CN109825599A - A method of identification Trachyostracous mussel and Mediterranean mussel hybrid individual genetic background - Google Patents
A method of identification Trachyostracous mussel and Mediterranean mussel hybrid individual genetic background Download PDFInfo
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- CN109825599A CN109825599A CN201811449272.4A CN201811449272A CN109825599A CN 109825599 A CN109825599 A CN 109825599A CN 201811449272 A CN201811449272 A CN 201811449272A CN 109825599 A CN109825599 A CN 109825599A
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Abstract
Of the invention provides a kind of method for identifying Trachyostracous mussel and Mediterranean mussel hybrid individual genetic background, the provided nucleic acid fragment for being used to distinguish Trachyostracous mussel and Mediterranean mussel, sequence in Trachyostracous mussel is SEQ ID NO:1, and the sequence in the mussel of Mediterranean is SEQ ID NO:2.The present invention, which screens, obtains the genome molecules label that can distinguish Trachyostracous mussel and Mediterranean mussel, in conjunction with the mitochondrial markers that can distinguish Trachyostracous mussel and Mediterranean mussel hybrid generation, on the basis of determining hybrid generation which, different dissolution peak temperatures is presented in the HRM curve expanded by mitochondrial markers, by the position of peak of curve, the mitochondria difference of two kinds of mussels is distinguished；And the dissolution peak value that filial generation can be consistent with its female parent, and then judge the maternal source of filial generation.
Description
Technical field
The invention belongs to shellfish Biotechnology in Genetic Breeding fields, and in particular to a kind of identification Trachyostracous mussel and Mediterranean mussel are miscellaneous
The method for handing over individual inheritance background.
Background technique
Trachyostracous mussel (Mytilus coruscus) is under the jurisdiction of Mollusca Mollusca, Bivalvia Bivalvia, makes a gift of
Shellfish mesh Mytilodia, Mytilidae Mytilidae, nutritive value is high, wide market.Trachyostracous mussel forms product, head
Greatly, dressing percentage is high, full of nutrition, delicious flavour, deep by domestic and international consumers.Zhoushan sea area is originating in for Trachyostracous mussel
Ground is local important economic shellfish；Zhoushan sea area only has Trachyostracous mussel before the 1970s, without Mediterranean mussel
Distribution.Mediterranean mussel (Mytilus galloprovincialis) was introduced from Shandong, Liaoning and other places from 1973 to carry out manually
After cultivation succeeds, Mediterranean mussel culture is quickly grown in Zhoushan.It is reported that there are Mediterranean mussels and thickness for Zhoushan sea area
The hybrid Population of shell mussel, referred to as " hybrid ".Show that there is introgressions between Mediterranean mussel and rear shell mussel
The phenomenon that.For the angle of conservation biology, interspecific hybridization and introgression between invasive species and indigenous species would generally be led
Population structure is caused to change.
With the expansion of Trachyostracous mussel consumption market, the demand to Trachyostracous mussel seed also rises year by year, due to mussel seedling
Kind is difficult to be identified from the appearance, it is necessary to develop the method for carrying out the identification of mussel seed based on molecular level.
Summary of the invention
The object of the present invention is to provide it is a kind of identify Trachyostracous mussel and Mediterranean mussel hybrid individual genetic background method,
So as to effectively identify to Trachyostracous mussel and Mediterranean mussel and hybrid individual.
Present invention firstly provides a kind of for distinguishing the nucleic acid fragment of Trachyostracous mussel and Mediterranean mussel, wherein the core
Acid fragment expands in Trachyostracous mussel and obtains 199bp segment, and in Mediterranean, mussel amplification obtains 133bp segment, the nucleic acid piece
Sequence of the section in Trachyostracous mussel is SEQ ID NO:1, and the sequence in the mussel of Mediterranean is SEQ ID NO:2,
A method of it for identifying Trachyostracous mussel and Mediterranean mussel, is realized by detecting above-mentioned nucleic acid fragment
's；
The detection method, one of which are that above-mentioned nucleic acid fragment is expanded using PCR amplification primer,
One of primer pair, sequence information are as follows:
Upstream primer: CTAGAACCAGTATACAAACCTGTGAA (SEQ ID NO:3),
Downstream primer: CTAGAGTCTTAATGCTTTGTATGATGA (SEQ ID NO:4)；
Above-mentioned nucleic acid fragment detect again hybrid individual whether be pure lines in application.
Another aspect of the present invention provides a kind of detection and determines Trachyostracous mussel and Mediterranean mussel hybrid individual female parent source
Method；It comprises the following steps that
1) first using molecular labeling that can be above-mentioned come whether determine individual to be detected be hybrid generation；
2) to the individual for being determined as hybrid generation, its maternal source is determined by detection mitochondrial molecule mark；
The mitochondrial molecule mark；Wherein the nucleic acid sequence in Trachyostracous mussel is SEQ ID NO:5；In Mediterranean
The sequence of mussel is SEQ ID NO:6；
The wherein survey mitochondrial molecule mark, is detected by high-resolution solubility curve method；
In high-resolution solubility curve method primer sequence YB-16S-4, using used in high-resolution solubility curve method
Primer YB-16S-4 sequence information it is as follows:
Upstream primer: GCTTTGGCTTGCTTAAGTAGTTT (SEQ ID NO:7)；
Downstream primer: ACTAAGCACAACTTAAGAGAA (SEQ ID NO:8).
The present invention, which screens, obtains the genome molecules label that can distinguish Trachyostracous mussel and Mediterranean mussel, in conjunction with can distinguish
The mitochondrial markers of Trachyostracous mussel and Mediterranean mussel hybrid generation, which, passes through mitochondria on the basis of determining hybrid generation
Different dissolution peak temperatures is presented in the HRM curve that label amplification obtains, and by the position of peak of curve, distinguishes two kinds of mussels
Mitochondria difference；And the dissolution peak value that filial generation can be consistent with its female parent, and then judge the maternal source of filial generation.
Detailed description of the invention
Fig. 1: two kinds of mussel gene magnification sequence alignment figures are wherein the identical base sequence of two kinds of mussels in box；
Fig. 2: CG hybrid generation and parent's qualification figure, wherein M:marker；A1, A2: Trachyostracous mussel is maternal；B1, B2: ground
Middle sea mussel male parent；1-10:CG hybrid generation；
Fig. 3: GC hybrid generation and parent's qualification figure, wherein M:marker；B3, B4: Mediterranean mussel is maternal；A3, A4: thick
Shell mussel male parent；1-10:GC hybrid generation；
Fig. 4: CG hybrid generation and parent detect figure, and wherein red represents Trachyostracous mussel female parent；Green represents Mediterranean and makes a gift of
Shellfish male parent；Blue represents CG hybrid generation；
Fig. 5: GC hybrid generation and parent detect figure, and it is maternal that Green represents Mediterranean mussel；Red represents thick shell and makes a gift of
Shellfish male parent；Purple represents G C hybrid generation.
Specific embodiment
Applicant screens the line grain for obtaining the maternal source for being able to detect Trachyostracous mussel and Mediterranean mussel filial generation
Can body molecular labeling mark in conjunction with the karyogene that distinguish Trachyostracous mussel and Mediterranean mussel, to facilitate the present invention.
Wherein the construction method of hybrid generation is as follows in the present invention:
Using Trachyostracous mussel female as female parent, for Mediterranean mussel male as male parent, the stimulation by running water that heats up obtains smart ovum,
Hybridized, obtains the filial generation of CG Orthogonal Composite.
Using Mediterranean mussel female as female parent, for Trachyostracous mussel male as male parent, the stimulation by running water that heats up obtains smart ovum,
Hybridized, obtains GC reciprocal cross and combine filial generation.
Take Selecting Parents of Hybrid Combination Based muscle, 100% alcohol fixation.60 days after Mytilus Edulis Larva hatching, 100% alcohol of sampling is solid
It is fixed.Parent and each 10 individual DNA of reciprocal cross filial generation are extracted using phenol chloroform method.The DNA of extraction is diluted to 100ng/
Microlitre, and detect its integrality with 1.5% agarose gel electrophoresis, be stored in -20 DEG C it is spare.
Embodiment 1: the screening and identification of karyogene molecular labeling
It is found by the applicant that there are the genomic fragment segments of difference in size in Trachyostracous mussel and Mediterranean mussel, wherein
Segment nucleotides sequence is classified as in Trachyostracous mussel
TCTAGAACCAGTATACAAACCTGTGAATAAGATTCCAACACCATATATATCC AAGAAAAGTTATCCG
GCACCATATAAACCGAAAGGCTATTATCCTACGAAA CGTTATCAGCCAACATATGGATCAAAGACAAACTATCCG
CCAATATATAAGC CcAATTGCAAAGAAGCTATCATCATACAAAGCATTAAGACTCTAGAAG(SEQ ID NO:1)
Sequence in the mussel of Mediterranean are as follows:
TCTAGAACCAGTATACAAACCTGTGAAGACAAGTTATCATCCTACgGAATA GTTATCCGCCAACATA
TGGATCAAAGACAAACTATCTGCCACTTGCAAAGA AGCTGTCATCATACAAAGCATTAAGACTCTAGA(SEQ ID
NO:2)
Comparison result shows that corresponding nucleotide fragments length in Trachyostracous mussel is 199bp segment, in Mediterranean mussel
It is 133bp segment in amplification, the two differs 66bp (Fig. 1)；Therefore available to differentiate Trachyostracous mussel and Mediterranean mussel.
Designed for detecting the PCR amplification primer of above-mentioned segment, one of primer pair Myti-2, sequence information is such as
Under:
Upstream primer: CTAGAACCAGTATACAAACCTGTGAA (SEQ ID NO:3)
Downstream primer: CTAGAGTCTTAATGCTTTGTATGATGA (SEQ ID NO:4)；
PCR reaction system is 25 μ L, comprising: 100ng DNA, 2 × Es TaqMasterMix (Dye) (health is century, in
State).
52 DEG C of annealing 1min, 72 DEG C of extension 1min；Last 72 DEG C of extensions 10min.PCR product is solidifying using 2.5% agarose
Gel electrophoresis detection.Two Selecting Parents of Hybrid Combination Based and the agarose amplification of filial generation are as shown in Fig. 2 and Fig. 3.CG hybridization group is maternal
For two Trachyostracous mussels, male parent is two Mediterranean mussels, and respectively distinctive single tape, hybrid generation are presented respectively has two simultaneously
The characteristic bands of a parent, show as biobelt.GC hybridization group female parent is two Mediterranean mussels, and male parent is two Trachyostracous mussels,
Respectively distinctive single tape is presented respectively, hybrid generation has the characteristic bands there are two parent simultaneously, shows as biobelt.The result shows that
The label of screening can effectively judge the kind of mussel seedling, and identify the Parent source of hybrid generation.
Embodiment 2: screening mitochondrial markers
The F mtDNA sequence for screening the Trachyostracous mussel and Mediterranean mussel that obtain, by using AlignX (a
Component of Vector NTI Suite 7.1) software progress sequence alignment, search single nucleotide polymorphism (single
Nucleotide polymorphism, SNP) region, screening obtains that there are the 84bp segments in 6 sites snp, in thick shell
Mussel F mtDNA sequence are as follows:
GCTTTGGCTTGCTTAAGTAGTTTAGGAAAAACGTAAGATTTTCATTCTTAAT TCAGAAATTATTTCT
CTTAAGTTGTGCTTAGT(SEQ ID NO:5)
In Mediterranean mussel F mtDNA sequence are as follows:
GCTTTGGCTTGCTTAAGTAGTTTAGGGAAAACATAAGATTTTCATTCTTAAG TCAGAAAGCAGTTCT
CTTAAGTTGTGCTTAGT(SEQ ID NO:6)
Wherein 6 sites snp are located at amplicon 27 (A-G), 33 (G-A), 52 (T-G), 60 (T-G), 61 (T-C),
63 positions (T-G).
The primer sequence YB-16S-4 for detecting above-mentioned segment is devised according to design of primers principle, primer sequence is as follows:
Upstream primer: GCTTTGGCTTGCTTAAGTAGTTT (SEQ ID NO:7)；
Downstream primer: ACTAAGCACAACTTAAGAGAA (SEQ ID NO:8).
The maternal source of seed is identified using high-resolution solubility curve method using above-mentioned primer.It uses480 saturated fluorescence dyestuff HRM kits include: 10 μ L 1 × 480HRM
Master Mix withDye (Roche Diagnostics), positive anti-primer each 10 μm of ol, 100ng DNA
Template, 1.6 μ L Mgcl2, moisturizing to 20 μ L.The melting curve analysis of PCR reaction and product exists simultaneously480
It is carried out on real-time quantitative analysis instrument (Roche Diagnostics).Response procedures are as follows: 95 DEG C of initial denaturation 10min, 95 DEG C of denaturation
30s, Tm annealing 30s, 72 DEG C of 30s, 45 circulations.Amplified production rises to 95 DEG C of 5s by 72 DEG C with 4.4 DEG C/s, with 2.2 DEG C/s drop
To 65 DEG C of 1min, 95 degrees Celsius are risen to 0.11 DEG C/s.40 DEG C are cooled to 0.11 DEG C/s.Roche is used after reactionIncluded 1.5 software of Tm Calling and Gene Scanning Software of 480 softwares carries out Tm value
Analysis and Genotyping.In CG cross combination, the solubility curve peak value of two Trachyostracous mussel female parents is in 75.5 DEG C, two ground
The solubility curve peak value of extra large mussel male parent is located at 78.1 DEG C.The solubility curve figure of 10 hybrid generations and maternal Trachyostracous mussel one
It causes, is respectively positioned on 75.5 DEG C.Similarly, the solubility curve figure of 10 filial generations of CG reciprocal cross combination is consistent with maternal Mediterranean mussel, equal position
In 78.1 DEG C.Amplification curve is shown in Fig. 4 and Fig. 5.Show the dissolution peak curve of two groups of hybrid generations from the testing result of Fig. 4 and Fig. 5
Figure is consistent with female parent, shows the matrilinear inheritance rule for meeting F mitochondria according to the primer amplification result, can be used as identification filial generation
The label in maternal source.
Embodiment 3: to the qualification result of 4 nursery source mussel seedlings
The Trachyostracous mussel seed of hatching 60-120 days is acquired from 4, Zhejiang Province mussel nursery (being named as A, B, C, D)
Totally 103 individuals, are identified using the above method.After being individually homogenized using spat, phenol chloroform method extracts the DNA of individual.
The DNA of extraction is diluted to 100ng/ microlitres, and detects its integrality with 1.5% agarose gel electrophoresis, is stored in -20 DEG C
It is spare.
Determine whether detection individual is hybrid generation first, karyogene sequence is expanded using primer Myti-2, carries out agar
Sugar detection, it is determined whether there is hybridization, testing result shows 29 detection spats of nursery B, 30 inspections with nursery C
Surveying spat is single band, and feature is consistent with Trachyostracous mussel band, is determined as purebred Trachyostracous mussel seedling.
26 detection spats of nursery A have 23 the single band of Trachyostracous mussel characteristic occur, and 3 individuals go out simultaneously
Two characteristic bands of existing Trachyostracous mussel and Mediterranean mussel, are determined as hybrid individual.
18 detection spats of nursery D, have 1 the single band of Mediterranean mussel characteristic occur, 17 individuals are simultaneously
There are two characteristic bands of Trachyostracous mussel and Mediterranean mussel, is determined as hybrid individual.
For the parent source for further determining that hybrid individual, and to being determined as that purebred mussel seed confirms again,
I has carried out solubility curve analysis to all 103 individuals, using F mitochondria primer YB-16S-4.The result shows that: nursery B
29 detection spats, be respectively positioned on 75.5 DEG C with 30 detection spat solubility curve peaks of nursery C, be Trachyostracous mussel feature
Peak.Female parent is determined as Trachyostracous mussel.
, there is Trachyostracous mussel characteristic peak value in 26 detection spats of nursery A, show that this 26 individual female parents are
Trachyostracous mussel.Syncaryon genetic test is as a result, can determine that, wherein 23 individuals are purebred Trachyostracous mussel seedling, 3 hybrid individuals are
Trachyostracous mussel female parent and Mediterranean mussel paternal hybrid obtain.
18 detection spats of nursery D, show Mediterranean mussel characteristic peak value, show that this 18 individuals are female
It originally is Mediterranean mussel.Syncaryon genetic test is as a result, can determine that, wherein 1 individual is purebred Mediterranean mussel seedling, 17
Hybrid individual is that Mediterranean mussel female parent and Trachyostracous mussel paternal hybrid obtain.
To sum up, this all 103 spat obtains effective identification, identifies that success rate is 100%.Wherein nursery B and C
It is Trachyostracous mussel seedling；A factory 88.5% is Trachyostracous mussel seedling, and 11.5% is Trachyostracous mussel ♀ × Mediterranean mussel ♂ cross hybrid seedling；
C factory 5.6% is Mediterranean mussel seedling, and 94.4% is Mediterranean mussel ♀ × Trachyostracous mussel ♂ cross hybrid seedling.
Qualification result shows that in nursery A and D production process, the female parent used in the parent of A factory is that thick shell is made a gift of
Shellfish, but a small amount of close shellfish of Mediterranean mussel male is mixed in male parent, lead to the hybrid seedlings for occurring a small amount of；D factory is then to use
Female parent be Mediterranean mussel, but be mixed with the close shellfish of a large amount of Trachyostracous mussel male in male parent, lead to the major part of production
Seed is cross hybrid seedling and the purebred seedling of Mediterranean mussel, not purebred Trachyostracous mussel seed.Qualification result embodies thick shell and makes a gift of
The importance that parent selects in shellfish seedling raising process, and the mistake of female and the close shellfish selection of male, the Trachyostracous mussel seed produced
There is also differences for the difference of ratio.
Claims (8)
1. a kind of for distinguishing the nucleic acid fragment of Trachyostracous mussel and Mediterranean mussel, which is characterized in that the nucleic acid fragment exists
Sequence in Trachyostracous mussel is SEQ ID NO:1, and the sequence in the mussel of Mediterranean is SEQ ID NO:2.
2. a kind of for identifying the method for Trachyostracous mussel and Mediterranean mussel, which is characterized in that the method is to pass through detection
Nucleic acid fragment described in claim 1 is realized.
3. method according to claim 2, which is characterized in that the method is to expand right using PCR amplification primer
It is required that nucleic acid fragment described in 1.
4. method as claimed in claim 3, which is characterized in that the primer, the sequence of upstream primer are SEQ ID
NO:3, the sequence of downstream primer are SEQ ID NO:4.
5. nucleic acid fragment described in claim 1 detection hybrid individual whether be pure lines in application.
6. a kind of detect the method for determining Trachyostracous mussel and Mediterranean mussel hybrid individual female parent source；It is characterized in that, described
Method comprises the following steps that
1) determine whether individual to be detected is hybrid generation using nucleic acid fragment described in claim 1 first；
2) to the individual for being determined as hybrid generation, its maternal source is determined by detection mitochondrial molecule mark；
The mitochondrial molecule mark；Wherein the nucleic acid sequence in Trachyostracous mussel is SEQ ID NO:5；In Mediterranean mussel
Sequence be SEQ ID NO:6.
7. method as claimed in claim 6, which is characterized in that the detection detects mitochondrial molecule mark, wherein being to make
It is detected with high-resolution solubility curve method.
8. the method for claim 7, which is characterized in that the method, used in primer pair, upstream primer
Sequence be SEQ ID NO:7, the sequence of downstream primer is SEQ ID NO:8.
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|---|---|---|---|---|
| WO2016207857A1 (en) * | 2015-06-24 | 2016-12-29 | Universidad De Chile | Set of primers and method for detecting and identifying mussel species of the genus mytilus, using high-resolution melting and pcr |
| CN106701948A (en) * | 2016-12-30 | 2017-05-24 | 青岛农业大学 | Identifying method for purple scallop, bay scallop and filial-generation female parent source |
| CN108456735A (en) * | 2018-04-03 | 2018-08-28 | 宁波大学 | A kind of Mytilus galloprovincialis microsatellite locus and its application in hybrid generation identification |
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2018
- 2018-11-28 CN CN201811449272.4A patent/CN109825599B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016207857A1 (en) * | 2015-06-24 | 2016-12-29 | Universidad De Chile | Set of primers and method for detecting and identifying mussel species of the genus mytilus, using high-resolution melting and pcr |
| CN106701948A (en) * | 2016-12-30 | 2017-05-24 | 青岛农业大学 | Identifying method for purple scallop, bay scallop and filial-generation female parent source |
| CN108456735A (en) * | 2018-04-03 | 2018-08-28 | 宁波大学 | A kind of Mytilus galloprovincialis microsatellite locus and its application in hybrid generation identification |
Non-Patent Citations (1)
| Title |
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| 申望等: "舟山海域外来物种地中海贻贝自然分布现状及生态影响", 《台湾海峡》 * |
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