CN106701948A - Identifying method for purple scallop, bay scallop and filial-generation female parent source - Google Patents
Identifying method for purple scallop, bay scallop and filial-generation female parent source Download PDFInfo
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Abstract
The invention relates to an identifying method for a purple scallop, bay scallop and filial-generation female parent source. The identifying method is characterized in that a primer adopted is a labeled primer mtDNA-Z which is designed based on difference of a mitochondria DNA sequence, and the labeled primer is composed of a left end primer sequence mtDNA-Z-F: 5'-TATGAGGTGTCCCCCAAGTC-3' and a right end primer sequence mtDNA-Z-R: 5'-ACTGGCAGACAAACAAATCGT-3'; extracted DNA of purple scallops, bay scallops and hybrid scallops is taken as a template; the primer is adopted to perform PCR (Polymerase Chain Reaction) amplification on the template; if a PCR special band appears near 460 bp, the female parent mitochondria gene source is determined as the purple scallops; and if no PCR special band appears near the 460 bp, the female parent mitochondria gene source is determined as the bay scallops. According to the identifying method disclosed by the invention, influences of nuclear genome DNA are eliminated, and mitochondria DNA does not need to be independently extracted for identifying, so that the purple scallop, bay scallop and filial-generation female parent source can be quickly and efficiently determined, and therefore, the method is simple and convenient, is short in periodic time, can be performed on a large scale, and provides technical support and guarantee for smoothly performing a breeding process and ensuring unborn pedigree.
Description
Technical field
The invention belongs to descendant's identification technology of scallop crossbreeding, more particularly, to purple scallop, bay scallop and hybridization
The authentication method in the maternal source of offspring.
Background technology
Since bay scallop (Argopecten irradians) is introduced from nineteen eighty-two from the U.S., yield increases year by year,
As one of Chinese most important cultivated shellfish, but the germplasm degenerate problem for occurring in recent years turns into the Chinese scallop culture of restriction
The serious hindrance of industry development.
Purple scallop (Argopecten purpuratus) is the excellent economic shellfish of South America Pacific coast, in size
Deng, cultivate extensively in Chile and Peru, and make Chile turn into be only second to China scallop culture big country.In order to improve bay scallop
Germplasm, improve the economic flow rate of scallop culture industry, Wang Chunde is equal to 2008 and introduces purple scallop and and bay scallop from Peru
Successful cross, cultivates growth vigor highly significant two kinds of hybridization first filial generation, i.e. purple sea hybrid scallop (purple scallop ovum × sea
Gulf scallop sperm) and extra large purple scallop hybrid (filial generation of bay scallop ovum × purple scallop sperm) (Wang Chunde etc., 2009；Wang et al,
2011).But purple scallop and bay scallop are hermaphroditic animal, when breeding, two kinds of scallops discharge sperm and ovum simultaneously
Son, has sperm or ovum pollution, to ensure being smoothed out and confirming offspring for breeding process in the practices of breeding
Pedigree, it is often necessary to determine whether a certain offspring is real filial generation and its Parent source, it is therefore desirable to set up one
Kind of method comes Rapid identification its parental source especially maternal source.
It is miscellaneous identification although to be may also be used for using molecular labeling (such as microsatellite marker) authentication method from Matrix attachment region
Hand over whether offspring is real cenospecies, but cannot distinguish that its Parent is originated, cause that filial generation pedigree is chaotic, it is right to be difficult to
The problems such as excellent strain is screened, and found in cross experiment, the parent as hybridization is different, its hybridization first filial generation
The performance of proterties is also different, proterties having or even showing as parent completely, therefore the maternal of Rapid identification scallop hybrid is originated
It is very important.
The content of the invention
It is an object of the invention to provide a kind of authentication method in the maternal source of purple scallop, bay scallop and filial generation,
The method is the otherness based on mtdna sequence and the labeled primer that designs, and purple using the labeled primer Sequence Identification
The maternal source of scallop, bay scallop and filial generation, to make up the deficiencies in the prior art.
Research lacks genetic recombination it has been found that mitochondrial genomes follow strictly matrilinear inheritance rule, the line grain of filial generation
Body must be maternal from it, therefore by identifying that the source of its mitochondrial DNA is assured that the maternal source of cenospecies.This hair
It is bright according to bay scallop and purple scallop mitochondrial genomes otherness feature, a pair of specific primers are specially devised, to lead to
The type that PCR method identifies filial generation chondriogen is crossed, so as to realize the maternal source of Rapid identification scallop hybrid.
The present invention is based on following design：There are some researches show, the mitochondrial genomes of scallop follow strict matrilinear inheritance,
I.e. the chondriogen of offspring is identical with its maternal chondriogen type, and unrelated with the chondriogen type of male parent；Tool
Body is the sibling species of Argopecten category to bay scallop and purple scallop, although its mitochondrial genomes sequence similarity is up to
82.34%, but still special primer can be designed according to a little difference of both Mitochondrial gene sequences, so as to distinguish hybridization
The source of offspring's chondriogen, be breeding work be smoothed out provide technical support or guarantee.
The present invention is achieved by the following technical solution：The maternal source of purple scallop, bay scallop and filial generation
Authentication method, labeled primer used is mtDNA-Z, respectively by left end primer sequence mtDNA-Z-F：5’-
TATGAGGTGTCCCCCAAGTC-3 ' and right-hand member primer sequence mtDNA-Z-R：5 '-ACTGGCAGACAAACAAATCGT-3 ' groups
Into.
The present invention is identified the maternal source of purple scallop, bay scallop and its filial generation using above-mentioned labeled primer
Method is as follows：
(1) purple scallop to be identified, bay scallop and filial generation purple sea hybrid scallop, the closed shell of sea purple scallop hybrid are taken respectively
Flesh, directly extracts the complete genome DNA of above-mentioned scallop with conventional method, and being diluted to final concentration 100ng/ μ L with TE buffer solutions protects
It is standby in the presence of -20 DEG C；
(2) DNA of above-mentioned scallop respectively to extract is template, and entering performing PCR to it with above-mentioned primer mtDNA-Z expands, reaction
System is 10 μ L, including 0.25U Taq DNA enzymatics (Takara Inc., Shiga, Japan), 1 × PCR buffer (contain 1.5mM
MgCl2) and 0.2mM dNTP mix, each 1 μM of upstream and downstream primer, 50ngDNA templates；Response procedures are：94 DEG C of predegeneration 3min,
94 DEG C of 1min, 56 DEG C of 1min, 72 DEG C of 1min, 30 circulations, 72 DEG C of extension 10min；PCR primer through 1% agarose gel electrophoresis,
EB is dyeed, gel imaging system photograph observation；For purple scallop and bay scallop, if there is PCR specificity bars at nearly 460bp
Band occurs, then prove that this scallop is purple scallop, if no PCR specific bands occur at nearly 460bp, proves this scallop
It is bay scallop；For purple sea hybrid scallop and sea purple scallop hybrid, if there is PCR specific bands to occur at nearly 460bp,
Then determine that its female parent is purple scallop, if no PCR specific bands occur at nearly 460bp, it is determined that its female parent is bay
Scallop.
The invention has the advantages that：
(1) design of primers of the present invention based on mtdna sequence eliminates the influence of nuclear DNA, therefore need not be single
Solely extract mitochondrial DNA to be identified, using a small amount of mitochondrial DNA in the full-length genome of traditional extraction, you can with mark
Primer mtDNA-Z enters performing PCR amplification, and qualification result can be just obtained after being detected through agarose electrophoresis, therefore cycle time is short, side
Method is easy, can in high volume carry out, rapidly and efficiently.
(2) after specific primer of the present invention based on mtdna sequence design can quickly and easily identify two hybridization
Generation and two parent.
Brief description of the drawings
Fig. 1 is the purple scallop of labeled primer mtDNA-Z PCR amplifications used, bay scallop product in the embodiment of the present invention 1
Electrophoretogram, wherein 1-6：Bay scallop, 7-12:Purple scallop, M:DL 2000.
Fig. 2 is labeled primer mtDNA-Z PCR amplified hybridizations offspring purple escallop used, sea purple scallop in the embodiment of the present invention 2
The electrophoretogram of product, wherein 1-8：Extra large purple scallop, 9-16：Purple escallop, M:Marker.
Specific embodiment
Embodiment 1 by taking purple scallop, bay scallop as an example, the result identified.
(1) 6 purple scallops and 6 closed shell fleshes of bay scallop are taken respectively, and directly with Tiangeng DNA extraction kit, (Tiangeng is biochemical
Science and Technology Ltd., Beijing) extract complete genome DNA, with TE buffer solutions be diluted to final concentration 100ng/ μ L be stored in -20 DEG C it is standby
With, then through 1.2% agarose gel electrophoresis, EB dyeing, full-length genome of the gel imaging system photograph observation to ensure to extract
The purity and satisfactory quality of DNA.
(2) DNA with the purple scallop of said extracted and bay scallop enters performing PCR to it and expands as template with above-mentioned primer mtDNA-Z
Increase, reaction system is 10 μ L, including 0.25U Taq DNA enzymatics (Takara Inc., Shiga, Japan), MgCl containing 1.5mM2
1 × PCR buffer, 0.2mM dNTP mix, each 1 μM of upstream and downstream primer, 50ngDNA templates.Response procedures are：94 DEG C pre-
Denaturation 3min, 94 DEG C of 1min, 56 DEG C of 1min, 72 DEG C of 1min, 30 circulations；72 DEG C of extension 10min.PCR primer is through 1% agarose
Gel electrophoresis, EB dyeing, gel imaging system photograph observation electrophoresis result.
(3) electrophoresis result differentiates and shows, does not amplify band (see Fig. 1) in all of bay scallop, and in all of purple
Scallop amplifies obvious band near 460bp, and size is consistent with expected 463bp.By the amplification in above-mentioned purple scallop
Sequencing result size is 460bp after product purification, carries out DNAMAN with the mtdna sequence of purple scallop and compares, and measures sequence
It is 97.81% (being shown in Table 1) with the segment-similarity of purple scallop mitochondrial DNA, so as to eliminate mtDNA-Z primers by Matrix attachment region
Influence, be defined as the pcr amplification reaction carried out as template using purple scallop mitochondrial DNA.Obviously the primer that the present invention is designed
Purple scallop and bay scallop can be identified.
The sequencing result deck watch of table 1
The result that embodiment 2 is identified by taking purple sea hybrid scallop, sea purple scallop hybrid as an example.
(1) purple sea hybrid scallop (purple scallop (ovum) × bay scallop (essence)) and sea purple scallop hybrid (bay scallop are taken respectively
(ovum) × purple scallop (essence)) the scallop closed shell flesh of each 8, with Tiangeng DNA extraction kit (Tiangeng biochemical technology Co., Ltd,
Beijing) extract complete genome DNA respectively, with TE buffer solutions be diluted to final concentration 100ng/ μ L be stored in -20 DEG C it is standby, extraction
Complete genome DNA is through 1.2% agarose gel electrophoresis, EB dyeing, full base of the gel imaging system photograph observation to ensure to extract
Because of the purity and satisfactory quality of group DNA.
(2) DNA with the purple sea hybrid scallop of said extracted and sea purple scallop hybrid enters performing PCR as template with above-mentioned primer pair
Amplification, reaction system is 10 μ L, including 0.25U Taq DNA enzymatics (Takara Inc., Shiga, Japan), 1 × PCR
Buffer (MgCl containing 1.5mM2), 0.2mM dNTP mix, each 1 μM of upstream and downstream primer, 50ngDNA templates.Response procedures are：
94 DEG C of predegenerations 3min, 94 DEG C of 1min, 56 DEG C of 1min, 72 DEG C of 1min, 30 circulations；72 DEG C of extension 10min.PCR primer is through 1%
Agarose gel electrophoresis, EB dyeing, gel imaging system photograph observation electrophoresis result.
(3) electrophoresis result differentiates and shows that all of purple sea hybrid scallop amplifies obvious band, size near 460bp
It is consistent with expected 463bp, and band (see Fig. 2) is not amplified in all of sea purple scallop hybrid, illustrates that the present invention sets
The purple scallop of primer pair identification of meter and the maternal source of Argopecten irradians irradians offspring are errorless.
SEQUENCE LISTING
<110>Qingdao Agricultural University
<120>The authentication method in the maternal source of purple scallop, bay scallop and filial generation
<160> 1
<210> 1
<211> 463
<212> DNA
<213>Purple scallop（Argopecten purpuratus）
<220>
<221> misc_feature
<400> 1
tatgaggtgt cccccaagtc ttgggttttt gggggaggtt ataataggga tagggatttg 60
tagaattttt ccacaaggct atattttttg ttttattcta ctattctttt tcggtggtgc 120
aagaataatg gtgctttaca ccagggtaat acacggaagg ttttcgactg ccctggttcc 180
tggggggtct ggaattacga agtgtagtta tcttggtctt ttccatgggg tgccgttaat 240
tattttgttt tttttacctg cgttcttgtc tttgcgggct cttcggagcg gtctttaaga 300
agtagggcta aagcccacgc actgttgaca gagtaggaga ggaggaagta gaaaaccaga 360
ttaaatgggt ggcgagagcg atgagattgt acaagacccg ggataaaggc taagagaagg 420
ggtctcccga tcgaattagg gtacgatttg tttgtctgcc agt 463
Claims (4)
1. the authentication method that a kind of purple scallop, bay scallop and filial generation female parent are originated, it is characterized in that labeled primer used is
MtDNA-Z, is made up of left end primer sequence mtDNA-Z-F and right-hand member primer sequence mtDNA-Z-R respectively.
2. the authentication method that purple scallop as claimed in claim 1, bay scallop and filial generation female parent are originated, it is characterized in that on
Stating left end primer sequence mtDNA-Z-F is：5’-TATGAGGTGTCCCCCAAGTC-3’.
3. the authentication method that purple scallop, bay scallop and filial generation female parent are originated as claimed in claim 1, it is characterized in that above-mentioned
Right-hand member primer sequence mtDNA-Z-R is：5’-ACTGGCAGACAAACAAATCGT-3’.
4. the authentication method that purple scallop as claimed in claim, bay scallop and filial generation female parent are originated, it is characterised in that
Comprise the following steps that：
(1) purple scallop to be identified, bay scallop and the purple scallop hybrid in sea, the closed shell flesh of purple sea hybrid scallop are taken, directly with often
The method of rule extracts complete genome DNA, with TE buffer solutions be diluted to final concentration 100ng/ μ L be stored in -20 DEG C it is standby；
(2) it is template with the purple scallop, bay scallop and the DNA of above-mentioned scallop hybrid that extract, with above-mentioned primer mtDNA-Z to it
Enter performing PCR amplification, reaction system is 10 μ L, including 0.25U Taq DNA enzymatics, MgCl containing 1.5mM21 × PCR buffer,
0.2mM dNTP mix, each 1 μM of left and right end primer, 50ngDNA templates；Response procedures are：94 DEG C of predegeneration 3min, 94 DEG C
1min, 56 DEG C of 1min, 72 DEG C of 1min, 30 circulations, 72 DEG C of extension 10min；PCR primer is through 1% agarose gel electrophoresis, EB dyes
Color, gel imaging system photograph observation electrophoresis result；If thering are PCR specific bands to occur at nearly 460bp, it is determined that maternal
Chondriogen source is purple scallop, if no PCR specific bands occur at nearly 460bp, it is determined that its maternal line grain
Body gene source is bay scallop.
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| CN201611253679.0A CN106701948B (en) | 2016-12-30 | 2016-12-30 | Method for identifying female parent sources of purple scallops, bay scallops and hybrid offspring |
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| CN201611253679.0A CN106701948B (en) | 2016-12-30 | 2016-12-30 | Method for identifying female parent sources of purple scallops, bay scallops and hybrid offspring |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109207613A (en) * | 2018-11-28 | 2019-01-15 | 宁波大学 | A kind of molecular labeling for identifying Trachyostracous mussel and Mediterranean mussel hybrid individual female parent source |
| CN109430126A (en) * | 2018-11-06 | 2019-03-08 | 广东海洋大学 | A kind of breeding method of the black scallop hybrid new varieties in purple sea |
| CN109825599A (en) * | 2018-11-28 | 2019-05-31 | 宁波大学 | A method of identification Trachyostracous mussel and Mediterranean mussel hybrid individual genetic background |
| CN115807109A (en) * | 2022-12-20 | 2023-03-17 | 中国海洋大学三亚海洋研究院 | PCR technology-based scallop variety identification method and specific primers thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109430126A (en) * | 2018-11-06 | 2019-03-08 | 广东海洋大学 | A kind of breeding method of the black scallop hybrid new varieties in purple sea |
| CN109207613A (en) * | 2018-11-28 | 2019-01-15 | 宁波大学 | A kind of molecular labeling for identifying Trachyostracous mussel and Mediterranean mussel hybrid individual female parent source |
| CN109825599A (en) * | 2018-11-28 | 2019-05-31 | 宁波大学 | A method of identification Trachyostracous mussel and Mediterranean mussel hybrid individual genetic background |
| CN109207613B (en) * | 2018-11-28 | 2022-03-04 | 宁波大学 | Molecular marker for identifying female parent source of hybrid individual of mytilus coruscus and Mediterranean mussel |
| CN109825599B (en) * | 2018-11-28 | 2022-08-09 | 宁波大学 | Method for identifying individual genetic background of hybrid of mytilus coruscus and Mediterranean mussel |
| CN115807109A (en) * | 2022-12-20 | 2023-03-17 | 中国海洋大学三亚海洋研究院 | PCR technology-based scallop variety identification method and specific primers thereof |
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