loading buffer, adopts the polyacrylamide gel electrophoresis of 10%, carries out cma staining, until there is clear band to occur after electrophoresis.
8. method according to claim 7, is characterized in that: the step of cma staining is: 1. fix 5 ~ 8 min with 10% ethanol and 0.5% acetic acid; 2. 5 ~ 8 min are again fixed with 10% ethanol and 0.5% acetic acid; 3. 0.2%AgNO is used
3dyeing 10 ~ 15 min; 4. the distilled water flushing several seconds is used; 5. with 1.5%NaOH and the colour developing of 0.5% formaldehyde.
9. method according to claim 7 or 8, is characterized in that: 1,0 data are converted to the rule of letter and are: 1. have 2 sites, that only has first site by the size in site is encoded to A; That only has second site is encoded to B; What two sites had is encoded to C; What two sites did not all have is encoded to N; 2. have 3 sites, that only has 1 site is encoded to A, B, C by size successively; Only have 2 sites, be encoded to D, E, F successively by Sites Combination; What three sites had is encoded to G; If three sites are not all encoded to N; 3. have 4 sites, that only has 1 site is encoded to A, B, C, D by size successively; Only have 2 sites then to encode letter from E, be encoded to E, F, G, H, I, J successively by Sites Combination; That only has 3 sites is encoded to K, L by Sites Combination successively from K; What 4 sites had is encoded to M, and what 4 sites did not all have is encoded to N.
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Cited By (7)
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| CN106591460A (en) * | 2016-12-27 | 2017-04-26 | 中国农业科学院茶叶研究所 | Method for identifying variety of 'Chinese Tea 302' tea tree by adopting SSR molecular marker and applications of SSR molecular marker |
| CN107180165A (en) * | 2017-05-27 | 2017-09-19 | 云南省农业科学院质量标准与检测技术研究所 | A kind of method for setting up Yunrui series corn variety DNA molecular label |
| CN108998551A (en) * | 2018-07-23 | 2018-12-14 | 湖南省茶叶研究所（湖南省茶叶检测中心） | Tea tree quadruple fluorescence SSR molecular marker detection method |
| CN109337997A (en) * | 2018-09-20 | 2019-02-15 | 江西省林业科学院 | A kind of Camellia polymorphism Chloroplast gene microsatellite molecular marker primer and screening and the method for screening sibling species |
| CN110592253A (en) * | 2019-09-23 | 2019-12-20 | 云南农业大学 | DNA combined bar code for identifying Yunnan tea tree variety and identification method thereof |
| CN113652498A (en) * | 2021-09-13 | 2021-11-16 | 中国农业科学院茶叶研究所 | MNP (MNP) molecular marker combination for identifying albino tea tree varieties, method and application |
| CN115896333A (en) * | 2022-11-24 | 2023-04-04 | 安徽农业大学 | Method for identifying Jinyu No. 1 tea tree strain by using SSR fingerprint |
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| CN106591460A (en) * | 2016-12-27 | 2017-04-26 | 中国农业科学院茶叶研究所 | Method for identifying variety of 'Chinese Tea 302' tea tree by adopting SSR molecular marker and applications of SSR molecular marker |
| CN106591460B (en) * | 2016-12-27 | 2020-04-28 | 中国农业科学院茶叶研究所 | Method for identifying variety of 'Zhongcha 302' tea tree by SSR molecular marker and application |
| CN107180165A (en) * | 2017-05-27 | 2017-09-19 | 云南省农业科学院质量标准与检测技术研究所 | A kind of method for setting up Yunrui series corn variety DNA molecular label |
| CN108998551A (en) * | 2018-07-23 | 2018-12-14 | 湖南省茶叶研究所（湖南省茶叶检测中心） | Tea tree quadruple fluorescence SSR molecular marker detection method |
| CN109337997A (en) * | 2018-09-20 | 2019-02-15 | 江西省林业科学院 | A kind of Camellia polymorphism Chloroplast gene microsatellite molecular marker primer and screening and the method for screening sibling species |
| CN109337997B (en) * | 2018-09-20 | 2020-05-22 | 江西省林业科学院 | Camellia polymorphism chloroplast genome microsatellite molecular marker primer and method for screening and discriminating kindred species |
| CN110592253A (en) * | 2019-09-23 | 2019-12-20 | 云南农业大学 | DNA combined bar code for identifying Yunnan tea tree variety and identification method thereof |
| CN113652498A (en) * | 2021-09-13 | 2021-11-16 | 中国农业科学院茶叶研究所 | MNP (MNP) molecular marker combination for identifying albino tea tree varieties, method and application |
| CN113652498B (en) * | 2021-09-13 | 2024-06-04 | 中国农业科学院茶叶研究所 | Tea tree MNP molecular marker combination, method and application for identifying albino tea tree varieties |
| CN115896333A (en) * | 2022-11-24 | 2023-04-04 | 安徽农业大学 | Method for identifying Jinyu No. 1 tea tree strain by using SSR fingerprint |
| CN115896333B (en) * | 2022-11-24 | 2024-06-07 | 安徽农业大学 | Method for identifying Jinyu No. 1 tea tree strain by utilizing SSR fingerprint |
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Application publication date: 20150930 |