CN104357547B - A kind of construction method of plectropomus leopardus Microsatellite DNA molecular marker - Google Patents
A kind of construction method of plectropomus leopardus Microsatellite DNA molecular marker Download PDFInfo
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- 108091092878 Microsatellite Proteins 0.000 title claims abstract description 82
- 241000321455 Plectropomus leopardus Species 0.000 title claims abstract description 54
- 239000003147 molecular marker Substances 0.000 title claims abstract description 23
- 238000010276 construction Methods 0.000 title claims abstract description 12
- 238000013461 design Methods 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 12
- 230000003252 repetitive effect Effects 0.000 claims abstract description 7
- 230000002068 genetic effect Effects 0.000 claims abstract description 4
- 238000011160 research Methods 0.000 claims abstract description 4
- 108020004414 DNA Proteins 0.000 claims description 10
- 238000012408 PCR amplification Methods 0.000 claims description 7
- 108020004999 messenger RNA Proteins 0.000 claims description 7
- 238000000137 annealing Methods 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 4
- 210000002966 serum Anatomy 0.000 claims description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- 241000269799 Perca fluviatilis Species 0.000 claims description 3
- 239000002299 complementary DNA Substances 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 239000000284 extract Substances 0.000 claims description 2
- 238000013467 fragmentation Methods 0.000 claims description 2
- 238000006062 fragmentation reaction Methods 0.000 claims description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 2
- 238000012163 sequencing technique Methods 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 claims description 2
- 241000282373 Panthera pardus Species 0.000 claims 2
- 208000031481 Pathologic Constriction Diseases 0.000 claims 2
- 210000001215 vagina Anatomy 0.000 claims 2
- 238000009395 breeding Methods 0.000 claims 1
- 230000001488 breeding effect Effects 0.000 claims 1
- 238000012772 sequence design Methods 0.000 claims 1
- 238000012214 genetic breeding Methods 0.000 abstract description 3
- 239000003550 marker Substances 0.000 abstract description 3
- 238000002372 labelling Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 241000276618 Perciformes Species 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 241000218636 Thuja Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
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- 238000010438 heat treatment Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
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Abstract
The invention discloses the construction method of a kind of plectropomus leopardus Microsatellite DNA molecular marker, the method screens the Unigene containing plectropomus leopardus microsatellite repetitive sequence by transcript profile order-checking, then microsatellite locus detected in Unigene sequence；According to the specific primer of two ends, site sequential design microsatellite marker, and detect its polymorphism, the microsatellite marker of exploitation plectropomus leopardus rich polymorphism.A step of going forward side by side demonstrates wherein 7 plectropomus leopardus Microsatellite DNA molecular markers, and the plectropomus leopardus Microsatellite DNA molecular marker in the present invention can be applicable to the researchs such as Genetic Constitution of Population and the genetic breeding of plectropomus leopardus population.
Description
Technical field
The invention belongs to microsatellite molecular marker technical field, be specifically related to a kind of plectropomus leopardus microsatellite DNA molecule
The construction method of labelling.
Background technology
Plectropomus leopardus (Plectropomus leopardus) is under the jurisdiction of Perciformes, section, gill spine perch belongs to, and is China's weight
Want the cultured fishes that south is important.In the last few years, the pressure of fishing for of high intensity had made plectropomus leopardus resource be in excessively
Fish for state.In order to protect and manage plectropomus leopardus fishery resources, study its Genetic Constitution of Population and hereditary variation level is
The most necessary.Microsatellite molecular marker has become as current seawater fish colony because having the advantages such as polymorphism height, codominance
The means that genetic analysis is the most frequently used.But, the microsatellite molecular marker for plectropomus leopardus exploitation only has 35 up to now
(Molecular Ecology Resources 9:1485–1487；Conservation genetics Resources5:
1067–1069；Conservation genetics Resources 2:101 103), quantity is very limited, it would be highly desirable to exploitation is more
The newest plectropomus leopardus microsatellite molecular marker.On the other hand, the exploitation of current plectropomus leopardus microsatellite mainly uses enrichment
Library method, needs to carry out the steps such as DNA extraction, genomic library construction, probe hybridization enrichment, colony screening, wastes time and energy, logical
Often can only obtain dozens of labelling.Therefore a kind of plectropomus leopardus efficient microsatellite molecular marker development technique it is badly in need of.
Summary of the invention
It is an object of the invention to provide the construction method of a kind of plectropomus leopardus Microsatellite DNA molecular marker, the method
Efficiently, easy, the shortest, can primary development more plectropomus leopardus microsatellite molecular marker.
The above-mentioned purpose of the present invention is achieved through the following technical solutions: a kind of plectropomus leopardus microsatellite DNA divides
The construction method of sub-labelling, containing following steps:
(1) total serum IgE of plectropomus leopardus sample is extracted in the order-checking of plectropomus leopardus transcript profile, through containing mRNA enrichment, fragmentation
MRNA, synthesis cDNA, end reparation and add " A " and connect operation, set up sequencing library, use Illumina Hi SeqTM2000
Order-checking, it is thus achieved that plectropomus leopardus original series；
(2) the original sequence that transcript profile order-checking is obtained by the acquisition of plectropomus leopardus Unigene and the detection of microsatellite locus
Obtaining high-quality sequence after the low quality segment sequence of row removal joint and Q-value≤10, from the beginning high-quality sequence assembles
To the Unigene of plectropomus leopardus, in Unigene sequence, then carry out microsatellite locus lookup, obtain multiple microsatellite position
Point；
(3) the part microsatellite locus chosen in multiple microsatellite locus is verified, according to this part microsatellite locus
The specific primer of two ends this microsatellite locus of sequential design, extract plectropomus leopardus genomic DNA, use this microsatellite position
The specific primer of point carries out PCR amplification, uses software Cervus2.0 to detect, and the part microsatellite locus detected is i.e.
For the part Microsatellite DNA molecular marker of plectropomus leopardus, design different specific primers according to different microsatellite locus,
The DNA molecular marker of different microsatellite locus can be verified one by one.
As the present invention one preferred embodiment, step of the present invention (2) uses software Trinity to high-quality
From the beginning sequence assembles the Unigene obtaining 88813 plectropomus leopardus, then exists with software MicroSAtellite
Unigene sequence carries out microsatellite locus lookup, obtains 14649 microsatellite locus, repeat secondary when microsatellite locus is searched
Number is 4～39, and wherein 3716 is mononucleotide repeat sequence, and 6332 is dinucleotide repetitive sequence, and 4020 is three cores
Thuja acid repetitive sequence, 310 is TTTC, and 153 is fermentation by five tubes, and 118 is Hexanucleotide
Repetitive sequence.
It practice, have according to the quantity of different software or different order-checking conditions Unigene and microsatellite locus
Change somewhat, these are only the one using the software in the present invention and order-checking condition and obtain preferred embodiment.
7 microsatellite locus preferably chosen in step of the present invention (3) in multiple microsatellite locus are verified, these 7
The specific primer of microsatellite locus is as shown in SEQ ID:1～14.
It practice, have thousands of according to 14649 the site design specific primers detected, the present invention provides this
7 pairs of primers are simply verified, checking can design primer according to these sites detected and it is expanded out, thus proves this
The method of kind can detect substantial amounts of exploitation microsatellite locus and can design specific primer according to these sites.
The present invention is for other sites remaining, it is also desirable to design other primer amplifications out, and the present invention is only with machine examination
Survey 7 therein, it is simply that illustrate to use the method in the present invention, all of microsatellite locus such as 14649 has been carried out
Confirm.
In step of the present invention (3), during the design of specific primer, primer length is preferably 20～25bp, and annealing temperature is preferred
Being 50～60 degree, pcr amplification product length is preferably 100～400bp.
The reaction system used during PCR amplification in step of the present invention (3) is preferably: include in 20 μ L reaction systems: 0.5
μM specific primer, 0.2mM dNTP, 1.5mM MgCl2, 1 × PCR buffer, 1U Taq archaeal dna polymerase and 50ng mould
Plate DNA, response procedures: 94 DEG C of 5min, 94 DEG C of 30s, annealing temperature 30s of each microsatellite locus, 72 DEG C of 30s, finally extend
5min。
The plectropomus leopardus Microsatellite DNA molecular marker obtained in step of the present invention (3) can be applicable to plectropomus leopardus kind
The Genetic Constitution of Population of group and genetic breeding research.
Compared with prior art, present invention have the advantage that the present invention can disposably detect that substantial amounts of exploitation is micro-
Specific primer also can be designed according to these sites in satellite site, and the method is efficient, easy, the shortest.
Accompanying drawing explanation
Fig. 1 is plectropomus leopardus Unigene that the order-checking of embodiment 1 transcription group obtains；
Fig. 2 is the plectropomus leopardus microsatellite locus detected in Unigene sequence in embodiment 1；
Fig. 3 is that in embodiment 1, site DX1～DX7 divides at the MICROSATELLITE of 30 tail plectropomus leopardus genomic DNAs
Type figure.
Detailed description of the invention
The present invention is further described below in conjunction with the accompanying drawings with specific embodiment.
Embodiment 1
The construction method of plectropomus leopardus Microsatellite DNA molecular marker, containing following steps:
1, plectropomus leopardus transcript profile order-checking:
Using conventional Trizol method to extract sample total serum IgE, the DNA in DNase I enzymic digestion total serum IgE, by with Oligo
(dT) 3'polyA of magnetic bead and mRNA is in mutually absorption thus is enriched with mRNA, under heating by mRNA with interrupt reagent effect after,
Reclaim product, synthetic double chain cDNA in PCR instrument on ice by ethanol precipitation, Thermomixer carries out end reparation, makes
Repairing product at 3' end plus base A, then make adapter and add the connection of " A " product, purification reclaims amplification and builds library, makes
With Illumina Hi SeqTM2000 order-checkings.
2, the acquisition of plectropomus leopardus Unigene and the detection of microsatellite locus
After original series removal joint and low quality (mass value Q≤10) fragment sequence that transcript profile order-checking is obtained
Obtain high-quality sequence, re-use software Trinity and from the beginning sequence is assembled obtain plectropomus leopardus 88813
Unigene (Fig. 1), then detects 14649 microsatellite positions with software MicroSAtellite (MISA) in Unigene sequence
Point, wherein, 3716 is mononucleotide repeat sequence, and 6332 is dinucleotide repetitive sequence, and 4020 is trinucleotide weight
Complex sequences, 310 is TTTC, and 153 is fermentation by five tubes, and 118 are repeated sequence for Hexanucleotide
Row, number of repetition 4-39 (table 1, Fig. 2), choose 7 microsatellite locus therein, more micro-defend according to these 7 with software Primer3
The specific primer of championship point two ends sequential design microsatellite marker.
3, the checking in plectropomus leopardus polymorphic micro-satellite site
Extracting 30 tail plectropomus leopardus genomic DNAs, with 7, specific primer is carried out PCR amplification, reaction system is 20 μ
L:0.5 μM of primer, 0.2mM dNTP, 1.5mM MgCl2,1 × PCR buffer, 1U Taq archaeal dna polymerase and 50ng template
DNA.Response procedures: 94 DEG C of 5min, 94 DEG C of 30s, each SSR site optimum annealing temperature 30s, 72 DEG C of 30s, finally extend
5min.PCR primer ABI PRISM 3730 automatic dna sequencer separates, and fragment length software GeneMapper is according to interior
Mark ROX-500 determines.The allele number of software Cervus2.0 7 microsatellite locus (DX1～DX7) of detection is 4-12, sees
Survey heterozygosity and expectation heterozygosity is respectively 0.1180-0.2432 and 0.7568-0.8820 (table 2, Fig. 3).
The microsatellite locus that the order-checking of table 1 plectropomus leopardus transcript profile detects
The allele number of software Cervus2.0 7 microsatellite locus (DX1～DX7) of detection is 4-12, observes heterozygosis
Degree and expectation heterozygosity are respectively 0.1180-0.2432 and 0.7568-0.8820 (table 2, Fig. 3), and these numerical value show this 7 positions
Point has more much higher state property.
The present invention uses above-mentioned 7 pairs of primers to verify, these 7 pairs of primers can amplify this 7 microsatellite DNA molecule marks
Note, checking can design primer according to these 14649 microsatellite locus detected and it is expanded out, thus proving this
The method of kind can detect substantial amounts of exploitation microsatellite locus and can design specific primer according to these sites.
The plectropomus leopardus Microsatellite DNA molecular marker that the present invention obtains can be applicable to the population of plectropomus leopardus population and loses
Pass in the research such as structure and genetic breeding.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limit, the change made under other any spirit without departing from the present invention and principle, modify, substitute, combine, simplify,
All should be the substitute mode of equivalence, be included in protection scope of the present invention.
Claims (4)
1. a construction method for plectropomus leopardus Microsatellite DNA molecular marker, is characterized in that containing following steps:
(1) total serum IgE of plectropomus leopardus sample is extracted in the order-checking of plectropomus leopardus transcript profile, through containing mRNA enrichment, fragmentation
MRNA, synthesis cDNA, end reparation and add " A " and connect operation, set up sequencing library, use Illumina Hi Seq
2000 order-checkings, it is thus achieved that plectropomus leopardus original series；
(2) original series that transcript profile order-checking obtains is gone by the acquisition of plectropomus leopardus Unigene and the detection of microsatellite locus
Except obtaining high-quality sequence after the low quality segment sequence of joint and Q-value≤10, from the beginning high-quality sequence assembles and obtains leopard
The Unigene of stricture of vagina gill spine perch, then carries out microsatellite locus lookup in Unigene sequence, obtains multiple microsatellite locus；
(3) the part microsatellite locus chosen in multiple microsatellite locus is verified, according to the two of this part microsatellite locus
Terminal sequence designs the specific primer of this microsatellite locus, extracts plectropomus leopardus genomic DNA, uses this microsatellite locus
Specific primer carries out PCR amplification, uses software Cervus2.0 to detect, and the part microsatellite locus detected is leopard
The part Microsatellite DNA molecular marker of stricture of vagina gill spine perch, according to the specific primer that different microsatellite locus designs is different,
The DNA molecular marker of the different microsatellite locus of checking one by one；
Step (2) uses software Trinity from the beginning high-quality sequence is assembled and obtain 88813 plectropomus leopardus
Unigene, then carries out microsatellite locus lookup in Unigene sequence with software MicroSAtellite, obtains 14649
Microsatellite locus, when microsatellite locus is searched, number of repetition is 4 ~ 39, and wherein 3716 is mononucleotide repeat sequence, 6332
For dinucleotide repetitive sequence, 4020 is trinucleotide repeats sequence, and 310 is TTTC, and 153 is five
Trinucleotide repeat sequence, 118 is Hexanucleotide repetitive sequence；
7 microsatellite locus chosen in step (3) in multiple microsatellite locus are verified, the spy of these 7 microsatellite locus
Specific primer is as shown in SEQ ID:1 ~ 14.
The construction method of plectropomus leopardus Microsatellite DNA molecular marker the most according to claim 1, is characterized in that: step
(3) primer length 20 ~ 25bp during the design of specific primer in, annealing temperature 50 ~ 60 degree, pcr amplification product length 100 ~
400bp。
3. according to the construction method of the plectropomus leopardus Microsatellite DNA molecular marker described in claim 1, it is characterized in that: step (3)
The reaction system used during middle PCR amplification: include in 20 μ L reaction systems: 0.5 μM of specific primer, 0.2mM dNTP,
1.5mM MgCl2, 1 × PCR buffer, 1U Taq archaeal dna polymerase and 50ng template DNA, response procedures: 94 DEG C of 5min,
94 DEG C of 30s, annealing temperature 30s of each microsatellite locus, 72 DEG C of 30s, finally extend 5min.
4. according to the construction method of the plectropomus leopardus Microsatellite DNA molecular marker described in claim 1, it is characterized in that: step (3)
The plectropomus leopardus Microsatellite DNA molecular marker of middle acquisition can be applicable to Genetic Constitution of Population and the heredity of plectropomus leopardus population
Breeding research.
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| CN104988136A (en) * | 2015-06-22 | 2015-10-21 | 红河学院 | Method for developing microsatellite markers of bagarius yarrelli sykes fishes and application of the method |
| CN105349533A (en) * | 2015-12-21 | 2016-02-24 | 生工生物工程（上海）股份有限公司 | Method for constructing strand-specific transcriptome library |
| CN105695572B (en) * | 2016-02-02 | 2021-02-23 | 中国水产科学研究院南海水产研究所 | Method for developing molecular markers in large scale and efficiently based on Indel and SSR site technology |
| CN114015789A (en) * | 2021-12-06 | 2022-02-08 | 中国水产科学研究院黄海水产研究所 | Genome selection method for cultivating disease-resistant improved Dongxiang spots |
| CN114717326B (en) * | 2022-03-10 | 2024-03-19 | 中国科学院南海海洋研究所 | SSR (simple sequence repeat) marker of Perch gill and amplification primer and application thereof |
| CN115820868B (en) * | 2022-07-27 | 2023-08-29 | 中国水产科学研究院南海水产研究所 | Polymorphic primer for amplifying SNP molecular markers of oplegnathus ruticosus and application thereof |
| CN116004848B (en) * | 2022-09-20 | 2024-02-23 | 广东海洋大学 | Leptoradix leopariae internal reference gene ef2, primer and application thereof |
| CN116516028B (en) * | 2023-06-27 | 2023-09-15 | 中国海洋大学三亚海洋研究院 | SNP locus related to anti-nervous necrosis virus character of leopard gill-acanthus japonicus and application thereof |
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