CN105734141A - Molecular biology method for identifying purity of tobacco varieties - Google Patents
Molecular biology method for identifying purity of tobacco varieties Download PDFInfo
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Abstract
The invention relates to a molecular biology method for identifying purity of tobacco varieties. The method comprises the following main steps: respectively extracting total genome DNA of a control group and a to-be-detected tobacco variety, performing whole genome sequencing of proper depth by adopting the latest sequencing technology, performing sequence splicing, assembling and whole genome sequence comparison on the control group and the to-be-detected tobacco variety based on a tobacco genome reference sequence by utilizing bioinformatics means, counting base differences of the two groups, and calculating the purity percentage of the to-be-detected tobacco variety relative to the control tobacco variety. The method disclosed by the invention is not influenced by environmental conditions and seasons, is accurate and reliable in result, can accurately identify the purity of the tobacco varieties from a single base variation level of the smallest hereditary unit, can be used for parent purification and authenticity identification of the tobacco varieties and has great significances for guaranteeing safety of tobacco production varieties, maintaining economic benefits of tobacco growers and effectively solving intellectual property disputes of the tobacco varieties.
Description
Technical field
The present invention relates to the authentication method of a kind of purity of plant variety, particularly to a kind of molecular biology method adopting gene order-checking and gene comparision that tobacco bred purity carries out precise Identification, belong to Nicotiana tabacum L. technical field of molecular biology.
Background technology
Variety (varietalpurity) refers to the degree that in batch of seeds, (or in breeding breeding field) is individual with typical consistent in feature, characteristic between individuality, and namely the seed number (strain, spike number) of this kind accounts for the percentage rate for inspection this crop (kind) sample number.The purity requirement that People's Republic of China's tobacco business standard tobacco Seed Inspection code (YC/T20-1994) defines, original seed requires that purity is not less than 99.9%, and one-level breeding is not less than 99.5%, and two grades of breedings are not less than 99.0%.
The verity of seed and purity are to evaluate the important indicator that seed quality is good and bad.The lower general who has surrendered of tobacco bred purity causes the obvious reduction of yield of tobacco and quality, such as upper leaf hacks not good, plant type change, leaves number minimizing, pitch reduction, quality decline and pest and disease damage resistance reduction etc., have a strong impact on the economic benefit of Tobacco Leaf Industry and tobacco grower, leaf tobacco production is constituted safely significant threat.In consideration of it, at present, production mainly takes following five kinds of effective measures and improves purity: (1) prevents mechanical admixture.(2) strict quarantine measures, it is prevented that biology hybrid.(3) original seed is purified, it is prevented that the inherited character of kind own changes.(4) natural mutation is prevented.(5) correct artificial selection.
At present, the main following four of the method for identification of species purity: (1) seed morphology identification method.Main according to the qualification such as the morphological characteristic such as size, shape of seed, color, gloss, seed coat lines.(2) seedling morphology identification method.According to the configuration of seedling, external structure, root system, blade, main occur that quantity (dividing unifacial leaf and dicotyledonous), cotyledon be unearthed/stay the assessment seed purity such as great soil group type, seedling defect.(3) field plot field plot test method.According to the field management that the strict field test arranged is consistent with execution, find out and do not meet this kind typicality shape or there is the plant of significant variation, calculate purity.(4) molecular markers for identification method.Utilize conventional molecular marker as: some samples are detected by RFLP, RAPD, AFLP, SRAP, SSR and InDel labelling one by one, and statistics is present in the sample size of the molecular marker polymorphism compareed between material to be checked, calculates purity.Wherein, integrated marker development cost and identification result, SSR marker is most widely used, such as patent of invention " test kits (CN104164513B) of new No. 19 varieties of certain herbaceous plants with big flowers of a kind of Rapid identification oil certain herbaceous plants with big flowers ", " a kind of method (CN104059992B) of Fructus Melo cenospecies Purity Identification based on EST-SSR labelling ", " a kind of Arillus Longan filial generation Purity method (CN103627799B) based on EST-SSR labelling " etc. all employ SSR molecular marker, also there is the patent of invention " molecular marking technique application (CN1110248C) in the Rapid identification hybrid rice seed true and false and purity " etc. using other Marker Identification varieties.First three Purity method belongs to the phenotypic evaluation of macroscopic view above, the accuracy of result is subject to people, season, the such environmental effects such as weather are bigger, 4th kind of method belongs to the inherent hereditary material qualification of microcosmic, not by such environmental effects, accuracy is greatly improved, but the method also can only carry out pcr amplification DNA fragmentation length out according to molecular marker or judge breed difference and purity height with or without polymorphic differences, but the base difference as hereditary material least unit cannot be detected, in other words, the kind being usually present Individual base difference cannot by above-mentioned Markers for Detection out, therefore, being developed to this minimum hereditary unit level of base the method for identification of species purity seems very necessary.
2010, Chinese tobacco genome plan formally starts, 2013 the end of the year countries tobacco cara gene obtain Nicotiana tabacum L. full-length genome reference sequences, this is splicing of tobacco bred genome weight sequencing sequence in the present invention, assemble and compare and provide sequence reference template, it is greatly saved the time, reduces development cost.
Summary of the invention
The present invention solves that the deficiencies in the prior art provide a kind of new method identifying tobacco bred purity, it is specially a kind of molecular biology method identifying tobacco bred purity, tobacco bred purity can be identified from the base level of minimum hereditary unit, relatively the molecular marker identification method of macrophenotypic class and microcosmic class is more accurate, more can reflect essential difference, it is greatly improved the accuracy of tobacco bred Purity result, can as " final judgement " of tobacco bred Purity, not by environmental influence, to ensureing leaf tobacco production kind of a safety, promote Tobacco Leaf Industry development, ensure that tobacco grower's economic interests and solution tobacco bred IP dispute are significant.
Described a kind of molecular biology method identifying tobacco bred purity, it is characterised in that concrete steps are as follows successively:
A, by comparison with tobacco bred seed to be checked be seeded in identified nutritive cube respectively, it is placed on illumination cultivation frame and cultivates, in 24 hours, auxiliary light is designed as and turns on light 8 ~ 10 hours, turn off the light 14 ~ 16 hours, indoor temperature is maintained at 25 ~ 28 DEG C, keeps the skin wet according to nutritive cube Middle nutrition soil dry and wet situation；
B, grow after 7 ~ 10 true leaves until tobacco seedlings, randomly select the equal comparison of quantity and tobacco bred tobacco seedlings to be checked, every strain tobacco seedlings gathers 1 ~ 3 true leaf, CTAB small-sample method is adopted to extract and purified genes group STb gene, being adjusted to identical by every part of DNA concentration, be generally 100 ~ 200ng/ μ l, the comparison each tobacco seedlings DNA mixed in equal amounts of tobacco bred is configured to comparison DNA mixing pit, tobacco bred to be checked each tobacco seedlings DNA mixed in equal amounts is configured to DNA mixing pit to be checked, and-20 DEG C save backup；
C, utilize genomic sequencing technique that comparison and tobacco bred DNA mixing pit to be checked are carried out the sequence of resurveying of 40 ~ 50 times of degree of depth respectively, obtain weight sequencing data, bioinformatics method is adopted to carry out genome sequence splicing and assemble according to tobacco gene group reference sequences, it is thus achieved that comparison and tobacco bred whole genome sequence to be checked；
D, utilize bioinformatics method comparative control and the base difference of tobacco bred whole genome sequence to be checked, calculate tobacco bred purity to be checked, in this, as the foundation judging tobacco bred purity to be checked height.
In stepb, choose comparison equal with tobacco bred tobacco seedlings quantity to be checked, it is generally 10-15 strain, the DNA concentration of the every strain tobacco seedlings extracted and after purification is adjusted to identical, being generally 100 ~ 200ng/ μ l, comparison is configured to tobacco bred to be checked each tobacco seedlings DNA mixed in equal amounts respectively and compares DNA mixing pit and DNA mixing pit to be checked.
In step C, the tobacco gene group used derives from countries tobacco cara gene with reference to sequence, and comparison and the genomic sequence degree of depth of resurveying of tobacco bred to be checked are 40 ~ 50 times.
In step D, tobacco bred genome sequence percent purity computing formula to be checked is: purity=100* identical base number/(identical base number+distinguishing base number).
Beneficial effect
A kind of molecular biology method identifying tobacco bred purity involved in the present invention, have precisely efficiently, the advantage such as not affected by environment, identifying, accuracy is substantially better than conventional phenotype and molecular marker identification method, for preventing and guaranteeing that leaf tobacco production kind safety, reduction production risk are significant.The present invention is mainly suitable for the aspects such as the Purity of other common cultivation tobacco breds such as flue-cured tobacco, burley tobaccos, parent's purification and true and false Seed Identification.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1, flue-cured tobacco main breed cloud and mist 87 Purity, its concrete steps are as follows successively:
A, by comparison with cloud and mist 87 variety seeds to be checked be seeded in identified nutritive cube respectively, sow 5 alms bowls respectively, it is placed on illumination cultivation frame and cultivates, in 24 hours, auxiliary light is designed as and turns on light 8 hours, turn off the light 16 hours, indoor temperature is maintained at 25 ~ 28 DEG C, keeps the skin wet according to nutritive cube Middle nutrition soil dry and wet situation, generally observes once every 2 ~ 3 days；
B, grow after 7 ~ 10 true leaves until tobacco seedlings, randomly select the equal comparison of quantity and cloud and mist 87 kind tobacco seedlings to be checked, respectively take 10 strains, every strain tobacco seedlings gathers 1 ~ 3 true leaf, CTAB small-sample method is adopted to extract and purified genes group STb gene, every part of DNA concentration is adjusted to identical 150ng/ μ l, 10 strains are compareed cloud and mist 87 tobacco seedlings DNA mixed in equal amounts and is configured to comparison cloud and mist 87DNA mixing pit, 10 strain cloud and mist to be checked 87 tobacco seedlings DNA mixed in equal amounts are configured to cloud and mist 87DNA mixing pit to be checked, and-20 DEG C save backup；
C, utilize genomic sequencing technique (order-checking platform is Hiseq4000) that two DNA mixing pits that step B builds are carried out the sequence of resurveying of 40 times of degree of depth respectively, obtain weight sequencing data, bioinformatics method is adopted to carry out genome sequence splicing and assemble according to the tobacco gene group reference sequences that countries tobacco cara gene provides, it is thus achieved that the whole genome sequence of comparison and cloud and mist 87 kind to be checked；
D, utilize bioinformatics method more to be checked with the base difference compareing tobacco bred whole genome sequence, calculate cloud and mist 87 variety to be checked, in this, as the foundation judging cloud and mist 87 variety to be checked height.
Whole genome sequence comparative result shows, the distinguishing base number of comparison and cloud and mist 87 kind to be checked is 1, 771, 450, 6, identical base number is 441, 091, 502, 3, according to purity computing formula: purity=100* identical base number/(identical base number+distinguishing base number) is known, purity=the 100*4410915023/(17714506+4410915023 of cloud and mist 87 kind to be checked)=99.6%, specify according to People's Republic of China's tobacco business standard tobacco Seed Inspection code (YC/T20-1994), the purity of cloud and mist 87 kind to be checked is higher than one-level breeding purity, leaf tobacco production can be used safely in.
Example 2, burley tobaccos kind Burley37 Purity, its concrete steps are as follows successively:
A, by comparison with Burley37 variety seeds to be checked be seeded in identified nutritive cube respectively, sow 7 alms bowls respectively, it is placed on illumination cultivation frame and cultivates, in 24 hours, auxiliary light is designed as and turns on light 10 hours, turn off the light 14 hours, indoor temperature is maintained at 25 ~ 28 DEG C, keeps the skin wet according to nutritive cube Middle nutrition soil dry and wet situation, generally observes once every 2 ~ 3 days；
B, grow after 7 ~ 10 true leaves until tobacco seedlings, randomly select the equal comparison of quantity and Burley37 kind tobacco seedlings to be checked, respectively take 15 strains, every strain tobacco seedlings gathers 1 ~ 3 true leaf, CTAB small-sample method is adopted to extract and purified genes group STb gene, every part of DNA concentration is adjusted to identical 180ng/ μ l, 15 strains are compareed Burley37 tobacco seedlings DNA mixed in equal amounts and is configured to comparison Burley37DNA mixing pit, 15 strain Burley37 to be checked tobacco seedlings DNA mixed in equal amounts is configured to Burley37DNA mixing pit to be checked, and-20 DEG C save backup；
C, utilize genomic sequencing technique (order-checking platform is Hiseq4000) that two DNA mixing pits that step B builds are carried out the sequence of resurveying of 50 times of degree of depth respectively, obtain weight sequencing data, bioinformatics method is adopted to carry out genome sequence splicing and assemble according to the tobacco gene group reference sequences that countries tobacco cara gene provides, it is thus achieved that the whole genome sequence of comparison and Burley37 kind to be checked；
D, utilize bioinformatics method more to be checked with the base difference compareing tobacco bred whole genome sequence, calculate Burley37 variety to be checked, in this, as the foundation judging Burley37 variety to be checked height.
Whole genome sequence comparative result shows, the distinguishing base number of comparison and Burley37 kind to be checked is 1, 327, 246, 1, identical base number is 441, 088, 147, 3, according to purity computing formula: purity=100* identical base number/(identical base number+distinguishing base number) is known, purity=the 100*4410881473/(13272461+4410881473 of cloud and mist 87 kind to be checked)=99.7%, specify according to People's Republic of China's tobacco business standard tobacco Seed Inspection code (YC/T20-1994), the purity of Burley37 kind to be checked is higher than one-level breeding purity, leaf tobacco production can be used safely in.
Claims (5)
1. the molecular biology method identifying tobacco bred purity, it is characterised in that concrete steps are as follows successively:
A, by comparison with tobacco bred seed to be checked be seeded in identified nutritive cube respectively, it is placed on illumination cultivation frame and cultivates, in 24 hours, auxiliary light is designed as and turns on light 8 ~ 10 hours, turn off the light 14 ~ 16 hours, indoor temperature is maintained at 25 ~ 28 DEG C, keeps the skin wet according to nutritive cube Middle nutrition soil dry and wet situation；
B, grow after 7 ~ 10 true leaves until tobacco seedlings, randomly select the equal comparison of quantity and tobacco bred tobacco seedlings to be checked, every strain tobacco seedlings gathers 1 ~ 3 true leaf, CTAB small-sample method is adopted to extract and purified genes group STb gene, being adjusted to identical by every part of DNA concentration, be 100 ~ 200ng/ μ l, the comparison each tobacco seedlings DNA mixed in equal amounts of tobacco bred is configured to comparison DNA mixing pit, tobacco bred to be checked each tobacco seedlings DNA mixed in equal amounts is configured to DNA mixing pit to be checked, and-20 DEG C save backup；
C, utilize genomic sequencing technique that the comparison in step B and tobacco bred DNA mixing pit to be checked are carried out the sequence of resurveying of 40 ~ 50 times of degree of depth respectively, obtain weight sequencing data, bioinformatics method is adopted to carry out genome sequence splicing and assemble according to tobacco gene group reference sequences, it is thus achieved that comparison and tobacco bred whole genome sequence to be checked；
D, utilize the base difference of bioinformatics method comparative control and tobacco bred whole genome sequence to be checked, calculate tobacco bred purity to be checked, in this, as judging tobacco bred purity to be checked foundation just.
2. a kind of molecular biology method identifying tobacco bred purity according to claim 1, it is characterised in that: in stepb, choose comparison equal with tobacco bred tobacco seedlings quantity to be checked, for 10-15 strain.
3. a kind of molecular biology method identifying tobacco bred purity according to claim 1, it is characterised in that: in stepb, the DNA concentration of the every strain tobacco seedlings extracted and after purification is adjusted to identical, is 100 ~ 200ng/ μ l.
4. a kind of molecular biology method identifying tobacco bred purity according to claim 1, it is characterised in that: in stepb, comparison is configured to tobacco bred to be checked each tobacco seedlings DNA mixed in equal amounts respectively and compares DNA mixing pit and DNA mixing pit to be checked.
5. a kind of molecular biology method identifying tobacco bred purity according to claim 1, it is characterised in that: in step C, comparison and the genomic sequence degree of depth of resurveying of tobacco bred to be checked are 40 ~ 50 times.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106755490A (en) * | 2017-01-23 | 2017-05-31 | 云南省烟草质量监督检测站 | A kind of junior tobacco leaf variety sampling Detection method based on DNA molecular marker |
| CN107354205A (en) * | 2017-07-10 | 2017-11-17 | 中国烟草总公司郑州烟草研究院 | Primer for identifying flue-cured tobacco Zhongyan-100 combines and kit, application and detection method |
| CN107354206A (en) * | 2017-07-10 | 2017-11-17 | 中国烟草总公司郑州烟草研究院 | Primer for identifying flue-cured tobacco south river 3 combines and kit, application and detection method |
| CN111898807A (en) * | 2020-07-14 | 2020-11-06 | 云南省烟草农业科学研究院 | Tobacco yield prediction method based on whole genome selection and application |
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| CN102965443A (en) * | 2012-12-10 | 2013-03-13 | 山东农业大学 | Method for identifying purity of tobacco variety Zhongyan 90 by using specific molecular marker method |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755490A (en) * | 2017-01-23 | 2017-05-31 | 云南省烟草质量监督检测站 | A kind of junior tobacco leaf variety sampling Detection method based on DNA molecular marker |
| CN107354205A (en) * | 2017-07-10 | 2017-11-17 | 中国烟草总公司郑州烟草研究院 | Primer for identifying flue-cured tobacco Zhongyan-100 combines and kit, application and detection method |
| CN107354206A (en) * | 2017-07-10 | 2017-11-17 | 中国烟草总公司郑州烟草研究院 | Primer for identifying flue-cured tobacco south river 3 combines and kit, application and detection method |
| CN107354205B (en) * | 2017-07-10 | 2019-12-27 | 中国烟草总公司郑州烟草研究院 | Primer combination and kit for identifying tobacco 100 in flue-cured tobacco, application and detection method |
| CN107354206B (en) * | 2017-07-10 | 2019-12-27 | 中国烟草总公司郑州烟草研究院 | Primer combination and kit for identifying No. 3 of flue-cured tobacco Nanjiang, application and detection method |
| CN111898807A (en) * | 2020-07-14 | 2020-11-06 | 云南省烟草农业科学研究院 | Tobacco yield prediction method based on whole genome selection and application |
| CN111898807B (en) * | 2020-07-14 | 2024-02-27 | 云南省烟草农业科学研究院 | Tobacco leaf yield prediction method based on whole genome selection and application |
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