CN102181535A - Scylla paramamosain microsatellite DNA marker and screening method thereof - Google Patents
Scylla paramamosain microsatellite DNA marker and screening method thereof Download PDFInfo
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- CN102181535A CN102181535A CN 201110068886 CN201110068886A CN102181535A CN 102181535 A CN102181535 A CN 102181535A CN 201110068886 CN201110068886 CN 201110068886 CN 201110068886 A CN201110068886 A CN 201110068886A CN 102181535 A CN102181535 A CN 102181535A
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- scylla paramamosain
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- microsatellite dna
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Abstract
The invention relates to the technology of the DNA molecular marker and provides a Scylla paramamosain microsatellite DNA marker and a screening method thereof. The Scylla paramamosain microsatellite DNA marker can be numbered to be A508. The screening method comprises the following steps: constructing a Scylla paramamosain microsatellite enriched library, selecting monoclone for sequencing; using a microsatellite DNA locus searching software to obtain the positive clone containing the Scylla paramamosain microsatellite sequence, and determining the Scylla paramamosain microsatellite DNA marker. The microsatellite marker A508 can be used for the genetic structure analysis of Scylla paramamosain, idioplasm resource protection and molecular marker-assisted selection, have good repeatability and be a reliable and effective molecular marker. The obtained Scylla paramamosain microsatellite DNA marker lays the foundation for the genetic structure analysis of Scylla paramamosain, idioplasm resource protection, molecular marker-assisted selection and the like.
Description
Technical field
The present invention relates to the dna molecular marker technology, particularly Scylla paramamosain (Scylla paramamosain) microsatellite DNA mark and screening method thereof.
Background technology
Microsatellite sequence (microsatellite sequences) claims that again (simple sequence repeats SSRs), is meant the section of DNA sequence that is repeated repeatedly to constitute by 2～6 nucleotidyl our units in the genome to simple repeated sequence.The high mutagenicity of microsatellite DNA, codominance are expressed and the ubiquity in the eukaryotic gene group, make it become s-generation molecule marker, analysis of genetic diversity (1, Mc Connel S, 1995, Aquaculture, 137:19-30), genetic linkage maps makes up and molecular marking supplementary breeding (2, Cnaani A., 2003, Aquaculture is widely used in 223:117-128).
Scylla paramamosain (Scylla paramamosain) is under the jurisdiction of Arthropoda (Arthropoda), Crustachia (Crustacea), Decapoda (Decapoda), Portumidae (Portunidae), mud crab genus (Scylla).Coastally on the south China the Changjiang river all produce, in the majority with Fujian, Guangdong, Guangxi, Hainan, be one of important marine economy crab class of China's southeastern coast.China's blue crab cultivation industry still is in initial stage at present, and most seeds are from natural water area.Because aquaculture is huge to the demand of seed, causes the seed in nature marine site also to be faced with the huge pressure of fishing for.This nonstandard industry pattern makes wild population face the risk that germplasm is degenerated and genetic diversity is lost.For guaranteeing the healthy and rapid development of mud crab industry, China has now carried out the genetic improvement work of Scylla paramamosain, but the Scylla paramamosain microsatellite DNA mark of report is very rare at present.Therefore it is significant to the genetic construction and the reinforcement molecular marking supplementary breeding of research Scylla paramamosain germ plasm resource to develop the Scylla paramamosain microsatellite marker.
Summary of the invention
The object of the present invention is to provide Scylla paramamosain microsatellite DNA mark and screening method thereof.
Described Scylla paramamosain microsatellite DNA mark can be numbered A508, and its nucleotides sequence is classified as:
tcctgagtaa?cagcatacac?cctacctaac?agtgccagca?tgaccattag?agataagagt 60
cagaataccg?ataaaagctg?aaggccatgg?atccgccaaa?aataactatg?atgacgccag 120
cagccctgag?actgacgaca?actacacttc?gattttcgtc?gctaaaataa?caatgtggac 180
agaataaggt?gcgtgcgtga?tacggctggc?gtgctaagcc?tgtgttacta?taaccactac 240
caagccacgc?accgcagcct?tccttcctta?gccgccagat?acttcacagc?cgacgctaga 300
cacataccac?ggccaccacc?accaccacaa?ccactacatc?caccacacta?ccacgcacca 360
ctgctcccat?cagctgcaca?caatcgctcc?aggaattctc?tctctgcttc?acccctcacc 420
cttgtgctgt?gacggtgcct?ccagcttcca?cacacacaca?cagtcactca?cacacagaaa 480
cacacatagc?cggaggtctt?ccaacaggag?gcgaacaaaa?cgcaagccaa?caaaaataac 540
gctaagtcct?cagtggtacc?tatgttttta?gccggccgat?gctattatgt?gtttgagtcc 600
ctttat 606。
The screening method of described Scylla paramamosain microsatellite DNA mark may further comprise the steps:
1) makes up the Scylla paramamosain enriched microsatellite library, select mono-clonal and check order;
2) use the microsatellite DNA site to search the positive colony that software obtains to contain the Scylla paramamosain microsatellite sequence, determine the Scylla paramamosain microsatellite DNA mark.
The invention provides primer sequence and the amplification method of Scylla paramamosain microsatellite DNA mark A508 and amplification Scylla paramamosain microsatellite marker A508.The A508 microsatellite marker can be used for the analysis of Scylla paramamosain genetic construction, plasm resource protection and molecular marking supplementary breeding, and its repeatability is good, is a kind of reliable and effective molecule marker.The Scylla paramamosain microsatellite DNA mark that the present invention obtains is that genetic construction analysis, plasm resource protection and the molecular marking supplementary breeding etc. of Scylla paramamosain lay the foundation.
Description of drawings
Fig. 1 is the STR somatotype figure with the individual DNA of 20 Scylla paramamosains of primer amplified in Scylla paramamosain microsatellite DNA mark A508 site.In Fig. 1,1～20 is 20 Scylla paramamosain individualities.
Embodiment
The structure of embodiment 1 Scylla paramamosain enriched microsatellite library
1) adopts ordinary method to extract 10 Scylla paramamosain chelicera muscle tissue genomic dnas and balanced mix structure Scylla paramamosain genomic dna pond, utilize restriction enzyme Mse I that enzyme is carried out in the genomic dna pond and cut.Enzyme is cut product electrophoresis on 1.5% sepharose, cuts the dna fragmentation of 500～1000bp under the UV-light and reclaims the enzyme that test kit (available from Qiagen company) purifying reclaims with gel and cut product.
2) with the joint of above-mentioned recovery fragment and 5 ' end phosphorylation, connect with the T4DNA ligase enzyme, sequence first chain of described joint is 5 '-GACGATGAGTCCTGAG-3 ', and second chain is 5 '-TACTCAGGACTCAT-3 '.
3) be template with above-mentioned connection product, with the long-chain in the joint (sequence is 5 '-GACGATGAGTCCTGAG-3 ') is primer, utilize PCR method to increase, and use PCR product cleaning agents box (available from Omega company) that amplified production is concentrated into about 120ng/ μ L.
4) biotin labeled oligonucleotide probe (CT) 15 and (CA) 15 with the hybridization of PCR product.Concrete operations are: get the PCR product 5 μ L (about 600ng) after the above-mentioned cleaning, add 495 μ L sterilization ultrapure water, 10min unwinds in 95 ℃ of water-baths, change mixture of ice and water cooling 3min rapidly over to, in ice bath, add 2 μ L (CA) 15 and (CT) the plain label probe (about 200pmol) of 15 mixed biologics, and add 13 μ L, 20 * SSC damping fluid.Placement one is added with the beaker of deionized water in 55 ℃ of water-baths, will hybridize mixed system and put into beaker, hatches 5min in 55 ℃.Beaker is taken out together with the hybridization mixed system, slowly cool off in room temperature.
5) get a pipe 600 μ L Streptomycin sulphate avidin magnetic bead (Streptavidin MagneSphere Paramagnetic Particles, Promega company), be resuspended in 100 μ L, 0.5 * SSC damping fluid after the balance, and with above-mentioned hybridization complex thorough mixing, under room temperature, place 10min.
6) adsorb magnetic bead with magnet, abandon supernatant.Magnetic bead at room temperature washs 3 times with 300 μ L, 0.1 * SSC damping fluid, each 5min.
7) adsorb magnetic bead with magnet, abandon supernatant.Magnetic bead at room temperature expresses 2 times with 100 μ L sterilized waters.
8) add 100 μ L sterilized waters at last, be incubated 10min down, the careful supernatant of drawing under the magnet effect at 94 ℃.Add the dehydrated alcohol precipitation of 0.3M sodium-acetate (pH5.2) and 2 times of volumes in supernatant liquor, 70% washing with alcohol 1 time is dissolved in 30 μ L sterilized waters.And be that primer increases to it with the long-chain in the joint.
9) will be connected to pMD 18-T carrier (TaKaRa company) behind the amplified production purifying, will connect product then and be transformed into intestinal bacteria Top10 competent cell, at the dull and stereotyped enterprising row filter of Amp+LB.
1) the mono-clonal bacterium colony on the picking Amp+LB flat board is cultivated at Amp+LB liquid nutrient medium test tube.
2) being forward and reverse primer with the long-chain in the above-mentioned joint (GACGATGAGTCCTGAG), is that template is carried out PCR with bacterium liquid.PCR system 20 μ L, 55 ℃ of annealing temperatures are extended time 30S, 30 circulations.
3) bacterium liquid PCR product is carried out agarose electrophoresis and detect, the amplified production band be the clone of successful connection 500～1000bp's, the amplified production band about 100bp oneself connects for carrier.
4) bacterium liquid PCR is identified the clone with fragment insertion who obtains checks order.Use the microsatellite DNA site to search software SSRhunter sequencing result is carried out little satellite screening, obtain positive colony and sequence information.
5) use 5 pairs of positive sequences of Primer to carry out design of primers, and synthetic general primer, be that template is carried out the PCR detection with 10 mud crab genomes that make up the library, filter out the A508 microsatellite locus that can stablize amplification.
Embodiment 3 utilizes the Scylla paramamosain microsatellite DNA mark to carry out the population genetics analysis
The fluorescent dye primer of synthetic A508 microsatellite marker is used for the analysis of Scylla paramamosain population genetics.The concrete operations step is as follows:
1) use test kit (available from sky root company) to extract the genomic dna of 20 Scylla paramamosain individualities of a wild population.
2) with the primer (referring to table 1) in A508 site this 20 DNA of individual that increase.
Table 1 A508 site primer property list
Annotate: F represents forward primer, and R represents reverse primer.
3) each PCR product is used mark and GENEMAPPER version 3.2 softwares in the ROX-500 molecular weight carry out the mensuration of fragment length at ABIPRISM 3730 sequenators.
4) interpretation of result: carry out the calculating of allelotrope number, effective number of allele order, observation heterozygosity, expection heterozygosity, Hardy-Weinberg balance check (p value) by PopGene32 software.
(short tandem repeat, STR) the somatotype result shows: the sequence length of A508 microsatellite locus of the present invention polymorphism (referring to Fig. 1) occurs to STR in 20 individualities that detect.A508 microsatellite marker of the present invention is applicable to the genetic construction analysis of Scylla paramamosain germ plasm resource.
The invention provides the dna sequence dna of Scylla paramamosain A508 microsatellite locus and the primer sequence and the amplification method of amplification Scylla paramamosain A508 microsatellite locus.The A508 microsatellite locus is used for the analysis of Scylla paramamosain genetic construction, plasm resource protection and molecular marking supplementary breeding, and good reproducibility is a kind of reliably molecule marker effectively.
Claims (3)
1. the Scylla paramamosain microsatellite DNA mark is characterized in that described Scylla paramamosain microsatellite DNA mark is numbered A508, and nucleotides sequence is classified as:
tcctgagtaa?cagcatacac?cctacctaac?agtgccagca?tgaccattag?agataagagt 60
cagaataccg?ataaaagctg?aaggccatgg?atccgccaaa?aataactatg?atgacgccag 120
cagccctgag?actgacgaca?actacacttc?gattttcgtc?gctaaaataa?caatgtggac 180
agaataaggt?gcgtgcgtga?tacggctggc?gtgctaagcc?tgtgttacta?taaccactac 240
caagccacgc?accgcagcct?tccttcctta?gccgccagat?acttcacagc?cgacgctaga 300
cacataccac?ggccaccacc?accaccacaa?ccactacatc?caccacacta?ccacgcacca 360
ctgctcccat?cagctgcaca?caatcgctcc?aggaattctc?tctctgcttc?acccctcacc 420
cttgtgctgt?gacggtgcct?ccagcttcca?cacacacaca?cagtcactca?cacacagaaa 480
cacacatagc?cggaggtctt?ccaacaggag?gcgaacaaaa?cgcaagccaa?caaaaataac 540
gctaagtcct?cagtggtacc?tatgttttta?gccggccgat?gctattatgt?gtttgagtcc 600
ctttat 606。
2. the screening method of Scylla paramamosain microsatellite DNA mark as claimed in claim 1 is characterized in that it may further comprise the steps:
1) makes up the Scylla paramamosain enriched microsatellite library, select mono-clonal and check order;
2) use the microsatellite DNA site to search the positive colony that software obtains to contain the Scylla paramamosain microsatellite sequence, determine the Scylla paramamosain microsatellite DNA mark.
3. the application of Scylla paramamosain microsatellite DNA mark as claimed in claim 1 in genetic construction analysis, plasm resource protection and the molecular marking supplementary breeding of Scylla paramamosain.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586239A (en) * | 2012-02-23 | 2012-07-18 | 山东省农业科学院蔬菜研究所 | Co-segregation SSR (Simple Sequence Repeat) codominant molecular marker of onion male sterility gene Ms site and application thereof |
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2011
- 2011-03-21 CN CN 201110068886 patent/CN102181535A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 《2009年中国水产学会学术年会论文摘要集》 20091231 马洪雨等 拟穴青蟹（Scylla paramamosain）部分基因组富集文库构建及多态微卫星标记制备 19 3 , * |
| 《Genbank》 20100404 XU,X.J, et al GU059599 1,3 , * |
| 《水产学报》 20090131 路心平等 利用线粒体DNA标记分析中国东南沿海拟穴青蟹种群遗传结构 15-23 1－3 第33卷, 第1期 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586239A (en) * | 2012-02-23 | 2012-07-18 | 山东省农业科学院蔬菜研究所 | Co-segregation SSR (Simple Sequence Repeat) codominant molecular marker of onion male sterility gene Ms site and application thereof |
| CN102586239B (en) * | 2012-02-23 | 2013-09-04 | 山东省农业科学院蔬菜研究所 | Co-segregation SSR (Simple Sequence Repeat) codominant molecular marker of onion male sterility gene Ms site and application thereof |
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Application publication date: 20110914 |