CN104004756A - SSR (Simple Sequence Repeat) core primer group and method thereof for identifying tea variety - Google Patents

SSR (Simple Sequence Repeat) core primer group and method thereof for identifying tea variety Download PDF

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Publication number
CN104004756A
CN104004756A CN201410249326.8A CN201410249326A CN104004756A CN 104004756 A CN104004756 A CN 104004756A CN 201410249326 A CN201410249326 A CN 201410249326A CN 104004756 A CN104004756 A CN 104004756A
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China
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tea tree
core primers
primers designed
pairs
ssr
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张成才
成浩
王丽鸳
韦康
吴立赟
刘圆
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Tea Research Institute Chinese Academy of Agricultural Sciences
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Tea Research Institute Chinese Academy of Agricultural Sciences
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Abstract

The invention relates to an SSR (Simple Sequence Repeat) core primer group and a method thereof for identifying a tea variety, and belongs to the technical field of biology. The method is characterized by comprising the following steps: (1) material selection; (2) genome DNA extraction; (3) PCR (Polymerase Chain Reaction) amplification of a target product; (4) electrophoretic separation of a PCR product; (5) silver staining development; (6) data analysis; and (7) result judgment. The SSR core primer group and the method thereof for identifying the tea variety, which are disclosed by the invention, have the characteristics of easiness and convenience for operation, accuracy in result, high repeatability, high identification capacity, low cost and the like and are suitable for identifying the tea variety.

Description

A kind of method of SSR core primers group and evaluation tea tree breed thereof
Technical field
The invention belongs to biological technical field, be specially a kind of method of SSR core primers group and evaluation tea tree breed thereof.
Background technology
The cash crop that Cha Shi China is important, by the end of the year 2010,121 of the total national authorization kinds of China, more than 100 of provincial authorization kinds.Realize each interracial effective evaluation, contribute to protect kind power, prevent that fake and forged or impure kind from coming into the market.At present, conventionally according to the Morphological Differences between tea tree breed, carry out kind discriminating, but same kind may show the difference of some proterties under different land occupation conditions, between different varieties, also may there is some proterties more similar, according to Morphological Differences, be difficult to obtain conclusion accurately.And different tea tree breeds can be accurately differentiated in new tea varietY specificity, consistence and stability (be called for short DUS) test, but need test plant, newly slightly, 35 proterties such as blade, waste time and energy.Workload that at present tea tree breed exists in identifying is large, efficiency is low and the problem such as qualification result poor accuracy.
Molecular marking technique is widely used in recent years the germ plasm resource of plant and differentiates field, wherein, that SSR mark has is reproducible, polymorphism is high, codominance, be easy to detect, distinguishing ability is subject to the advantages such as environmental influence by force and not, is that a kind of effective kind is differentiated means.At present, in corn and paddy rice, China has promulgated the cultivar identification technology national standard based on SSR mark.Therefore, SSR technology has broad application prospects in tea tree breed protection, false kind differentiation, purity detecting and strain evaluation field.
Summary of the invention
For the above-mentioned problems in the prior art, the object of the invention is to the technical scheme that design provides a kind of SSR core primers group and identifies the method for tea tree breed, quantity and two indexs of interracial genetic distance in conjunction with difference primer pair, tea tree breed is identified, can improve efficiency and the accuracy rate of evaluation, have easy and simple to handle, result accurately, the feature such as repeatability is strong, distinguishing ability is strong and cost is low, be suitable for the evaluation of tea tree breed.
Described a kind of SSR core primers group, it is characterized in that comprising 10 pairs of core primers designed, 24 pairs of standby primers designed, 10 pairs of described core primers designed are 1057,1604,1622,1624,1633,1651,1696,5053,5077,5094, as shown in table 1; The standby primers designed of described 24 couple is 1052,1056,1058,1088,1605,1608,1616,1628,1639,1683,1701,3011,3110,4012,4042,4048,4102,5033,5043,5062,5082,5091,5102,5110, as shown in table 2;
10 pairs of core primers designed information of table 1
Sequence number Numbering Upstream primer (5`-3`) Downstream primer (5`-3`)
1 1057 AACCACACACACAACAATCACTC TTCACCCCAGAACTGGAAAC
2 1604 AGGGTTTTAGGGCTACGAGG TGATTGGAGAACACAACCGA
3 1622 CTCTCGGCACTGCTTCTTCT GAGGACAACGAGGAAGTGGA
4 1624 GAGGCACTCACTACTCCTCCA GTTGTGGGGAGACTCCAAGA
5 1633 AACCGAACAACAGACCCTTG GCTGAGAGAGGAGCGAGAAA
6 1651 GATCGAAATCACCAATGCCT GAGAGAGAAGTCCGTGCGTC
7 1696 TCCAAAAGATTCGGTTCGTC TCGATATTTTCCTTCCGTCG
8 5053 GACGATGGACCCTTCTTTGA CATCATCATCATCCTCACCG
9 5077 TTCACCCTCAGACCAAAAGG TCGATCAATTGCAAAACACA
10 5094 CGAGACATCGAACACCACAG CGTATCGTAGCGGTGAAGGT
24 pairs of standby primers designed information of table 2
Sequence number Numbering Upstream primer (5`-3`) Downstream primer (5`-3`)
1 1052 TAGCCTTGGGTGGATTTCAG TGAATTTGCAGAAGTGAGAGAA
2 1056 AATGGCGTCTCCAATTTGTC CGGAAGAAGAGACGGTGAAG
3 1058 CCCCTCCATCTATCTCCCTC GGACTGTTGTTGGGGAAGAA
4 1088 GGCTGTATGATGGCTTGGTT TAAAATGTGGGTCTCCAGGC
5 1605 TGTAAGTGGCCGGAGTTTTC ACCACAGCAACAGTCACAGC
6 1608 CTTGTGAGGGAGTGACGGAT ATGCCTGCTCGATATTACGG
7 1616 TTCATTCATCAATGGCGGTA CAAAGGGCTTTGTTTTGAGG
8 1628 TGGAGGCATCATTACCAGAA CGTCTCAGACCACTCGTCAA
9 1639 GTGCAAACCGTTGGATTCTT CGATGAGGATGATGATGGTG
10 1683 AGTTGTTCATGGAGGCAAGG ACTTGGCCCTCTTCTTAGGC
11 1701 TTGTCTCTCACCGCCTTTCT CCGAAACTACCAAAACTCCG
12 3011 ATTGCGGGGAAAAGAGAAAT TTGACAAGACTCGTCGATGG
13 3110 TGGGCCAGAAGAGAAAAGAA GGTGTTCCTGGCACTTCAAT
14 4012 ACTCACCATTCGACACCCTC GATCTGGAGACCGATTTGGA
15 4042 AAGCCTTCAAGAACCAGCAA TCTTAATAGCATTATTTGCAGCCA
16 4048 CTCTCGAGGGAAAAGCAATG CAGCATCAGCACTTCCAAGA
17 4102 GTCCGGCAAGATTGGATTTA TCGGTCGATCATCACTGTGT
18 5033 TGCTTCCATGGTGATTTTGA AATGGTGGCTTCAACAAAGG
19 5043 AATTGTAGCAGTTGCCTCCG GAGTAGGTCGATTTGGCGAG
20 5062 TCATTTGAAGTGCAACTGGG TAAGGCGATGATTAGGGTGG
21 5082 CCGATTCCAGGAGGGATTAT TTTGGAATCCGGTTGAAATC
22 5091 TTCGATTGAAATCGGAGGAC CACAACCTCAAGCAAGCAAA
23 5102 TCAATTGCGTCACAAACACA TTTTGTTCATGACGGAGCAC
24 5110 CCTTGCTTTGCTTCTCTCTTTC ATCGGAGGGAGGTCTGAATC
Described SSR core primers group is identified the method for tea tree breed, it is characterized in that comprising the following steps:
1) take the tender leaf of different varieties of tea plant is material;
2) extraction of tea tree genomic dna;
3) pcr amplification of target product:
1. take step 2) gained genomic dna is template;
2. use 10 pairs of core primers designed to carry out pcr amplification;
3. use 10 μ L PCR reaction systems, specifically comprise: each 0.2 μ L of upstream and downstream primer, 10 * Buffer (Mg 2+plus) 1 μ L, dNTP 0.2 μ L, rTaq enzyme 0.5U, adds ddH 2o to 10 μ L;
4. PCR response procedures is: first, and 94-95 ℃ of sex change 2-4 minute; Then, according to following program, carry out 30 circulation: 94-95 ℃ of sex change 25-35 seconds, 55-60 ℃ annealing 25-30 second, 72 ℃ and extend 25-30 second; Then, 72 ℃ are extended 2-4 minute; Finally, 3-5 ℃ of preservation;
4) electrophoretic separation of PCR product
1. in step 3) gained PCR reaction product, add 2-3 μ L 6 * loading buffer, mix;
2. with pipettor, inhaling 2-3 μ L mixed solution is added in the point sample hole of 10% polyacrylamide gel preparing;
3. 160-180V voltage, electrophoresis 110-130 minute;
5) silver dyes colour developing
1. after step 4) finishes, carefully take off film, put into the special-purpose plastics casing of dyeing;
2. add 400-450mL stationary liquid, shaking table 65-75rpm shakes 5-7 minute, pours out stationary liquid, distilled water flushing 2-3 time;
3. add 350-450mL staining fluid, 65-75rpm shakes 8-12 minute, pours out AgNO 3solution, distilled water flushing 2-3 time;
4. add 400-450mL nitrite ion, 70-75rpm vibrates high-visible to band, uses distilled water flushing 2-3 time;
5. Taking Pictures recording;
6) data analysis
1. the band on artificial interpretation electrophorogram, the descending assignment successively such as A, B, C, D, E of press, disappearance band, with " .. " expression, is made genotype matrix;
2. compare interracial core primers designed difference logarithm;
3. use Popgene computed in software genetic distance;
4. use Mega5.0 software building dendrogram;
7) result is judged
When core primers designed difference logarithm >=2 of two kinds, be judged to be different varieties, form result of determination; When core primers designed difference logarithm≤1 of two kinds, be judged to be same breed or fairly similar kind, now, should be according to investigator need to introduce 24 pairs of standby primers designed, carry out above 3), 4), 5), 6) step, and the data of comprehensive 10 pairs of core primers designed calculate genetic distance and differentiate, when two kind genetic distance >0.140 are judged to be different varieties, genetic distance≤0.140 is judged to be same kind.
Described SSR core primers group is identified the method for tea tree breed, it is characterized in that step 2) in the extraction of tea tree genomic dna adopt following methods:
1) get 100 mg tea tree tender leafs, add liquid nitrogen grinding;
2) use sky to take root in thing genome DNA extracting reagent kit and extract tea tree genomic dna;
3) use spectrophotometric determination genomic dna concentration, use ddH 2o is diluted to 28-32ng/ μ L, at-20 ℃, saves backup.
Described SSR core primers group is identified the method for tea tree breed, it is characterized in that in the 4. PCR response procedures of step 3): first, and 94-95 ℃ of sex change 3 minutes; Then, according to following program, carry out 28 seconds, 72 ℃ of 30 circulation: 94-95 ℃ of sex change 28-30 seconds, 58 ℃ of annealing and extend 26-28 second; Then, 72 ℃ are extended 3 minutes; Finally, 4 ℃ of preservations.
Described SSR core primers group is identified the method for tea tree breed, it is characterized in that step 4) 3. in: voltage is 170V, and electrophoresis time is 120 minutes.
Described SSR core primers group is identified the method for tea tree breed, it is characterized in that step 5) 2. in: described stationary liquid is settled to 1 L preparation according to 100ml ethanolic soln, 5mL Glacial acetic acid, adding distil water.
Described SSR core primers group is identified the method for tea tree breed, it is characterized in that step 5) 3. in: described staining fluid is according to 2g AgNO 3adding distil water is settled to 1L preparation.
Described SSR core primers group is identified the method for tea tree breed, it is characterized in that step 5) 4. in: described nitrite ion is according to 15g NaOH solution and add 5mL 37% formaldehyde adding distil water to be settled to 1 L preparation.
Described Popgene software, Mega5.0 software can directly be downloaded use from network.
The method of above-mentioned a kind of SSR core primers group and evaluation tea tree breed thereof, has the following advantages:
1) the present invention proposes 10 pairs of core primers designed, these 10 pairs of core primers designed, with traditional comparing based on morphologic discrimination method, can improve cultivar identification efficiency greatly;
2) use of 10 pairs of core primers designed that the present invention proposes, can avoid the limitation of single primer pair in cultivar identification, improve the ability of a large amount of kinds of SSR technical evaluation, the kind of avoiding the indivedual gene locuss of same kind to undergo mutation causing is differentiated mistake, improves and identifies accuracy rate;
3) the present invention proposes 24 pairs of standby primers designed, according to investigator's particular case choice for use whether the standby primers designed of this 24 couple can be, this overlaps standby primers designed and can further the qualification result of core primers designed be verified and be supplemented, when guaranteeing detection efficiency, further improve and identify accuracy rate;
4) the present invention improves Methods for Genomic DNA Extraction from Tea Plant, select common on the market genome DNA extracting reagent kit to carry out DNA extraction, reduced the consumption of sample tissue, saved reagent preparation complicated while using CTAB method to extract DNA, reduced the requirement to experimenter's operant level, improve DNA extraction efficiency, improved DNA extraction quality;
5) the present invention proposes 10 pairs of core primers designed and 24 pairs of standby primers designed, used the annealing temperature of 58 ℃ all can obtain expanding effect well, reached the unification of annealing temperature, can simplify PCR process;
6) the present invention revises the PCR response procedures of SSR technology, and the feature less according to object fragment reduced in the time of 72 ℃ of extensions, can effectively shorten the PCR reaction times, improves determination rates;
7) the invention provides a kind of standard of perfection of tea tree breed, in conjunction with quantity and two indexs of interracial genetic distance of difference primer pair, tea tree breed is identified, can improve efficiency and the accuracy rate of evaluation.
Accompanying drawing explanation
Fig. 1 is tea tree breed identity process figure of the present invention;
Fig. 2 is the electrophoretic band of primer 5077 and 5110 in each kind, and M is 20bp Marker, and K is blank;
Fig. 3 is the bands of a spectrum mimic diagrams of 10 pairs of core primers in 45 parts of tea tree materials, and numbering 1-45 represents different tea tree materials, with table 3;
Fig. 4 is the genetic cluster figure of 10 pairs of core primers and 46 parts of tea tree materials of 24 pairs of standby primer pairs.
Embodiment
Below in conjunction with Figure of description 1-4 and specific embodiment, the invention will be further described, but should not be understood as limitation of the present invention.
Embodiment 1
1) the using and preparing of main agents:
1. genome DNA extracting reagent kit: the agility Plant Genome that Beijing Tian Gen biochemical technology company limited produces is extracted test kit;
2. PCR reacts required reagent: the rTaq enzyme that uses Dalian precious biotechnology company limited to produce, comprises the 50 rTaq enzymes of μ L 5U/ μ L, 10 * Buffer of 1mL, 800 μ L dNTP Mixture and 1mL Loading Buffer;
3. the EDTA(pH 8.0 of 1L 5 * TBE:54g Tris, 27.5g boric acid, 20mL 0.5M);
4. the acrylamide of 500mL 30%: 145g acrylamide and 5g N, N-bis-methylene diacrylamides, add ddH 2o is settled to 500mL;
5. 50mL 10% ammonium persulphate: 5g ammonium persulphate, adds ddH 2o is settled to 50mL;
6. 1L stationary liquid: 100ml ethanolic soln, 5mL Glacial acetic acid, adding distil water is settled to 1L;
7. 1L staining fluid: 2g AgNO 3, adding distil water is settled to 1L;
8. 1L nitrite ion: use distilled water preparation, 15g NaOH solution also adds 5mL 37% formaldehyde;
9. 120ml 10% polyacrylamide gel: ddH 2o 55mL, 30% acrylamide 40mL, 5 * TBE 24mL, 10% ammonium persulphate 1mL, TEMED 100 μ L.
2) material selection
The tender leaf of different varieties of tea plant (table 3) of take is material;
45 parts of tea tree materials that table 3 is participated in the experiment
Numbering Title Type Numbering Title Type Numbering Title Type
1 The ancient tea of water Cultivars 16 Meet frost Cultivars 31 Apple cloud B Cultivars
2 Crow ox early Cultivars 17 Strength peak Cultivars 32 Zhejiang agriculture 12 B Cultivars
3 Apple cloud A Cultivars 18 Green cloud Cultivars 33 Luo She Cultivars
4 Zhejiang agriculture 121 Cultivars 19 Zhejiang agriculture 12 A Cultivars 34 No. 1, report good fortune Cultivars
5 Vine tea Cultivars 20 The chrysanthemum spring Cultivars 35 Cui Feng Cultivars
6 Zhejiang agriculture 21 Cultivars 21 Cold green Cultivars 36 The white gross tea in Ruian Cultivars
7 Zhejiang agriculture 25 Cultivars 22 Longjing Changye Cultivars 37 Middle tea 102 Cultivars
8 Yellow leaf early Cultivars 23 Zhejiang agriculture 113 A Cultivars 38 Hengshan Mountain early Cultivars
9 Ruian is early clear and bright Cultivars 24 Blue or green peak Cultivars 39 Dragon Well tea 43 B Cultivars
10 Tongue is fragrant purple Cultivars 25 Middle tea 108 Cultivars 40 Crow ox is B early Cultivars
11 Eyebrow peak Cultivars 26 Middle tea 302 Cultivars 41 Zhenong 117 Cultivars
12 Frost peak Cultivars 27 Zhenong 139 Cultivars 42 NH-01 Material to be identified
13 The white tea in Anji Cultivars 28 Luxuriant green Cultivars 43 NH-02 Material to be identified
14 Pingyang is early special Cultivars 29 Zhejiang agriculture 113 B Cultivars 44 NH-03 Material to be identified
15 Dragon Well tea 43 A Cultivars 30 Silver monkey tea Cultivars 45 NH-04 Material to be identified
3) extraction of genomic dna
1. get 100 mg tea tree tender leafs, add liquid nitrogen grinding;
2. use sky to take root in thing genome DNA extracting reagent kit (day root, Beijing) and extract tea tree genomic dna;
3. use spectrophotometric determination genomic dna concentration, use ddH 2o is diluted to 30ng/ μ L, at-20 ℃, saves backup.
4) pcr amplification of target product
1. the step 3) gained genomic dna of take is template;
2. use 10 pairs of core primers designed to carry out pcr amplification (table 1);
3. use 10 μ L PCR reaction systems, specifically comprise: each 0.2 μ L of upstream and downstream primer, 10 * Buffer (Mg 2+plus) 1 μ L, dNTP 0.2 μ L, rTaq enzyme 0.5U, adds ddH 2o to 10 μ L;
4. PCR response procedures is: first, and 94 ℃ of sex change 3 minutes; Then, according to following program, carry out 30 circulations: 30 seconds, 72 ℃ extensions of 30 seconds, 58 ℃ annealing of 94 ℃ of sex change 30 seconds; Then, 72 ℃ are extended 3 minutes; Finally, 4 ℃ of preservations.
5) electrophoretic separation of PCR product
1. in step 4) gained PCR reaction product, add 2 μ L 6 * Loading Buffer(sample-loading buffers), mix;
2. with pipettor, inhaling 2 μ L mixed solutions is added in the point sample hole of 10% polyacrylamide gel preparing;
3. 170V voltage, electrophoresis 120 minutes.
6) silver dyes and develops the color after 1. step 5) finishes, and carefully takes off film, puts into the special-purpose plastics casing of dyeing;
2. add 400mL stationary liquid, shaking table 70rpm concussion 6 minutes, pours out stationary liquid, distilled water flushing 2 times;
3. add 400mL staining fluid, 70rpm concussion 10 minutes, pours out AgNO 3solution, distilled water flushing 2 times;
4. add 400mL nitrite ion, 70rpm vibrates high-visible to band, uses distilled water flushing 2 times;
5. Taking Pictures recording.
7) data analysis
1. the band on artificial interpretation electrophorogram, the descending assignment successively such as A, B, C, D, E of press, disappearance band, with " .. " expression, is made genotype matrix;
2. compare interracial core primers designed difference logarithm, as Fig. 2, Fig. 3;
3. use Popgene computed in software genetic distance;
4. use Mega5.0 software building dendrogram, as Fig. 4.
8) result is judged: when core primers designed difference logarithm >=2 of two kinds, be judged to be different varieties, form result of determination; When core primers designed difference logarithm≤1 of two kinds, be judged to be same breed or fairly similar kind, now, should be according to investigator need to introduce 24 pairs of standby primers designed, carry out above 3), 4), 5), 6) step, and the data of comprehensive 10 pairs of core primers designed calculate genetic distance and differentiate, when two kind genetic distance >0.140 are judged to be different varieties, genetic distance≤0.140 is judged to be same kind;
1. NH-01 and black ox difference primer pair morning equal 1, are therefore judged to be fairly similar kind.Another all the other 24 pairs of primers of introducing also calculate genetic distance, find that genetic distance is 0.107, are less than 0.140, therefore, judge that NH-01 belongs to early kind of black ox;
2. tea shoot NH-02, NH-03 are consistent with NH-04 banding pattern, all large 2 with the difference primer logarithm of other kinds, therefore judge the not known kind in experiment of this three strains tea shoot.
Above-mentioned steps 4) in 4. PCR response procedures: first, 94 ℃ or 95 ℃ of sex change 2 minutes or 4 minutes; Then, according to following program, carry out 30 circulations: 94 ℃ or 95 ℃ of sex change 25 seconds, 28 seconds or 35 seconds, 58 ℃ of annealing 25-30 seconds, 72 ℃ were extended 25 seconds, 27 seconds or 28 seconds; Then, 72 ℃ are extended 2 minutes or 4 minutes; Finally, 4 ℃ of preservations; Above-mentioned steps 4) 3. in: voltage is 160 or 180V, and electrophoresis time is 110 or 130 minutes; The other the same as in Example 1, also can obtain beneficial effect of the present invention.

Claims (8)

1. a SSR core primers group, it is characterized in that comprising 10 pairs of core primers designed, 24 pairs of standby primers designed, 10 pairs of described core primers designed are 1057,1604,1622,1624,1633,1651,1696,5053,5077,5094, as shown in table 1; The standby primers designed of described 24 couple is 1052,1056,1058,1088,1605,1608,1616,1628,1639,1683,1701,3011,3110,4012,4042,4048,4102,5033,5043,5062,5082,5091,5102,5110, as shown in table 2;
10 pairs of core primers designed information of table 1
Sequence number Numbering Upstream primer (5`-3`) Downstream primer (5`-3`) 1 1057 AACCACACACACAACAATCACTC TTCACCCCAGAACTGGAAAC 2 1604 AGGGTTTTAGGGCTACGAGG TGATTGGAGAACACAACCGA 3 1622 CTCTCGGCACTGCTTCTTCT GAGGACAACGAGGAAGTGGA 4 1624 GAGGCACTCACTACTCCTCCA GTTGTGGGGAGACTCCAAGA 5 1633 AACCGAACAACAGACCCTTG GCTGAGAGAGGAGCGAGAAA 6 1651 GATCGAAATCACCAATGCCT GAGAGAGAAGTCCGTGCGTC 7 1696 TCCAAAAGATTCGGTTCGTC TCGATATTTTCCTTCCGTCG 8 5053 GACGATGGACCCTTCTTTGA CATCATCATCATCCTCACCG 9 5077 TTCACCCTCAGACCAAAAGG TCGATCAATTGCAAAACACA 10 5094 CGAGACATCGAACACCACAG CGTATCGTAGCGGTGAAGGT
24 pairs of standby primers designed information of table 2
Sequence number Numbering Upstream primer (5`-3`) Downstream primer (5`-3`) 1 1052 TAGCCTTGGGTGGATTTCAG TGAATTTGCAGAAGTGAGAGAA 2 1056 AATGGCGTCTCCAATTTGTC CGGAAGAAGAGACGGTGAAG 3 1058 CCCCTCCATCTATCTCCCTC GGACTGTTGTTGGGGAAGAA 4 1088 GGCTGTATGATGGCTTGGTT TAAAATGTGGGTCTCCAGGC 5 1605 TGTAAGTGGCCGGAGTTTTC ACCACAGCAACAGTCACAGC 6 1608 CTTGTGAGGGAGTGACGGAT ATGCCTGCTCGATATTACGG 7 1616 TTCATTCATCAATGGCGGTA CAAAGGGCTTTGTTTTGAGG 8 1628 TGGAGGCATCATTACCAGAA CGTCTCAGACCACTCGTCAA 9 1639 GTGCAAACCGTTGGATTCTT CGATGAGGATGATGATGGTG 10 1683 AGTTGTTCATGGAGGCAAGG ACTTGGCCCTCTTCTTAGGC 11 1701 TTGTCTCTCACCGCCTTTCT CCGAAACTACCAAAACTCCG 12 3011 ATTGCGGGGAAAAGAGAAAT TTGACAAGACTCGTCGATGG 13 3110 TGGGCCAGAAGAGAAAAGAA GGTGTTCCTGGCACTTCAAT 14 4012 ACTCACCATTCGACACCCTC GATCTGGAGACCGATTTGGA 15 4042 AAGCCTTCAAGAACCAGCAA TCTTAATAGCATTATTTGCAGCCA 16 4048 CTCTCGAGGGAAAAGCAATG CAGCATCAGCACTTCCAAGA 17 4102 GTCCGGCAAGATTGGATTTA TCGGTCGATCATCACTGTGT 18 5033 TGCTTCCATGGTGATTTTGA AATGGTGGCTTCAACAAAGG 19 5043 AATTGTAGCAGTTGCCTCCG GAGTAGGTCGATTTGGCGAG 20 5062 TCATTTGAAGTGCAACTGGG TAAGGCGATGATTAGGGTGG 21 5082 CCGATTCCAGGAGGGATTAT TTTGGAATCCGGTTGAAATC 22 5091 TTCGATTGAAATCGGAGGAC CACAACCTCAAGCAAGCAAA 23 5102 TCAATTGCGTCACAAACACA TTTTGTTCATGACGGAGCAC 24 5110 CCTTGCTTTGCTTCTCTCTTTC ATCGGAGGGAGGTCTGAATC
2. right to use requires the method for the SSR core primers group evaluation tea tree breed described in 1, it is characterized in that comprising the following steps:
1) take the tender leaf of different varieties of tea plant is material;
2) extraction of tea tree genomic dna;
3) pcr amplification of target product:
1. take step 2) gained genomic dna is template;
2. use 10 pairs of core primers designed to carry out pcr amplification;
3. use 10 μ L PCR reaction systems, specifically comprise: each 0.2 μ L of upstream and downstream primer, 10 * Buffer (Mg 2+plus) 1 μ L, dNTP 0.2 μ L, rTaq enzyme 0.5U, adds ddH 2o to 10 μ L;
4. PCR response procedures is: first, and 94-95 ℃ of sex change 2-4 minute; Then, according to following program, carry out 30 circulation: 94-95 ℃ of sex change 25-35 seconds, 55-60 ℃ annealing 25-30 second, 72 ℃ and extend 25-30 second; Then, 72 ℃ are extended 2-4 minute; Finally, 3-5 ℃ of preservation;
4) electrophoretic separation of PCR product
1. in step 3) gained PCR reaction product, add 2-3 μ L6 * loading buffer, mix;
2. with pipettor, inhaling 2-3 μ L mixed solution is added in the point sample hole of 10% polyacrylamide gel preparing;
3. 160-180V voltage, electrophoresis 110-130 minute;
5) silver dyes colour developing
1. after step 4) finishes, carefully take off film, put into the special-purpose plastics casing of dyeing;
2. add 400-450mL stationary liquid, shaking table 65-75rpm shakes 5-7 minute, pours out stationary liquid, distilled water flushing 2-3 time;
3. add 350-450mL staining fluid, 65-75rpm shakes 8-12 minute, pours out AgNO3 solution, distilled water flushing 2-3 time;
4. add 400-450mL nitrite ion, 70-75rpm vibrates high-visible to band, uses distilled water flushing 2-3 time;
5. Taking Pictures recording;
6) data analysis
1. the band on artificial interpretation electrophorogram, the descending assignment successively such as A, B, C, D, E of press, disappearance band, with " .. " expression, is made genotype matrix;
2. compare interracial core primers designed difference logarithm;
3. use Popgene computed in software genetic distance;
4. use Mega5.0 software building dendrogram;
7) result is judged
When core primers designed difference logarithm >=2 of two kinds, be judged to be different varieties, form result of determination; When core primers designed difference logarithm≤1 of two kinds, be judged to be same breed or fairly similar kind, now, should be according to investigator need to introduce 24 pairs of standby primers designed, carry out above 3), 4), 5), 6) step, and the data of comprehensive 10 pairs of core primers designed calculate genetic distance and differentiate, when two kind genetic distance >0.140 are judged to be different varieties, genetic distance≤0.140 is judged to be same kind.
3. SSR core primers group as claimed in claim 2 is identified the method for tea tree breed, it is characterized in that step 2) in the extraction of tea tree genomic dna adopt following methods:
1) get 100 mg tea tree tender leafs, add liquid nitrogen grinding;
2) use sky to take root in thing genome DNA extracting reagent kit and extract tea tree genomic dna;
3) use spectrophotometric determination genomic dna concentration, use ddH 2o is diluted to 28-32ng/ μ L, at-20 ℃, saves backup.
4. SSR core primers group as claimed in claim 2 is identified the method for tea tree breed, it is characterized in that in the 4. PCR response procedures of step 3): first, and 94-95 ℃ of sex change 3 minutes; Then, according to following program, carry out 28 seconds, 72 ℃ of 30 circulation: 94-95 ℃ of sex change 28-30 seconds, 56-58 ℃ annealing and extend 26-28 second; Then, 72 ℃ are extended 3 minutes; Finally, 4 ℃ of preservations.
5. SSR core primers group as claimed in claim 2 is identified the method for tea tree breed, it is characterized in that step 4) 3. in: voltage is 170V, and electrophoresis time is 120 minutes.
6. SSR core primers group as claimed in claim 2 is identified the method for tea tree breed, it is characterized in that step 5) 2. in: described stationary liquid is settled to 1 L preparation according to 100ml ethanolic soln, 5mL Glacial acetic acid, adding distil water.
7. SSR core primers group as claimed in claim 2 is identified the method for tea tree breed, it is characterized in that step 5) 3. in: described staining fluid is according to 2g AgNO 3adding distil water is settled to 1L preparation.
8. SSR core primers group as claimed in claim 2 is identified the method for tea tree breed, it is characterized in that step 5) 4. in: described nitrite ion is according to 15g NaOH solution and add 5mL 37% formaldehyde adding distil water to be settled to 1 L preparation.
CN201410249326.8A 2014-06-06 2014-06-06 SSR (Simple Sequence Repeat) core primer group and method thereof for identifying tea variety Pending CN104004756A (en)

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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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CN104894276A (en) * 2015-06-18 2015-09-09 福建省农业科学院茶叶研究所 Molecular marker method for rapidly identifying suitability of tea germplasms for manufacture of oolong
CN104894276B (en) * 2015-06-18 2017-06-27 福建省农业科学院茶叶研究所 A kind of Rapid identification tea germplasm oolong tea fits the molecule labelling method of property processed
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
CN108148922A (en) * 2018-02-02 2018-06-12 安徽农业大学 For the SSR primers and identification method of the identification of grain rains scented tea tree and application
CN108148922B (en) * 2018-02-02 2020-11-20 安徽农业大学 SSR primer for identifying Valley-flavored tea trees, identification method and application
CN111944917A (en) * 2019-05-16 2020-11-17 南京农业大学 Method for developing camellia plant SSR primers based on transcriptome sequencing
CN110564886A (en) * 2019-09-29 2019-12-13 中国林业科学研究院亚热带林业研究所 Golden camellia SSR (simple sequence repeat) marker primer and application thereof in hybrid identification
CN110564886B (en) * 2019-09-29 2023-01-03 中国林业科学研究院亚热带林业研究所 Golden camellia SSR (simple sequence repeat) marker primer and application thereof in hybrid identification
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: 20140827