CN104357547A - Construction method of Plectropomus microsatellite DNA molecular markers - Google Patents
Construction method of Plectropomus microsatellite DNA molecular markers Download PDFInfo
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Abstract
The invention discloses a construction method of Plectropomus microsatellite DNA molecular markers. According to the method, Unigene containing Plectropomus microsatellite repeat sequences is firstly screened out by transcriptome sequencing. Then microsatellite sites are detected in the Unigene sequences. Specific primers of microsatellite markers are designed according to the sequences of both ends of the microsatellite sites and are used for detecting polymorphism of the microsatellite sites. The microsatellite markers with abundant polymorphism of Plectropomus are exploited, and seven microsatellite DNA molecular markers of Plectropomus are verified further. The Plectropomus microsatellite DNA molecular markers provided by the invention can be applied to population genetic structure and genetic breeding study of Plectropomus population.
Description
Technical field
The invention belongs to microsatellite molecular marker technical field, be specifically related to a kind of construction process of leopard line gill sour jujube perch Microsatellite DNA molecular marker.
Background technology
Leopard line gill sour jujube perch (Plectropomus leopardus) is under the jurisdiction of Perciformes, Sushi section, gill sour jujube perch belongs to, and is the important cultured fishes in the important south of China.In the last few years, the pressure of fishing for of high strength had made leopard line gill sour jujube perch resource be in overfishing state.In order to protect and manage leopard line gill sour jujube perch fishing resources, study its Genetic Constitution of Population and heritable variation level is very important.Because having, the advantage such as polymorphism is high, codominance has become current seawater fish population genetic and has analyzed the most frequently used means microsatellite molecular marker.But the microsatellite molecular marker up to now for the exploitation of leopard line gill sour jujube perch only has 35 (Molecular Ecology Resources 9:1485 – 1487; Conservation genetics Resources5:1067 – 1069; Conservation genetics Resources 2:101 – 103), quantity is very limited, urgently develops how new leopard line gill sour jujube perch microsatellite molecular marker.On the other hand, the exploitation of current leopard line gill sour jujube perch micro-satellite is main adopts enriched library method, needs to carry out the steps such as DNA extraction, genomic library construction, probe hybridization enrichment, colony screening, wastes time and energy, usually can only obtain dozens of mark.Therefore the efficient microsatellite molecular marker development technique of a kind of leopard line gill sour jujube perch is badly in need of.
Summary of the invention
The object of the present invention is to provide a kind of construction process of leopard line gill sour jujube perch Microsatellite DNA molecular marker, the method is efficient, easy, consuming time short, can primary development more leopard line gill sour jujube perch microsatellite molecular marker.
Above-mentioned purpose of the present invention is achieved through the following technical solutions: a kind of construction process of leopard line gill sour jujube perch Microsatellite DNA molecular marker, containing following steps:
(1) total serum IgE of leopard line gill sour jujube perch sample is extracted in the order-checking of leopard line gill sour jujube perch transcript profile, connects operation, set up sequencing library, adopt Illumina Hi Seq through containing mRNA enrichment, fragmentation mRNA, synthesis cDNA, end reparation and adding " A "
tM2000 order-checkings, obtain leopard line gill sour jujube perch original series;
(2) high quality sequence is obtained after the original series that transcript profile order-checking obtains is removed the low quality segment sequence of joint and Q value≤10 by the acquisition of leopard line gill sour jujube perch Unigene and the detection of microsatellite locus, from the beginning high quality sequence assembles the Unigene obtaining leopard line gill sour jujube perch, then in Unigene sequence, carry out microsatellite locus search, obtain multiple microsatellite locus;
(3) the part microsatellite locus chosen in multiple microsatellite locus is verified, according to the Auele Specific Primer of two ends this microsatellite locus of sequences Design of this part microsatellite locus, extract leopard line gill sour jujube perch genomic dna, the Auele Specific Primer of this microsatellite locus is adopted to carry out pcr amplification, software Cervus2.0 is adopted to detect, the part microsatellite locus detected is the part Microsatellite DNA molecular marker of leopard line gill sour jujube perch, design different Auele Specific Primers according to different microsatellite locus, the DNA molecular marker of different microsatellite locus can be verified one by one.
As one of the present invention preferred embodiment, software Trinity is adopted from the beginning high quality sequence to be assembled to the Unigene obtaining 88813 leopard line gill sour jujube perch in step of the present invention (2), then in Unigene sequence, carry out microsatellite locus with software MicroSAtellite to search, obtain 14649 microsatellite locus, when microsatellite locus is searched, multiplicity is 4 ~ 39, wherein 3716 is mononucleotide repeat sequence, 6332 is dinucleotides tumor-necrosis factor glycoproteins, 4020 is trinucleotide repeats sequence, 310 is TTTC, 153 is fermentation by five tubes, 118 is Hexanucleotide tumor-necrosis factor glycoproteins.
In fact, have change a little according to the quantity of different software or different order-checking condition Unigene and microsatellite locus, these are only adopt the software in the present invention and order-checking condition and the one that obtains preferred embodiment.
7 microsatellite locus preferably chosen in step of the present invention (3) in multiple microsatellite locus are verified, the Auele Specific Primer of these 7 microsatellite locus is as shown in SEQ ID:1 ~ 14.
In fact, 14649 site design Auele Specific Primers according to detecting have thousands of, the invention provides these 7 pairs of primers just to verify, checking can design primer according to these sites detected and it is increased out, thus proves that this method can detect a large amount of exploitation microsatellite locus and can design Auele Specific Primer according to these sites.
The present invention, for other sites remaining, also needs to design other primer amplifications out, and the present invention be only random detection wherein 7, illustrate the method that can adopt in the present invention exactly, confirms as 14649 all microsatellite locus.
During the design of Auele Specific Primer, primer length is preferably 20 ~ 25bp in step of the present invention (3), and annealing temperature is preferably 50 ~ 60 degree, and pcr amplification product length is preferably at 100 ~ 400bp.
The reaction system adopted during pcr amplification in step of the present invention (3) is preferably: comprise in 20 μ L reaction systems: 0.5 μM of Auele Specific Primer, 0.2mM dNTP, 1.5mM MgCl
2, 1 × PCR buffer, 1U Taq archaeal dna polymerase and 50ng template DNA, response procedures: 94 DEG C of 5min, 94 DEG C of 30s, the annealing temperature 30s of each microsatellite locus, 72 DEG C of 30s, finally extend 5min.
The leopard line gill sour jujube perch Microsatellite DNA molecular marker obtained in step of the present invention (3) can be applicable to Genetic Constitution of Population and the genetic breeding research of leopard line gill sour jujube perch population.
Compared with prior art, tool of the present invention has the following advantages: the present invention disposablely can detect a large amount of exploitation microsatellite locus and can according to these sites design Auele Specific Primer, and the method is efficient, easy, consuming time short.
Accompanying drawing explanation
Fig. 1 is the leopard line gill sour jujube perch Unigene that the order-checking of embodiment 1 transcription group obtains;
Fig. 2 is the leopard line gill sour jujube perch microsatellite locus detected in Unigene sequence in embodiment 1;
Fig. 3 be in embodiment 1 site DX1 ~ DX7 at the MICROSATELLITE somatotype figure of 30 tail leopard line gill sour jujube perch genomic dnas.
Embodiment
The present invention is further described below in conjunction with the drawings and specific embodiments.
Embodiment 1
The construction process of leopard line gill sour jujube perch Microsatellite DNA molecular marker, containing following steps:
1, leopard line gill sour jujube perch transcript profile order-checking:
Conventional Trizol method is adopted to extract sample total serum IgE, DNA in DNase I enzymic digestion total serum IgE, by mutually adsorbing thus enrichment mRNA with the magnetic bead of Oligo (dT) and the 3'polyA of mRNA, by mRNA and after interrupting reagent effect under heating, product is reclaimed with alcohol settling on ice, synthetic double chain cDNA in PCR instrument, end reparation is carried out in Thermomixer, reparation product is made to add base A at 3' end, make adapter again and add " A " product to be connected, purifying reclaims amplification and builds library, uses Illumina Hi Seq
tM2000 order-checkings.
2, the acquisition of leopard line gill sour jujube perch Unigene and the detection of microsatellite locus
High quality sequence is obtained after the original series that transcript profile order-checking obtains is removed joint and inferior quality (mass value Q≤10) fragment sequence, re-use software Trinity from the beginning to assemble sequence and obtain leopard line gill sour jujube perch 88813 Unigene (Fig. 1), then software MicroSAtellite (MISA) is used to detect 14649 microsatellite locus in Unigene sequence, wherein, 3716 is mononucleotide repeat sequence, 6332 is dinucleotides tumor-necrosis factor glycoproteins, 4020 is trinucleotide repeats sequence, 310 is TTTC, 153 is fermentation by five tubes, 118 is Hexanucleotide tumor-necrosis factor glycoproteins, multiplicity 4-39 (table 1, Fig. 2), choose 7 microsatellite locus wherein, use software Primer3 according to the Auele Specific Primer of these 7 microsatellite locus two ends sequences Design microsatellite markers again.
3, the checking in leopard line gill sour jujube perch polymorphic micro-satellite site
Extract 30 tail leopard line gill sour jujube perch genomic dnas, carry out pcr amplification with 7 pairs of Auele Specific Primers, reaction system is 20 μ L:0.5 μM primers, 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 is separated, and fragment length software GeneMapper determines according to interior mark ROX-500.The allelotrope number that software Cervus2.0 detects 7 microsatellite locus (DX1 ~ DX7) is 4-12, and observation heterozygosity and expectation heterozygosity are respectively 0.1180-0.2432 and 0.7568-0.8820 (table 2, Fig. 3).
Table 1 leopard line gill sour jujube perch transcript profile checks order the microsatellite locus detected
The allelotrope number that software Cervus2.0 detects 7 microsatellite locus (DX1 ~ DX7) is 4-12, observation heterozygosity and expectation heterozygosity are respectively 0.1180-0.2432 and 0.7568-0.8820 (table 2, Fig. 3), these numerical value show that these 7 sites have more much higher state property.
The present invention adopts above-mentioned 7 pairs of primers to verify, these 7 pairs of primers can amplify this 7 Microsatellite DNA molecular markers, checking can design primer according to these 14649 microsatellite locus detected and it is increased out, thus proves that this method can detect a large amount of exploitation microsatellite locus and can design Auele Specific Primer according to these sites.
The leopard line gill sour jujube perch Microsatellite DNA molecular marker that the present invention obtains can be applicable in the researchs such as the Genetic Constitution of Population of leopard line gill sour jujube perch population and genetic breeding.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included in protection scope of the present invention.
Claims (6)
1. a construction process for leopard line gill sour jujube perch Microsatellite DNA molecular marker, is characterized in that containing following steps:
(1) total serum IgE of leopard line gill sour jujube perch sample is extracted in the order-checking of leopard line gill sour jujube perch transcript profile, connects operation, set up sequencing library, adopt Illumina Hi Seq through containing mRNA enrichment, fragmentation mRNA, synthesis cDNA, end reparation and adding " A "
tM2000 order-checkings, obtain leopard line gill sour jujube perch original series;
(2) high quality sequence is obtained after the original series that transcript profile order-checking obtains is removed the low quality segment sequence of joint and Q value≤10 by the acquisition of leopard line gill sour jujube perch Unigene and the detection of microsatellite locus, from the beginning high quality sequence assembles the Unigene obtaining leopard line gill sour jujube perch, then in Unigene sequence, carry out microsatellite locus search, obtain multiple microsatellite locus;
(3) the part microsatellite locus chosen in multiple microsatellite locus is verified, according to the Auele Specific Primer of two ends this microsatellite locus of sequences Design of this part microsatellite locus, extract leopard line gill sour jujube perch genomic dna, the Auele Specific Primer of this microsatellite locus is adopted to carry out pcr amplification, software Cervus2.0 is adopted to detect, the part microsatellite locus detected is the part Microsatellite DNA molecular marker of leopard line gill sour jujube perch, design different Auele Specific Primers according to different microsatellite locus, the DNA molecular marker of different microsatellite locus can be verified one by one.
2. the construction process of leopard line gill sour jujube perch Microsatellite DNA molecular marker according to claim 1, it is characterized in that: in step (2), adopt software Trinity from the beginning high quality sequence to be assembled to the Unigene obtaining 88813 leopard line gill sour jujube perch, then in Unigene sequence, carry out microsatellite locus with software MicroSAtellite to search, obtain 14649 microsatellite locus, when microsatellite locus is searched, multiplicity is 4 ~ 39, wherein 3716 is mononucleotide repeat sequence, 6332 is dinucleotides tumor-necrosis factor glycoproteins, 4020 is trinucleotide repeats sequence, 310 is TTTC, 153 is fermentation by five tubes, 118 is Hexanucleotide tumor-necrosis factor glycoproteins.
3. the construction process of leopard line gill sour jujube perch Microsatellite DNA molecular marker according to claim 1, it is characterized in that: 7 microsatellite locus chosen in step (3) in multiple microsatellite locus are verified, the Auele Specific Primer of these 7 microsatellite locus is as shown in SEQ ID:1 ~ 14.
4. the construction process of leopard line gill sour jujube perch Microsatellite DNA molecular marker according to claim 1, it is characterized in that: primer length 20 ~ 25bp during the design of Auele Specific Primer in step (3), annealing temperature 50 ~ 60 degree, pcr amplification product length is at 100 ~ 400bp.
5. according to the construction process of leopard line gill sour jujube perch Microsatellite DNA molecular marker according to claim 1, it is characterized in that: the reaction system adopted during pcr amplification in step (3): comprise in 20 μ L reaction systems: 0.5 μM of Auele Specific Primer, 0.2mM dNTP, 1.5mM MgCl
2, 1 × PCR buffer, 1U Taq archaeal dna polymerase and 50ng template DNA, response procedures: 94 DEG C of 5min, 94 DEG C of 30s, the annealing temperature 30s of each microsatellite locus, 72 DEG C of 30s, finally extend 5min.
6. according to the construction process of leopard line gill sour jujube perch Microsatellite DNA molecular marker according to claim 1, it is characterized in that: the leopard line gill sour jujube perch Microsatellite DNA molecular marker obtained in step (3) can be applicable to Genetic Constitution of Population and the genetic breeding research of leopard line gill sour jujube perch population.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105695572A (en) * | 2016-02-02 | 2016-06-22 | 中国水产科学研究院南海水产研究所 | Method for massively and efficiently developing molecular markers on basis of Indel and SSR (simple sequence repeat) site techniques |
| 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 |
| CN114717326A (en) * | 2022-03-10 | 2022-07-08 | 中国科学院南海海洋研究所 | SSR (simple sequence repeat) marker of plectropomus leopardus as well as amplification primer and application of SSR marker |
| CN115820868A (en) * | 2022-07-27 | 2023-03-21 | 中国水产科学研究院南海水产研究所 | Polymorphic primer for amplifying acanthopagrus schlegelii SNP molecular marker and application thereof |
| CN116004848A (en) * | 2022-09-20 | 2023-04-25 | 广东海洋大学 | Leptoradix leopariae internal reference gene ef2, primer and application thereof |
| CN116516028A (en) * | 2023-06-27 | 2023-08-01 | 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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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN105695572A (en) * | 2016-02-02 | 2016-06-22 | 中国水产科学研究院南海水产研究所 | Method for massively and efficiently developing molecular markers on basis of Indel and SSR (simple sequence repeat) site techniques |
| 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 |
| CN114717326A (en) * | 2022-03-10 | 2022-07-08 | 中国科学院南海海洋研究所 | SSR (simple sequence repeat) marker of plectropomus leopardus as well as amplification primer and application of SSR marker |
| 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 |
| CN115820868A (en) * | 2022-07-27 | 2023-03-21 | 中国水产科学研究院南海水产研究所 | Polymorphic primer for amplifying acanthopagrus schlegelii SNP molecular marker 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 |
| CN116004848A (en) * | 2022-09-20 | 2023-04-25 | 广东海洋大学 | Leptoradix leopariae internal reference gene ef2, primer 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 |
| CN116516028A (en) * | 2023-06-27 | 2023-08-01 | 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 |
| 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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