CN105483243A - Method for identifying pure and hybrid misgurnus anguillicaudatus as well as pure and hybrid paramisgurnus dabryanus - Google Patents
Method for identifying pure and hybrid misgurnus anguillicaudatus as well as pure and hybrid paramisgurnus dabryanus Download PDFInfo
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Abstract
The invention discloses a method for identifying pure and hybrid misgurnus anguillicaudatus as well as pure and hybrid paramisgurnus dabryanus. According to the method disclosed by the invention, a platform capable of identifying pure and hybrid misgurnus anguillicaudatus as well as pure and hybrid paramisgurnus dabryanus is built by means of screening out four specific SSR (Simple sequence repeat) markers (2N18, 2N19, 4N7 and 4N17) of the misgurnus anguillicaudatus as well as two specific SSR markers (PD30 and PD50) of the paramisgurnus dabryanus, combining a known mitochondria marker (MP) capable of differentiating the misgurnus anguillicaudatus from the paramisgurnus dabryanus, and combining a method for detecting misgurnus anguillicaudatus ploidy by utilization of fin rays simultaneously. The method disclosed by the invention plays a very important role in the work, such as breeding, selective breeding and cross breeding and the like of the misgurnus anguillicaudatus and the paramisgurnus dabryanus.
Description
Technical field
The present invention relates to a kind of method differentiating loach and the purebred and hybrid of Misgurnus auguillicaudatus, belong to technical field of aquatic organism.
Background technology
Loach (Misgurnusanguillicaudatus) and Misgurnus auguillicaudatus (Paramisgurnusdabryanus) loach be under the jurisdiction of respectively in Cypriniformes Cobitidae belongs to and Misgurnus auguillicaudatus belongs to.These two kinds of loach are widely distributed in China, and except the western plateau of China, certainly reaching north in the south all has distribution.Because there being higher edible and pharmaceutical use, they are more and more subject to liking of human consumer.In recent years, along with the continuous increase of market demand, the wild resource amount of these two kinds of bullheads declines serious, and the market value that result in them climbs up and up, and its market economy is worth and can not despises.At present, the domestic cultivation upsurge having started one loach and Misgurnus auguillicaudatus, large-scale production makes the demand of excellent loach and Misgurnus auguillicaudatus seed increase sharply.But loach germ plasm resource mixes on current fish market, be purebredly difficult to differentiate with hybrid.If use the lower hybrid of fertility as parent, from the angle of breeding, the financial loss that can directly cause breeding field huge.Meanwhile, identify the purebred and hybrid of loach and Misgurnus auguillicaudatus, be the important prerequisite of carrying out these two kinds of loach seed selections and cross-breeding work, this directly concerns the success or not of breeding work.
Microsatellite marker, also known as simple sequence repeats (Simplesequencerepeats, SSRs), is one of molecule marker be most widely used so far.Its application is mainly divided into three directions: genetic diversity Journal of Sex Research, cultivar identification and Relationship iden-tification and genetic linkage maps build and QTL location.Mitochondrial DNA mark, as a kind of novel molecular tool, has the feature of matrilinear inheritance and lacks reparation restructuring ability.Multiple karyotype is there is, based on Diploid and Tetraploid in loach in China.Therefore, when carrying out the breeding process of loach, Ploidy Identification is an important content always, at present, people utilize blood to carry out loach Ploidy detection more, this has certain requirement to the specification of loach and also has certain injury to loach, if judged loach ploidy by fin ray, just can solve the above problems well.
In recent years, domestic loach germ plasm resource mixes, and market has occurred a large amount of loach and Misgurnus auguillicaudatus hybrid.At present, by formalness, molecule marker etc., mirror method for distinguishing is carried out to loach and Misgurnus auguillicaudatus though have, there is no the discrimination method of loach and the purebred and hybrid of Misgurnus auguillicaudatus so far.For breeding with cultivate excellent loach fry, the present invention aims to provide and a kind ofly differentiates the purebred method with hybrid of loach and Misgurnus auguillicaudatus.
Summary of the invention
The present invention is directed to that current loach germ plasm resource mixes, present situation that loach and Misgurnus auguillicaudatus hybrid occur in a large number, provide and a kind ofly can differentiate loach and Misgurnus auguillicaudatus is purebred and the method for hybrid.
The present invention includes: filter out 4 distinctive micro-satellite SSR marker (2N18,2N19,4N7 and 4N17) of loach, 2 distinctive micro-satellite SSR marker (PD30 and PD50) of Misgurnus auguillicaudatus and 1 known mitochondrial markers (MP) distinguishing loach and Misgurnus auguillicaudatus, the primer of above 7 marks of design, and carry out pcr amplification with the genomic dna of this primer pair testing sample, obtain pcr amplification product and carry out gel electrophoresis analysis.Meanwhile, the present invention also carries out the Ploidy detection of testing sample with fin ray, comprehensively analyze ploidy result and Gel electrophoresis results, thus differentiates loach and Misgurnus auguillicaudatus is purebred and hybrid.
More detailed method is as follows:
(1) get sample to be tested, clip part tail fin tissue, is divided into two parts by tail fin tissue, and the tail fin tissue of a soya bean size is used for Ploidy detection; The tail fin organize of another part is in the centrifuge tube of 1.5ml filling 95% ethanol, ammonium acetate/primary isoamyl alcohol method is adopted to extract genomic dna, then use UV spectrophotometer measuring DNA concentration, and the mass concentration of sample is diluted to 100ng/ μ L ,-20 DEG C save backup;
(2) be organized as sample with the tail fin of the soya bean size in step (1), utilize the ploidy analyser to carry out Ploidy detection;
(3) with the DNA in step (1) for masterplate, with 7 mark primer carry out pcr amplification, obtain pcr amplification product, then carry out gel electrophoresis analysis;
Title and the primer sequence thereof of described 7 marks are:
(4) Gel electrophoresis results obtained in the ploidy result obtained in step (2) and step (3) is comprehensively analyzed, in diplontic sample, only 4 distinctive SSR marker of loach and mitochondrial markers have the sample of amplified production to be diploid loach ♀ × diploid loach ♂; Only 2 distinctive SSR marker of Misgurnus auguillicaudatus and mitochondrial markers have the sample of amplified production to be Misgurnus auguillicaudatus ♀ × Misgurnus auguillicaudatus ♂; When loach and the peculiar SSR marker of Misgurnus auguillicaudatus have amplified production, sized by the amplified production of mitochondrial markers, the sample of 285bp band is diploid loach ♀ × Misgurnus auguillicaudatus ♂, and sized by the amplified production of mitochondrial markers, the sample of 214bp band is Misgurnus auguillicaudatus ♀ × diploid loach ♂; In triploid sample, when loach and the peculiar SSR marker of Misgurnus auguillicaudatus have amplified production, sized by the amplified production of mitochondrial markers, the sample of 285bp band is Tetraploid Loach, Misgurnus ♀ × Misgurnus auguillicaudatus ♂, and sized by the amplified production of mitochondrial markers, the sample of 214bp band is Misgurnus auguillicaudatus ♀ × Tetraploid Loach, Misgurnus ♂; Tetraploid sample is Tetraploid Loach, Misgurnus ♀ × Tetraploid Loach, Misgurnus ♂, and only 4 distinctive SSR marker of loach and mitochondrial markers have amplified production.
The ploidy detection method of described step (2) is: add 200 μ L nucleus extraction liquid on the tail fin tissue samples of soya bean size, blade cuts for several times, then 800 μ L cell dyeing liquids are added, lucifuge dyeing 1min, then use 30 μm of strainer filterings, be then settled to the ploidy analyser on 800 μ L with PBS and detect ploidy.
The cumulative volume of the pcr amplification described in step (3) is 10 μ L, wherein containing the template DNA 0.5 μ L of l00ng/ μ L, the 10xBuffer of 1.0 μ L, the 10mmol/LdNTPs of 0.2 μ L, the Taq DNA polymerase of the 0.5IU/ μ L of 0.1 μ L, 10nmol/L forward and the oppositely each 0.25 μ L of SSR primer, surplus is distilled water.
The program of the pcr amplification described in step (3) is: PCR loop parameter is: 94 DEG C of 5min; Then 30 circulations, each circulation comprises 94 DEG C of 1min, 30s under annealing temperature, 72 DEG C of 1min; Last 72 DEG C of 5min extend; Then 4 DEG C of preservations.
Electrophoresis on the MetaPhor sepharose of the gel electrophoresis analysis employing 1.0%-2.0% described in step (3).
The present invention's advantage compared with prior art:
Method set forth in the present invention be based on 4 distinctive micro-satellite SSR marker (2N18,2N19,4N7 and 4N17) of loach, 2 distinctive micro-satellite SSR marker (PD30 and PD50) of Misgurnus auguillicaudatus, 1 known distinguish loach and Misgurnus auguillicaudatus mitochondrial markers (MP) and a kind set up by the method that fin ray carries out loach Ploidy detection.Utilizing present method, efficiently and rapidly to loach and Misgurnus auguillicaudatus is purebred and hybrid is differentiated, a kind of discrimination method of accurate quick can be provided for solving current loach market germ plasm resource trouble waters.
Accompanying drawing explanation
Fig. 1 is the purebred Ploidy detection figure of a diploid loach of Stochastic choice in embodiment one.
Fig. 2 is the PCR primer electrophoresis detection partial results of the distinctive SSR marker 4N7 of loach of embodiment one, wherein M is DNAMarkI, DD1-DD5 is the purebred sample of diploid loach, TT1-TT5 is the purebred sample of Tetraploid Loach, Misgurnus, PP1-PP5 is the purebred sample of Misgurnus auguillicaudatus, TP1-TP5 is hybrid TP sample, and PT1-PT5 is hybrid PT sample.
Fig. 3 is the PCR primer electrophoresis detection partial results of the distinctive SSR marker PD30 of Misgurnus auguillicaudatus in embodiment one, wherein M is DNAMarkI, DD1-DD5 is the purebred sample of diploid loach, and TT1-TT5 is the purebred sample of Tetraploid Loach, Misgurnus, and PP1-PP5 is the purebred sample of Misgurnus auguillicaudatus.
Fig. 4 is the PCR primer electrophoresis detection partial results of embodiment one Mitochondria mark MP, and wherein M is DNAMarkI, PD1-PD5 is hybrid PD sample, and DP1-DP5 is hybrid DP sample.
Embodiment
Below in conjunction with specific embodiment, the present invention is further elaborated.
Embodiment one:
The loach that this example is used and Misgurnus auguillicaudatus adult fish are bought from Wuhan City, Hubei Province Baishazhou fishery market, utilize the ploidy analyser to differentiate diploid loach and Tetraploid Loach, Misgurnus.Select ripe female (25.1-48.3g) and male (11.3-28.4g) diploid loach, Tetraploid Loach, Misgurnus and Misgurnus auguillicaudatus as parent, injection oxytocic drug.The injected dose of raun is LHRH-A
2(20 μ g/kg) and DOM (4mg/kg), milter dosage reduces by half, and injecting method is abdominal cavity isotope labeled leucine.The laggard row artificial insemination of about 12h, obtains loach and purebred (diploid loach ♀ × diploid loach ♂, the DD of Misgurnus auguillicaudatus; Tetraploid Loach, Misgurnus ♀ × Tetraploid Loach, Misgurnus ♂, TT; Misgurnus auguillicaudatus ♀ × Misgurnus auguillicaudatus ♂, PP)) and four kinds of hybrid (diploid loach ♀ × Misgurnus auguillicaudatus ♂, DP; Misgurnus auguillicaudatus ♀ × diploid loach ♂, PD; Tetraploid Loach, Misgurnus ♀ × Misgurnus auguillicaudatus ♂, TP; Misgurnus auguillicaudatus ♀ × Tetraploid Loach, Misgurnus ♂, PT).The rectangle plastic cistern that loach and the purebred and hybrid of Misgurnus auguillicaudatus are placed in 1.0m × 0.5m is respectively raised.The part tail fin tissue of random clip 5 tail diploid loach purebred (DD), 5 tail Tetraploid Loach, Misgurnus purebred (TT), 5 tail Misgurnus auguillicaudatus purebred (PP) and four kinds of each 5 tails of hybrid (DP, PD, TP and PT) respectively.The tail fin tissue of every tail fish is divided into two parts, and the tail fin of a soya bean size is used for Ploidy detection; The tail fin organize of another part is in the centrifuge tube of 1.5ml filling 95% ethanol.Extract genomic dna with reference to the ammonium acetate/primary isoamyl alcohol method after improving, then use UV spectrophotometer measuring DNA concentration, and the mass concentration of each sample is diluted to 100ng/ μ L ,-20 DEG C save backup;
Ploidy detection method is: add 200 μ L nucleus extraction liquid on soya bean size fin ray sample, blade cuts for several times, then adds 800 μ L cell dyeing liquids, lucifuge dyeing 1min, then use 30 μm of strainer filterings, be then settled to the ploidy analyser on 800 μ L with PBS and detect ploidy;
With the genomic dna extracted for masterplate, the upstream and downstream primer (as table 1) designing 7 marks carries out pcr amplification, and the cumulative volume of PCR reaction is 10 μ L, and wherein containing the template DNA 0.5 μ L of l00ng/ μ L, the 10xBuffer of 1.0 μ L is (containing Mg
2+), the Taq DNA polymerase of the 0.5IU/ μ L of the 10mmol/LdNTPs of 0.2 μ L, 0.1 μ L, 10nmol/L forward and the oppositely each 0.25 μ L of SSR primer, surplus is distilled water; Pcr amplification program is: PCR loop parameter is: 94 DEG C of 5min; Then 30 circulations, each circulation comprises 94 DEG C of 1min, (annealing temperature of each mark is in table 1) 30s, 72 DEG C of 1min under annealing temperature; Last 72 DEG C of 5min extend; Then 4 DEG C of preservations.Then electrophoretic analysis is carried out to the MetaPhor sepharose of PCR primer 1-2%.
Table 1 marks title and primer sequence thereof and PCR annealing temperature
The ploidy result (Fig. 1) obtained by Ploidy detection and to PCR primer carry out gel electrophoresis analysis acquisition Gel electrophoresis results (Fig. 2-4) display: 5 DD purebred sample Ploidy detection results are diploid, and only 4 distinctive SSR marker of loach and mitochondrial markers have amplified production; 5 TT purebred sample Ploidy detection results are tetraploid, and only 4 distinctive SSR marker of loach and mitochondrial markers all have amplified production; 5 PP purebred sample Ploidy detection results are diploid, and only 2 distinctive SSR marker of Misgurnus auguillicaudatus and mitochondrial markers have amplified production; 5 DP sample Ploidy detection results are diploid, and loach and the distinctive SSR marker of Misgurnus auguillicaudatus all have amplified production, simultaneously the amplified production of mitochondrial markers all sized by the band of 285bp; 5 PD sample Ploidy detection results are diploid, and loach and the distinctive SSR marker of Misgurnus auguillicaudatus all have amplified production, simultaneously the amplified production of mitochondrial markers all sized by the band of 214bp; 5 TP sample Ploidy detection results are triploid, and loach and the peculiar SSR marker of Misgurnus auguillicaudatus all have amplified production, simultaneously the amplified production of mitochondrial markers all sized by the band of 285bp, and PT and TP sample uniquely equal unlike the amplified production of mitochondrial markers sized by the band of 214bp.The analytical results utilizing this discrimination method to obtain and known loach and background information of hybrid completely the same (100% mate) purebred with Misgurnus auguillicaudatus, prove that present method accurately and reliably.
Claims (5)
1. differentiate a method for loach and the purebred and hybrid of Misgurnus auguillicaudatus, it is characterized in that comprising the following steps:
(1) get sample to be tested, clip part tail fin tissue, is divided into two parts by tail fin tissue, and the tail fin tissue of a soya bean size is used for Ploidy detection; The tail fin organize of another part is in the centrifuge tube of 1.5ml filling 95% ethanol, ammonium acetate/primary isoamyl alcohol method is adopted to extract genomic dna, then use UV spectrophotometer measuring DNA concentration, and the mass concentration of sample is diluted to 100ng/ μ L ,-20 DEG C save backup;
(2) be organized as sample with the tail fin of the soya bean size in step (1), utilize the ploidy analyser to carry out Ploidy detection;
(3) with the DNA in step (1) for masterplate, with 7 mark primer carry out pcr amplification, obtain pcr amplification product, then carry out gel electrophoresis analysis;
Title and the primer sequence thereof of described 7 marks are:
(4) Gel electrophoresis results obtained in the ploidy result obtained in step (2) and step (3) is comprehensively analyzed, in diplontic sample, only 4 distinctive SSR marker of loach and mitochondrial markers have the sample of amplified production to be diploid loach ♀ × diploid loach ♂; Only 2 distinctive SSR marker of Misgurnus auguillicaudatus and mitochondrial markers have the sample of amplified production to be Misgurnus auguillicaudatus ♀ × Misgurnus auguillicaudatus ♂; When loach and the peculiar SSR marker of Misgurnus auguillicaudatus have amplified production, sized by the amplified production of mitochondrial markers, the sample of 285bp band is diploid loach ♀ × Misgurnus auguillicaudatus ♂, and sized by the amplified production of mitochondrial markers, the sample of 214bp band is Misgurnus auguillicaudatus ♀ × diploid loach ♂; In triploid sample, when loach and the peculiar SSR marker of Misgurnus auguillicaudatus have amplified production, sized by the amplified production of mitochondrial markers, the sample of 285bp band is Tetraploid Loach, Misgurnus ♀ × Misgurnus auguillicaudatus ♂, and sized by the amplified production of mitochondrial markers, the sample of 214bp band is Misgurnus auguillicaudatus ♀ × Tetraploid Loach, Misgurnus ♂; Tetraploid sample is Tetraploid Loach, Misgurnus ♀ × Tetraploid Loach, Misgurnus ♂, and only 4 distinctive SSR marker of loach and mitochondrial markers have amplified production.
2. as claimed in claim 1 differentiate the purebred method with hybrid of loach and Misgurnus auguillicaudatus, it is characterized in that, the ploidy detection method of described step (2) is: add 200 μ L nucleus extraction liquid on the tail fin tissue samples of soya bean size, blade cuts for several times, then 800 μ L cell dyeing liquids are added, lucifuge dyeing 1min, then uses 30 μm of strainer filterings, is then settled to the ploidy analyser on 800 μ L with PBS and detects ploidy.
3. as claimed in claim 1 differentiate the purebred method with hybrid of loach and Misgurnus auguillicaudatus, it is characterized in that, the cumulative volume of the pcr amplification described in step (3) is 10 μ L, wherein containing the template DNA 0.5 μ L of l00ng/ μ L, the Taq DNA polymerase of the 0.5IU/ μ L of the 10xBuffer of 1.0 μ L, the 10mmol/LdNTPs of 0.2 μ L, 0.1 μ L, 10nmol/L forward and the oppositely each 0.25 μ L of SSR primer, surplus is distilled water.
4. as claimed in claim 1 differentiate the purebred method with hybrid of loach and Misgurnus auguillicaudatus, it is characterized in that, the program of the pcr amplification described in step (3) is: PCR loop parameter is: 94 DEG C of 5min; Then 30 circulations, each circulation comprises 94 DEG C of 1min, 30s under annealing temperature, 72 DEG C of 1min; Last 72 DEG C of 5min extend; Then 4 DEG C of preservations.
5. the as claimed in claim 1 method differentiating the purebred and hybrid of loach and Misgurnus auguillicaudatus, is characterized in that: electrophoresis on the MetaPhor sepharose of the gel electrophoresis analysis employing 1-2% described in step (3).
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| CN108841930A (en) * | 2018-06-15 | 2018-11-20 | 天津市水产研究所 | A kind of Misgurnus auguillicaudatus microsatellite Parentage determination method and its application |
| CN108841930B (en) * | 2018-06-15 | 2021-09-28 | 天津市水产研究所 | Paramisgurnus dabryanus microsatellite family identification method and application thereof |
| CN110663593A (en) * | 2019-10-28 | 2020-01-10 | 江西农业大学 | Construction method of hybrid loach with fast growth and good meat quality and suitable for paddy field cultivation |
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