CN103627807B - Sacalia bealei microsatellite marked specific primer and detection method - Google Patents
Sacalia bealei microsatellite marked specific primer and detection method Download PDFInfo
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Abstract
The invention discloses a Sacalia bealei microsatellite marked specific primer and a detection method. The detection method comprises the following steps: performing polymerase chain reaction (PCR) amplification on microsatellite site sequence by taking Sacalia bealei genome DNA as a template and using the microsatellite marked specific primer; performing non-denaturing polyacrylamide gel electrophoresis detection on the PCR amplification products; and performing polymorphic analysis on microsatellite sites according to the detection result. The Sacalia bealei microsatellite marked specific primer and the detection method thereof, provided by the invention, can be applied in the fields of genetic diversity level evaluation, population genetic structure analysis and relationship identification of Sacalia bealei.
Description
Technical field
The invention belongs to molecular marking technique field, be specifically related to a kind of Auele Specific Primer and detection method of Sacalis bealei microsatellite marker.
Background technology
Sacalis bealei (Sacalia bealei) is under the jurisdiction of Chelonia eye spot Chinemys, is Chinese unique wheat, is mainly distributed in the ground such as Guangdong, Guangxi, Fujian, Anhui, Jiangxi, Guizhou, Hong Kong, lives in streams, mountain area.Sacalis bealei has edible, medicinal and ornamental value, is a kind of important economic terrapin.In recent years; owing to excessively catching, trade, habitat destroy, wild Sacalis bealei resource reduces day by day, in order to scientific conservation Sacalis bealei germ plasm resource; realize the Substantial evaluation of resource, analysis of genetic diversity and the artificial breeding of carrying out this kind are significant.
Molecule marker is the important tool of carrying out species genetic analysis and marker assisted selection, especially microsatellite molecular marker, it is distributed widely in eukaryotic gene group, there is polymorphism high, specificity is good, codominant inheritance, the advantage such as easy to detect, has become current most widely used molecule marker.Exploitation Sacalis bealei microsatellite marker is for the analysis of its pedigree geographic patterns, group structure and genetic diversity, and the scientific conservation of germ plasm resource all has important using value with utilization.At present, there is not yet the relevant report of Sacalis bealei microsatellite marker exploitation.
Summary of the invention
First object of the present invention is to provide a kind of Auele Specific Primer of Sacalis bealei microsatellite marker, it is characterized in that, the Auele Specific Primer of described microsatellite marker is as follows:
Microsatellite locus tSb-SSR3 Auele Specific Primer: F:ACGCCTTCCCAGAGTGTC
R:ATGCCTATTTCCCCAGTG
Microsatellite locus tSb-SSR12 Auele Specific Primer: F:TCTGGAGTCCATAGCTGC
R:CTCACATTGACGAAAGGC
Microsatellite locus tSb-SSR15 Auele Specific Primer: F:AGAACCCAGGATTTCGGAG
R:AGGAGCAGTTAGCAGGCGT
Microsatellite locus tSb-SSR19 Auele Specific Primer: F:AACACTTGCTTCCCTACCG
R:GCCCCTTCTTACTGACCG
Microsatellite locus tSb-SSR30 Auele Specific Primer: F:TTGAGGGTTTAATGCCTTGT
R:GAATGGATTGGACCACAGG
Microsatellite locus tSb-SSR33 Auele Specific Primer: F:TGGGAGTTTTGTCATTGTCTTC
R:TGCCGTTCTTATGTTATGTTCG
Microsatellite locus tSb-SSR36 Auele Specific Primer: F:TTTGACAGTATGGTGGGTTT
R:GAAAGGAGTCAGATAGGTGGA
Microsatellite locus tSb-SSR48 Auele Specific Primer: F:AGGTCATTGGGCGGTTTG
R:CCACTGTTGCTCATGTTGGTT
Second object of the present invention is to provide a kind of detection method of Sacalis bealei microsatellite marker, it is characterized in that, comprises the following steps:
(1), Sacalis bealei genomic dna is extracted;
(2) genomic dna, with step (1) extracted is for template, and the Auele Specific Primer of each microsatellite locus in the Auele Specific Primer of the Sacalis bealei microsatellite marker described in utilization carries out pcr amplification respectively;
(3) native polyacrylamide gel electrophoresis, is utilized to carry out polymorphic detection to pcr amplification product.
The present invention is by building the micro-satellite of Sacalis bealei (GT) 12 enriched library, the positive colony of screening containing microsatellite sequence, the Auele Specific Primer of design microsatellite locus, and polymorphic detection is carried out to these microsatellite locus, develop the Sacalis bealei microsatellite locus of 8 rich polymorphism.The numbering of 8 Sacalis bealei microsatellite locus is respectively: tSb-SSR3, tSb-SSR12, tSb-SSR15, tSb-SSR19, tSb-SSR30, tSb-SSR33, tSb-SSR36, tSb-SSR48; Its nucleotide sequence is respectively as shown in SEQIDNO.1 ~ 8.
The Auele Specific Primer of the Sacalis bealei microsatellite marker in the present invention is utilized to detect 33 Sacalis bealei samples, result shows, 8 microsatellite locus can effectively increase in this colony, amplification efficiency reaches 100%, utilize native polyacrylamide gel electrophoresis to detect to show, all there is abundant polymorphism in 8 microsatellite locus, and all meet Hardy-Weinberg equilibrium in colony.Illustrate that the Auele Specific Primer of 8 pairs of microsatellite markers in the present invention can be used in the work such as level of genetic diversity assessment, Genetic Constitution of Population analysis, Relationship iden-tification of Sacalis bealei.
The invention discloses one group can the Auele Specific Primer of efficient amplification Sacalis bealei colony microsatellite marker and detection method, and microsatellite locus rich polymorphism of the present invention, therefore, the Auele Specific Primer of Sacalis bealei microsatellite marker provided by the invention and detection method can be applicable to assess in the level of genetic diversity of Sacalis bealei, Genetic Constitution of Population analysis, the field such as Relationship iden-tification.
Accompanying drawing explanation
Fig. 1 is the native polyacrylamide gel electrophoresis figure of 8 pairs of microsatellite locus primer amplified Sacalis bealei genomic dnas.Wherein swimming lane M is marker, and swimming lane 1 ~ 33 is the native polyacrylamide gel electrophoresis band of often pair of microsatellite locus primer amplified Sacalis bealei genomic dna.
Embodiment
Following examples further illustrate of the present invention, instead of limitation of the present invention.Unreceipted specific experiment condition and method in the following example, the technique means adopted is generally conventional means well-known to those skilled in the art.
Embodiment 1:
One, the extraction of Sacalis bealei genomic dna
1, preparation of reagents:
Digestion damping fluid: take Tris0.6057g, EDTA18.612g, SDS2.5g, being settled to 500mL(final concentration with sterilizing ultrapure water is Tris10mmol/L, EDTA0.1mol/L, SDS0.5%), adjust ph to 8.0, room temperature preservation is for subsequent use.
2, DNA extracting:
Clip 33 Sacalis bealei tail muscles are organized in 2mL centrifuge tube respectively, are shredded by tissue, add 800 μ L and digest damping fluid, 10 μ L Proteinase Ks (20mg/mL), in 55 DEG C of digestion 8 ~ 10h after mixing; After digestion clearly thoroughly, add the saturated phenol of 800 μ L, put upside down the centrifugal 10min of mixing 20min, 10000g gently, extracting supernatant; Add the saturated phenol of 400 μ L again, 400 μ L chloroform/primary isoamyl alcohol (24:1), put upside down the centrifugal 10min of mixing 20min, 10000g gently, extracting supernatant; Add 800 μ L chloroform/primary isoamyl alcohol (24:1) again, put upside down the centrifugal 10min of mixing 20min, 10000g gently, extracting supernatant; Add the dehydrated alcohol of 1mL precooling again, in-20 DEG C of centrifugal 15min of freezing 2h, 12000g, abandon supernatant; Add 70% ethanol of precooling again, careful washing DNA precipitates the centrifugal 5min of twice, 12000g, abandons supernatant; Dry ethanol, add 50 μ L ultrapure water dissolution precipitations, be placed in-20 DEG C and save backup.
Two, the structure of Sacalis bealei enriched microsatellite library
1, genome enzyme is cut and fragment recovery
Get the genomic dna 300ng of a Sacalis bealei sample, use restriction endonuclease MboI to carry out enzyme in 37 DEG C and cut, the laggard row agarose gel electrophoresis of 2h, detect enzyme and cut effect, reclaim the DNA fragmentation of 400 ~ 1000bp.
2, joint is prepared and is connected
By adapter-primer SAU-LA(5 '-GCGGTACCCGGGAAGCTTGG-3 ') and SAU-LB(5 '-PO
4-GATCCCAAGCTTCCCGGGTACCGC-3 ') be dissolved as concentration 100 μMs, respectively get 10 μ L, in 95 DEG C of heating 3min after mixing, naturally cool to room temperature, obtain double-stranded adapters.The DNA fragmentation reclaimed in step 1 is mixed with double-stranded adapters, with T4 ligase enzyme (purchased from Dalian TaKaRa company, article No.: D2011A) in 16 DEG C of connections of spending the night, obtains connecting product.To connect product for template, SAU-LA is that primer carries out pcr amplification, uses PCR primer purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd., article No.: SK8141) to carry out purifying.
3, probe hybridization and enrichment with magnetic bead
First with biotin labeled (GT)
12the PCR primer of the purifying that probe and step 2 obtain is hybridized, and 95 DEG C of water-bath 15min, take out immediately, ice bath 3min, and 65 DEG C of water-bath 2h, obtain hybrid product.Then hybrid product is added suspension containing magnetic beads (purchased from Promega company, article No.: Z5481), room temperature with gentle mixes, and shakes 30min slowly, on magnetic frame, sucks solution.After washing the DNA fragmentation of non-specific adsorption with 2 × SSC solution (sodium-chlor of 0.3mol/L, the Trisodium Citrate of 0.03mol/L, 1%SDS), add ultrapure water in 95 DEG C of wash-out 10min, rapid Aspirate supernatant on magnetic frame.
4, clone's preparation and screening
The supernatant liquor obtained with step 3 is template, and SAU-LA is that primer carries out pcr amplification.Amplified production PCR primer purification kit reclaims, and is connected to PMD18-T carrier (purchased from Dalian TaKaRa company, article No. D109A), is converted into DH5 α competent cell (purchased from Dalian TaKaRa company, article No. D109A).Converted product is coated the LBAmp containing X-gal and IPTG
+on flat board, be inverted in incubated overnight in 37 DEG C of incubators.
Picking white bacterial plaque, is inoculated into 500 μ L containing Amp
+lB liquid nutrient medium in, cultivate in 37 DEG C of incubator overnight.Then with bacterium liquid for template, utilize PMD18-T vector primer M13-47 and RV-M to be respectively upstream and downstream primer and carry out pcr amplification, detect positive colony.
Three, microsatellite sequence measures, analyzes and specific primer design
With PMD18-T vector primer M13-47 as sequencing primer, the positive colony of clip size between 300 ~ 800bp is checked order.Gained sequence is found micro-satellite with TRF software and is repeated after check and correction, choose core and repeat the equal >50bp of both sides sequence, and the microsatellite sequence of multiplicity <100 time is as alternative site, with Primer Primere5.0 software design micro-satellite primers.Primer annealing temperature controls more than 50 DEG C, and PCR primer cut to lengthen is between 150 ~ 500bp.
8 microsatellite locus tSb-SSR3, primer sequence, the core of tSb-SSR12, tSb-SSR15, tSb-SSR19, tSb-SSR30, tSb-SSR33, tSb-SSR36, tSb-SSR48 repeat, annealing temperature is as shown in table 1.
A table 1.8 microsatellite locus specific primer sequence, core repeat and annealing temperature
In table, F represents upstream primer, and R represents downstream primer.
Four, micro-satellite primers increases in Sacalis bealei colony
Microsatellite locus Auele Specific Primer of the present invention is utilized to carry out pcr amplification to 33 Sacalis bealei samples.PCR reaction system is as shown in table 2:
Table 2.PCR reaction system
PCR response procedures is as shown in table 3.
Table 3.PCR response procedures
Five, native polyacrylamide gel electrophoresis detects PCR primer
1, preparation of reagents:
10 × TBE: take 54.495gTris, 27.81g boric acid, 14.615gEDTA, pH are adjusted to 8.0 ~ 8.2, are settled to 500ml(final concentration 0.9mol/LTris, 0.9mol/L boric acid, 0.1mol/LEDTA), autoclaving, room temperature preservation is for subsequent use;
30% collagen solution: take acrylamide 29g, N, N ' methylene-bisacrylamide 1g, add ultrapure water and dissolve, be settled to 100mL, be placed in brown bottle and save backup;
10%APS: take ammonium persulphate 0.1g, adds 1mL ultrapure water and dissolves ,-20 DEG C of preservations, need Fresh within use one week;
Staining fluid: the Silver Nitrate taking 0.5g is dissolved in (final concentration 0.1%) in 500mL pure water, saves backup in brown bottle;
Nitrite ion: weighing sodium hydroxide 10g, is dissolved in (final concentration is 2%) in 500mL pure water, adds 0.5mL formaldehyde before using.
2, glue:
According to formulated 10% non-denaturing polyacrylamide gel of table 4.
The non-denaturing polyacrylamide gel formula of table 4.10%
3, electrophoresis
Loading after gel sets, electrophoresis.Voltage 10 ~ 15V/cm, constant voltage electrophoresis 1 ~ 2h.
4, dye
Gel distilled water rinsing 30s, then staining fluid is added, shaking table shakes 10 ~ 15min slowly, removes staining fluid, distilled water flushing 1 time, 1 time is rinsed again with a small amount of nitrite ion, then rejoin nitrite ion, it is clear that shaking table shakes slowly to band, removes nitrite ion, with distilled water flushing 1 ~ 2 time, the native polyacrylamide gel electrophoresis of 8 pairs of microsatellite locus primer amplified Sacalis bealei genomic dnas as shown in Figure 1.
Six, the calculating of genetic parameters
With POPGENE32 computed in software number of alleles, observation heterozygosity and expectation heterozygosity; Whether meet Hardy-Weinberg equilibrium with genepop software detection site, calculate P value; With the polymorphism information content of each microsatellite locus of Cervus3.0 computed in software.The present invention's aforesaid method obtains 8 microsatellite markers, the genetic parameters of each microsatellite locus is as shown in table 5, all sites is high polymorphic locus (polymorphism information content >0.5), and all meets Hardy-Weinberg equilibrium (P>0.05).
The genetic parameters of table 5. microsatellite locus in colony
Claims (2)
1. an Auele Specific Primer for Sacalis bealei microsatellite marker, is characterized in that, the Auele Specific Primer of described microsatellite marker is as follows:
Microsatellite locus tSb-SSR3 Auele Specific Primer: F:ACGCCTTCCCAGAGTGTC
R:ATGCCTATTTCCCCAGTG
Microsatellite locus tSb-SSR12 Auele Specific Primer: F:TCTGGAGTCCATAGCTGC
R:CTCACATTGACGAAAGGC
Microsatellite locus tSb-SSR15 Auele Specific Primer: F:AGAACCCAGGATTTCGGAG
R:AGGAGCAGTTAGCAGGCGT
Microsatellite locus tSb-SSR19 Auele Specific Primer: F:AACACTTGCTTCCCTACCG
R:GCCCCTTCTTACTGACCG
Microsatellite locus tSb-SSR30 Auele Specific Primer: F:TTGAGGGTTTAATGCCTTGT
R:GAATGGATTGGACCACAGG
Microsatellite locus tSb-SSR33 Auele Specific Primer: F:TGGGAGTTTTGTCATTGTCTTC
R:TGCCGTTCTTATGTTATGTTCG
Microsatellite locus tSb-SSR36 Auele Specific Primer: F:TTTGACAGTATGGTGGGTTT
R:GAAAGGAGTCAGATAGGTGGA
Microsatellite locus tSb-SSR48 Auele Specific Primer: F:AGGTCATTGGGCGGTTTG
R:CCACTGTTGCTCATGTTGGTT。
2. a detection method for Sacalis bealei microsatellite marker, is characterized in that, comprises the following steps:
(1), Sacalis bealei genomic dna is extracted;
(2) genomic dna, with step (1) extracted, for template, utilizes the Auele Specific Primer of each microsatellite locus in the Auele Specific Primer of the Sacalis bealei microsatellite marker described in claim 1 to carry out pcr amplification respectively;
Described PCR amplification system is: 10 × Buffer 2.5 μ L, 25mmol/L Mg
2+1.5 μ L, 10mmol/L dNTPs 0.5 μ L, 10 μm of ol/L forward primer 0.5 μ L, 10 μm of ol/L reverse primer 0.5 μ L, 5U/ μ L Taq Enzyme 0.1 μ L, 50ng/ μ L DNA 0.5 μ L, ddH
2o 18.9 μ L, amounts to 25 μ L;
Described pcr amplification program is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, annealing 30s, 72 DEG C extend 30s, 30 circulations; 72 DEG C extend 5min; 16 DEG C of preservations; The annealing temperature of the Auele Specific Primer of Sacalis bealei microsatellite marker is respectively: microsatellite locus tSb-SSR3:55 DEG C, microsatellite locus tSb-SSR12:55 DEG C, microsatellite locus tSb-SSR15:57 DEG C, microsatellite locus tSb-SSR19:57 DEG C, microsatellite locus tSb-SSR30:53 DEG C, microsatellite locus tSb-SSR33:56 DEG C, microsatellite locus tSb-SSR36:53 DEG C, microsatellite locus tSb-SSR48:57 DEG C;
(3), utilize native polyacrylamide gel electrophoresis to carry out polymorphic detection to pcr amplification product, described native polyacrylamide gel electrophoresis uses the gel strength of 10%.
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| CN112391480A (en) * | 2020-11-17 | 2021-02-23 | 广东省科学院动物研究所 | Mitochondrial marker-based assessment method for genetic diversity of ocellatus mystus population |
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| CN101705295A (en) * | 2009-11-20 | 2010-05-12 | 中国水产科学研究院黄海水产研究所 | Detection method of turbot FF0901 microsatellite marker by utilizing specific primers |
| CN102154500A (en) * | 2011-03-25 | 2011-08-17 | 中国水产科学研究院黄海水产研究所 | Sextuple PCR (polymerase chain reaction) detection method of portunus trituberculatus miers microsatellite marker |
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| CN101705296A (en) * | 2009-11-20 | 2010-05-12 | 中国水产科学研究院黄海水产研究所 | Detection method of turbot FF0922 microsatellite marker by utilizing specific primers |
| CN101705295A (en) * | 2009-11-20 | 2010-05-12 | 中国水产科学研究院黄海水产研究所 | Detection method of turbot FF0901 microsatellite marker by utilizing specific primers |
| CN102154500A (en) * | 2011-03-25 | 2011-08-17 | 中国水产科学研究院黄海水产研究所 | Sextuple PCR (polymerase chain reaction) detection method of portunus trituberculatus miers microsatellite marker |
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