CN1880479A - Turtle germplasm identification method based on mtDNA - Google Patents
Turtle germplasm identification method based on mtDNA Download PDFInfo
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- CN1880479A CN1880479A CN 200610050749 CN200610050749A CN1880479A CN 1880479 A CN1880479 A CN 1880479A CN 200610050749 CN200610050749 CN 200610050749 CN 200610050749 A CN200610050749 A CN 200610050749A CN 1880479 A CN1880479 A CN 1880479A
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Abstract
The invention discloses a Chinese soft-shelled turtle quality authenticating method based on mtDNA, which comprises the following steps: collecting and authenticating sample; extracting total DNA, detecting DNA quality; augmenting object gene segment; detecting augmented product; testing sequence of object gene; correcting sequence; building standard molecular system tree for Chinese soft-shelled turtle quality; collecting mtDNA sequence of detected Chinese soft-shelled turtle; comparing each kind system corresponding sequence of detecting sequence with the built Chinese soft-shelled turtle quality; establishing new system tree; analyzing akin relationship; judging strain of detected Chinese soft-shelled turtle. The invention provides more reasonable and precise strain detecting method without non-specific evidence problem due to morphology elementary.
Description
Technical field
The present invention relates to a kind of animal Idioplasm identification method, specifically a kind of Chinese turtle germplasm identification method based on mtDNA.
Background technology
Traditional Trionyx sinensis (Wiegmann) Idioplasm identification is that the morphological specificity according to Trionyx sinensis (Wiegmann) is carried out on morphologic basis.In recent years, along with the expansion of the market requirement and the fast development of foster soft-shelled turtle industry, being accompanied by the introduction of external kind and the non-country of origin of domestic each strain cultures, causing occurring interlinear cross, germplasm pollution and resource degradation phenomena takes place, propagate morphological differences reduction between the strain of back artificially, make to be difficult to carry out Idioplasm identification according to morphological specificity.
Summary of the invention
Technical problem to be solved by this invention is to propose a kind of application mtDNA molecular marking technique, the Chinese turtle germplasm identification method based on mtDNA that the germplasm of Trionyx sinensis (Wiegmann) is carried out rationally, accurately identifies.
For solving the problems of the technologies described above, a kind of Chinese turtle germplasm identification method based on mtDNA of the present invention comprises the steps:
1, set up the Idioplasm identification standard of Trionyx sinensis (Wiegmann), comprise step by step following based on mtDNA:
1.1, sample collection and evaluation: the original seed of getting each strain germplasm characteristic remarkable of Trionyx sinensis (Wiegmann) is made sample;
1.2, total DNA extraction: extract complete genome DNA;
1.3, the DNA quality examination: utilize ultraviolet spectrophotometer to survey concentration and the purity of DNA, and the concentration of electrophoresis detection DNA and purity;
1.4, the amplification of target gene fragment: adopt polymerase chain reaction technology to carry out the amplification of target gene fragment;
1.5, the detection of amplified production: the concentration of amplified production is identical with step 1.3 with the detection method of purity, but the electrophoretic time shorten in the 40min, to adapt to PCR product fragment features of smaller;
1.6, goal gene is carried out sequencing;
1.7, sequence check and correction: the sequencer address of obtaining is carried out artificial interpretation, uses Chromas that sequencer map is observed, undesired signal correction or removal; Result with two-way order-checking imports Genedoc then, carries out two-way alignment, to reduce error, uses default setting during alignment.
1.8, set up Trionyx sinensis (Wiegmann) variety standard molecular system tree: carry out the sibship analysis, make up Trionyx sinensis (Wiegmann) variety standard molecular system tree.
2, Trionyx sinensis (Wiegmann) Idioplasm identification comprises step by step following:
2.1, be object with Trionyx sinensis (Wiegmann) to be identified, carry out each operation step by step of 1.1--1.6 in the 1st step;
2.2, the Trionyx sinensis (Wiegmann) mtDNA sequence to be measured after will proofreading and correct compares with each the strain corresponding sequence that has made up Trionyx sinensis (Wiegmann) variety standard molecular system tree;
2.3, according to sequence relatively, set up new genealogical tree, carry out the sibship analysis, judge the strain of Trionyx sinensis (Wiegmann) to be identified.
Above-mentioned a kind of Chinese turtle germplasm identification method based on mtDNA, at the sample collection of the 1st big step with when identifying, blood, opotism, the muscle samples of gathering living body, each sample size are the 0.5-1.0 gram, directly use or-20 ℃ of refrigerators are preserved stand-by.
Above-mentioned a kind of Chinese turtle germplasm identification method based on mtDNA carries out the purifying of DNA after the DNA detection of the 1st big step or the 2nd big step.
Above-mentioned a kind of Chinese turtle germplasm identification method based on mtDNA after in the 1st big step or the 2nd big step amplified production being detected, carries out purifying to amplified production.
Above-mentioned a kind of Chinese turtle germplasm identification method based on mtDNA, adopt a kind of in following two kinds of methods to the amplified production purifying time: 1, only observing for electrophoresis result has the amplified production of a bright band to carry out purifying at predetermined length; 2, when a bright band is arranged except that the predetermined length place, when all the other positions also have band, the glue behind the electrophoresis tapped rubber to reclaim and utilize reclaim product and increase again.
Above-mentioned a kind of Chinese turtle germplasm identification method based on mtDNA, the 1st big step or the 2nd big step 1.2 step by step in, adopt SDS and protease K digesting, phenol/chloroform method to extract complete genome DNA.
Above-mentioned a kind of Chinese turtle germplasm identification method based on mtDNA, the 1st big step or the 2nd big step 1.6 step by step in, with two-way sequence measurement goal gene is carried out sequencing.
Because authentication method of the present invention is used the mtDNA molecular marking technique germplasm of culturing soft-shelled turtle is identified, Trionyx sinensis (Wiegmann) Idioplasm identification technology and germplasm Molecular Identification standard have been set up, thereby the evidence of problem when having avoided utilizing the morphology principle to identify is not had a problem, thus can carry out more rationally Trionyx sinensis (Wiegmann), Idioplasm identification accurately.
Description of drawings
Fig. 1 is the schema of the present invention China turtle germplasm identification method;
Fig. 2 is the constructed NJ of mtDNA 16S rRNA partial sequence of 8 soft-shelled turtle samples, the synoptic diagram of ME tree;
Fig. 3 is the synoptic diagram of the constructed NJ tree of the mtDNA COI partial sequence of 10 soft-shelled turtle samples.
Embodiment
As shown in Figure 1, a kind of Chinese turtle germplasm identification method based on mtDNA of the present invention comprises following two big steps:
1, set up the Idioplasm identification standard of Trionyx sinensis (Wiegmann), specifically comprise following ten step by step based on mtDNA:
1.1, sample collection and evaluation: get the original seed of each strain germplasm characteristic remarkable of Trionyx sinensis (Wiegmann), gather blood, opotism, the muscle samples of living body, each sample size is the 0.5-1.0 gram, directly uses or-20 ℃ of refrigerators are preserved stand-by;
1.2, total DNA extraction: adopt SDS and protease K digesting, phenol/chloroform method to extract complete genome DNA (this is the most basic a kind of and a extracting method that expense is minimum, also can adopt other extracting method);
1.3, the DNA quality examination: survey concentration and the purity of DNA, the concentration of electrophoresis detection DNA and purity with ultraviolet spectrophotometer;
1.4, the purifying of DNA: show that as detected result DNA purity crosses the low purifying of then can considering to carry out;
1.5, the amplification of target gene fragment (mtDNA-16S rRNA and COI): adopt polymerase chain reaction (Polymerase Chain Reaction; PCR) technology is carried out the amplification of target gene fragment;
1.6, the detection of amplified production: the concentration of PCR product and the detection method of purity and step 1.3,1.4 basic identical, but the electrophoretic time shortens to 40min with interior (voltage when specifically the time is according to electrophoresis and the factors such as clip size of electric current and amplified production are determined), to adapt to PCR product fragment features of smaller;
1.7, the purifying of amplified production: used two kinds of methods during the PCR product purification: 1, only observing for electrophoresis result has the PCR product of a bright band only to carry out purifying at predetermined length; 2, when a bright band is arranged except that the predetermined length place, when all the other positions also have band, the glue behind the electrophoresis tapped rubber to reclaim and utilize reclaim product and carry out PCR again;
1.8, the method that adopts two-way order-checking to goal gene carry out sequencing (such result than the individual event order-checking more accurately, science);
1.9, sequence check and correction: at first the sequencer address of obtaining is carried out artificial interpretation, uses Chromas that sequencer map is observed, undesired signal correction or removal.Result with two-way order-checking imports Genedoc then, carries out two-way alignment, to reduce error, uses default setting during alignment;
1.10, set up Trionyx sinensis (Wiegmann) variety standard molecular system tree: by adjacent method (Neighbor Joining method; NJ), minimum evolution method (Minimum Evolution method; ME) or other method (as maximum parsimony principle, non-weighted method or the like) carry out the sibship analysis, make up Trionyx sinensis (Wiegmann) variety standard molecular system tree.
2, Trionyx sinensis (Wiegmann) Idioplasm identification, comprise following three step by step:
2.1, be object with Trionyx sinensis (Wiegmann) to be identified, carry out the operation of above each step of 1.1-1.9;
2.2, the Trionyx sinensis (Wiegmann) mtDNA sequence to be measured after will proofreading and correct compares with each the strain corresponding sequence that has made up Trionyx sinensis (Wiegmann) variety standard molecular system tree;
2.3, according to sequence relatively, set up new genealogical tree, carry out the sibship analysis, judge the strain of Trionyx sinensis (Wiegmann) to be identified.
According to the aforesaid method applicant 8 soft-shelled turtle samples are identified, the constructed NJ of its mtDNA 16S rRNA partial sequence, the result of ME tree is as shown in Figure 2.The topological framework of two kinds of methods of NJ and ME is consistent in the segmental analytical results of 16S rRNA sequence part.This shows between the soft-shelled turtle of these several kinds of 16S rRNA sequence part fragment the relation, Taiwan soft-shelled turtle 1 (Tw1), Taiwan soft-shelled turtle 2 (Tw2) and Thailand soft-shelled turtle (Tg), sequence unanimity between Songyang flower soft-shelled turtle (Sy), the Trionyx sinensis (Wiegmann) cross-fertilize seed (Zz), get together earlier between Hunan soft-shelled turtle (Hn) and the Trionyx sinensis (Wiegmann) (Hn), meet with Taiwan soft-shelled turtle 1 (Tw1), Taiwan soft-shelled turtle 2 (Tw2) and Thailand soft-shelled turtle (Tg) again, meet with Songyang flower soft-shelled turtle (Sy), Trionyx sinensis (Wiegmann) cross-fertilize seed (Zz) then, and the sibship between the Japanese soft-shelled turtle (Rb) farthest.
10 soft-shelled turtle samples are identified that the result of the NJ tree that its mtDNA COI partial sequence is constructed as shown in Figure 3 according to the aforesaid method applicant.In the segmental analytical results of COI sequence part, show, three Japanese soft-shelled turtles (Rb1-Rb3) sample sequences unanimity, the sample sequences unanimity of other seven soft-shelled turtles is divided into two significantly.
By above comparison and analysis, the result who derives from different pieces of information and algorithms of different supports to some extent that all these ten soft-shelled turtle samples are divided into two to be propped up greatly: one is Japanese soft-shelled turtle, and another comprises Hunan soft-shelled turtle, Trionyx sinensis (Wiegmann), Taiwan soft-shelled turtle, Thailand soft-shelled turtle, Songyang flower soft-shelled turtle and Trionyx sinensis (Wiegmann) cross-fertilize seed.Wherein Taiwan soft-shelled turtle and Thailand soft-shelled turtle, Songyang flower soft-shelled turtle hybridizes the interspecies relation minimum with Trionyx sinensis (Wiegmann), is that 16S rRNA sequence or COI sequence are all in full accord; Relation is nearer between Hunan soft-shelled turtle and Trionyx sinensis (Wiegmann).By the molecular analysis methods of 16S rRNA and COI sequence, can distinguish the soft-shelled turtle of each kinds such as Japanese soft-shelled turtle, Hunan soft-shelled turtle, Trionyx sinensis (Wiegmann), Taiwan soft-shelled turtle, Songyang flower soft-shelled turtle.
Can find out that from above example application mtDNA can illustrate the sibship between the different strain Trionyx sinensis (Wiegmann) fully, and different strains can be made a distinction, and sets up rational Idioplasm identification standard.
Claims (7)
1, a kind of Chinese turtle germplasm identification method based on mtDNA is characterized in that it comprises the steps:
I, set up the Idioplasm identification standard of Trionyx sinensis (Wiegmann), comprise step by step following based on mtDNA:
1.1, sample collection and evaluation: the original seed of getting each strain germplasm characteristic remarkable of Trionyx sinensis (Wiegmann) is made sample;
1.2, total DNA extraction: extract complete genome DNA;
1.3, the DNA quality examination: utilize ultraviolet spectrophotometer to survey concentration and the purity of DNA, and the concentration of electrophoresis detection DNA and purity;
1.4, the amplification of target gene fragment: adopt polymerase chain reaction technology to carry out the amplification of target gene fragment;
1.5, the detection of amplified production: the concentration of amplified production is identical with step 1.3 with the detection method of purity, but the electrophoretic time shorten in the 40min, to adapt to PCR product fragment features of smaller;
1.6, goal gene is carried out sequencing;
1.7, sequence check and correction: the sequencer address of obtaining is carried out artificial interpretation, uses Chromas that sequencer map is observed, undesired signal correction or removal; Result with two-way order-checking imports Genedoc then, carries out two-way alignment, to reduce error, uses default setting during alignment.
1.8, set up Trionyx sinensis (Wiegmann) variety standard molecular system tree: carry out the sibship analysis, make up Trionyx sinensis (Wiegmann) variety standard molecular system tree.
II, Trionyx sinensis (Wiegmann) Idioplasm identification comprise step by step following:
2.1, be object with Trionyx sinensis (Wiegmann) to be identified, carry out each operation step by step of 1.1--1.6 in I the step;
2.2, the Trionyx sinensis (Wiegmann) mtDNA sequence to be measured after will proofreading and correct compares with each the strain corresponding sequence that has made up Trionyx sinensis (Wiegmann) variety standard molecular system tree;
2.3, according to sequence relatively, set up new genealogical tree, carry out the sibship analysis, judge the strain of Trionyx sinensis (Wiegmann) to be identified.
2, a kind of Chinese turtle germplasm identification method as claimed in claim 1 based on mtDNA, it is characterized in that, at the sample collection of the big step of I with when identifying, gather blood, opotism, the muscle samples of living body, each sample size is the 0.5-1.0 gram, and directly application or-20 ℃ of refrigerators are preserved stand-by.
3, a kind of Chinese turtle germplasm identification method based on mtDNA as claimed in claim 1 or 2 is characterized in that, carries out the purifying of DNA after the DNA detection of I big step or II big step.
4, a kind of Chinese turtle germplasm identification method based on mtDNA as claimed in claim 1 or 2 is characterized in that, after in the big step of I or II big step amplified production being detected, amplified production is carried out purifying.
5, a kind of Chinese turtle germplasm identification method as claimed in claim 4 based on mtDNA, it is characterized in that, adopt a kind of in following two kinds of methods to the amplified production purifying time: 1, only observing for electrophoresis result has the amplified production of a bright band to carry out purifying at predetermined length; 2, when a bright band is arranged except that the predetermined length place, when all the other positions also have band, the glue behind the electrophoresis tapped rubber to reclaim and utilize reclaim product and increase again.
6, a kind of Chinese turtle germplasm identification method based on mtDNA as claimed in claim 1 or 2 is characterized in that, big step of I or II big step 1.2 step by step in, adopt SDS and protease K digesting, phenol/chloroform method to extract complete genome DNA.
7, a kind of Chinese turtle germplasm identification method based on mtDNA as claimed in claim 1 or 2 is characterized in that, big step of I or II big step 1.6 step by step in, with two-way sequence measurement goal gene is carried out sequencing.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212518A (en) * | 2010-04-06 | 2011-10-12 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Standard molecule for wheat tilletia indica mitra detection and construction method thereof |
| CN106811514A (en) * | 2015-12-01 | 2017-06-09 | 中华人民共和国上海出入境检验检疫局 | Soft-shelled turtle subfamily biotic component specificity real-time fluorescence detection method and its kit |
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2006
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212518A (en) * | 2010-04-06 | 2011-10-12 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Standard molecule for wheat tilletia indica mitra detection and construction method thereof |
| CN102212518B (en) * | 2010-04-06 | 2013-06-05 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Standard molecule for wheat tilletia indica mitra detection and construction method thereof |
| CN106811514A (en) * | 2015-12-01 | 2017-06-09 | 中华人民共和国上海出入境检验检疫局 | Soft-shelled turtle subfamily biotic component specificity real-time fluorescence detection method and its kit |
| CN106811514B (en) * | 2015-12-01 | 2020-10-16 | 中华人民共和国上海出入境检验检疫局 | Specific real-time fluorescence detection method for biological components in Amydae and kit thereof |
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