CN103627807A - Sacalia bealei microsatellite marked specific primer and detection method - Google Patents
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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 endemic species, is mainly distributed in the ground such as Guangdong, Guangxi, Fujian, Anhui, Jiangxi, Guizhou, Hong Kong, lives in streams, mountain area.Edible, medicinal and ornamental value that Sacalis bealei has 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, for science protection Sacalis bealei germ plasm resource; realize the sustainability utilization 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 protection of the science 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), extract Sacalis bealei genomic dna;
(2), the genomic dna that extracts of the step (1) of take is template, utilizes the Auele Specific Primer of each microsatellite locus in the Auele Specific Primer of described Sacalis bealei microsatellite marker to carry out respectively pcr amplification;
(3), utilize native polyacrylamide gel electrophoresis to carry out polymorphic detection to pcr amplification product.
The present invention is by building the micro-satellite of Sacalis bealei (GT) 12 enriched libraries, the positive colony that screening contains microsatellite sequence, the Auele Specific Primer of design microsatellite locus, and these microsatellite locus are carried out to polymorphic detection, developed 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.
Utilize the Auele Specific Primer of the Sacalis bealei microsatellite marker in the present invention to detect 33 Sacalis bealei samples, result shows, in 8 microsatellite locus Gai colonies, can effectively increase, amplification efficiency reaches 100%, utilizing native polyacrylamide gel electrophoresis to detect shows, 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 the level of genetic diversity assessment of Sacalis bealei, Genetic Constitution of Population analysis, sibship evaluation.
The invention discloses Auele Specific Primer and the detection method of one group of Sacalis bealei colony microsatellite marker that can efficiently increase, 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 the fields such as level of genetic diversity assessment at Sacalis bealei, Genetic Constitution of Population analysis, sibship evaluation.
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 every pair of microsatellite locus primer amplified Sacalis bealei genomic dna.
Embodiment
Following examples are to further illustrate of the present invention, rather than limitation of the present invention.Unreceipted specific experiment condition and method in the following example, the technique means adopting is generally conventional means well-known to those skilled in the art.
Embodiment 1:
One, the extraction of Sacalis bealei genomic dna
1, reagent preparation:
Digestion damping fluid: take Tris0.6057g, EDTA18.612g, SDS2.5g, with sterilizing ultrapure water, being settled to 500mL(final concentration is Tris10mmol/L, EDTA0.1mol/L, SDS0.5%), regulating pH value to 8.0, room temperature preservation is standby.
2, DNA extracting:
33 Sacalis bealei tail muscles of clip are organized in 2mL centrifuge tube respectively, and tissue is shredded, and add 800 μ L digestion damping fluids, and 10 μ L Proteinase Ks (20mg/mL), digest 8～10h in 55 ℃ after mixing; After digestion clearly thoroughly, add the saturated phenol of 800 μ L, put upside down gently and mix 20min, the centrifugal 10min of 10000g, extracting supernatant; Add the saturated phenol of 400 μ L, 400 μ L chloroform/primary isoamyl alcohol (24:1), put upside down gently and mix 20min, the centrifugal 10min of 10000g, extracting supernatant again; Add again 800 μ L chloroform/primary isoamyl alcohol (24:1), put upside down gently and mix 20min, the centrifugal 10min of 10000g, extracting supernatant; The dehydrated alcohol that adds again 1mL precooling, in-20 ℃ of freezing 2h, the centrifugal 15min of 12000g, abandons supernatant; 70% ethanol that adds again precooling, carefully washs the centrifugal 5min of DNA precipitation twice, 12000g, abandons supernatant; Dry ethanol, add 50 μ L ultrapure water dissolution precipitations, be placed in-20 ℃ 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 ℃ and cut, the laggard row agarose gel electrophoresis of 2h, detects enzyme and cuts effect, reclaims the DNA fragmentation of 400～1000bp.
2, joint is prepared and is connected
By joint primer SAU-LA(5 '-GCGGTACCCGGGAAGCTTGG-3 ') and SAU-LB(5 '-PO
4-GATCCCAAGCTTCCCGGGTACCGC-3 ') be dissolved as concentration 100 μ M, respectively get 10 μ L, after mixing, in 95 ℃ of heating 3min, naturally cool to room temperature, obtain double-stranded joint.The DNA fragmentation reclaiming in step 1 is mixed with double-stranded joint, and use T4 ligase enzyme (purchased from Dalian TaKaRa company, article No.: D2011A), in 16 ℃ of connections of spending the night, obtain connecting product.The connection product of take is template, and SAU-LA is that primer carries out pcr amplification, and use PCR product purification test kit (purchased from Sangon Biotech (Shanghai) Co., Ltd., article No.: SK8141) carry out purifying.
3, probe hybridization and enrichment with magnetic bead
First use biotin labeled (GT)
12the PCR product of the purifying that probe and step 2 obtain is hybridized, and 95 ℃ of water-bath 15min take out immediately, ice bath 3min, and 65 ℃ of water-bath 2h, obtain hybridizing product.Then hybridization product is added magnetic bead suspension (purchased from Promega company, article No.: Z5481), mix gently under room temperature, shake slowly 30min, suck solution on magnetic frame.With 2 * SSC solution, (sodium-chlor of 0.3mol/L, the Trisodium Citrate of 0.03mol/L 1%SDS), after the DNA fragmentation of washing non-specific adsorption, add ultrapure water in 95 ℃ of wash-out 10min, draw rapidly supernatant liquor on magnetic frame.
4, clone's preparation and screening
The supernatant liquor that the step 3 of take obtains is template, and SAU-LA is that primer carries out pcr amplification.Amplified production reclaims with PCR product purification test kit, 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 to the LBAmp containing X-gal and IPTG
+on flat board, be inverted in incubated overnight in 37 ℃ of incubators.
Picking white bacterial plaque, is inoculated into 500 μ L containing Amp
+lB liquid nutrient medium in, in 37 ℃ of shaking table incubated overnight.Then take bacterium liquid as template, utilize PMD18-T carrier primer M13-47 and RV-M to be respectively upstream and downstream primer and carry out pcr amplification, detect positive colony.
Three, microsatellite sequence mensuration, analysis and Auele Specific Primer design
With PMD18-T carrier primer M13-47, as sequencing primer, the positive colony by clip size between 300～800bp checks order.Institute's calling 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 that multiplicity is <100 time is as alternative site, with Primer Primere5.0 software design micro-satellite primers.Primer annealing temperature is controlled at more than 50 ℃, and PCR product length is controlled between 150～500bp.
8 microsatellite locus tSb-SSR3, tSb-SSR12, tSb-SSR15, tSb-SSR19, tSb-SSR30, tSb-SSR33, tSb-SSR36, the primer sequence of tSb-SSR48, core 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
Utilize microsatellite locus Auele Specific Primer of the present invention 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 product
1, reagent preparation:
10 * TBE: take 54.495gTris, 27.81g boric acid, 14.615gEDTA, pH is adjusted to 8.0～8.2, is settled to 500ml(final concentration 0.9mol/LTris, 0.9mol/L boric acid, 0.1mol/LEDTA), and autoclaving, room temperature preservation is standby;
30% collagen solution: take acrylamide 29g, N, N ' methylene-bisacrylamide 1g, adds ultrapure water and dissolves, and is settled to 100mL, is placed in brown bottle and saves backup;
10%APS: take ammonium persulphate 0.1g, add 1mL ultrapure water and dissolve ,-20 ℃ of preservations, need to used fresh preparation within a week;
Staining fluid: the Silver Nitrate that takes 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 use.
2, glue:
According to the formulated of table 4 10% non-denaturing polyacrylamide gel.
The non-denaturing polyacrylamide gel formula of table 4.10%
3, electrophoresis
Gel solidifies rear loading, electrophoresis.Voltage 10～15V/cm, constant voltage electrophoresis 1～2h.
4, dyeing
Distilled water rinsing 30s for gel, then add staining fluid, on shaking table, shake slowly 10～15min, remove staining fluid, distilled water flushing 1 time, with a small amount of nitrite ion, rinse 1 time again, then rejoin nitrite ion, it is clear on shaking table, to shake 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 genetics parameter
With POPGENE32 computed in software number of alleles, observation heterozygosity and expectation heterozygosity; With genepop software detection site, whether meet Hardy-Weinberg equilibrium, calculate P value; Polymorphism information content with each microsatellite locus of Cervus3.0 computed in software.The present invention obtains 8 microsatellite markers with aforesaid method, the genetics parameter 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 genetics parameter of table 5. microsatellite locus in colony
Claims (5)
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), extract Sacalis bealei genomic dna;
(2), the genomic dna that extracts of the step (1) of take is template, utilizes the Auele Specific Primer of each microsatellite locus in the Auele Specific Primer of described Sacalis bealei microsatellite marker to carry out respectively pcr amplification;
(3), utilize native polyacrylamide gel electrophoresis to carry out polymorphic detection to pcr amplification product.
3. the detection method of Sacalis bealei microsatellite marker according to claim 2, is characterized in that, described pcr amplification system is: 10 * Buffer2.5 μ L, 25mmol/L Mg
2+1.5 μ L, 10mmol/L dNTPs0.5 μ L, 10 μ mol/L Forward Primer0.5 μ L, 10 μ mol/LReverse Primer0.5 μ L, 5U/ μ L Taq Enzyme0.1 μ L, 50ng/ μ LDNA0.5 μ L, ddH
2o18.9 μ L, amounts to 25 μ L.
4. the detection method of Sacalis bealei microsatellite marker according to claim 2, is characterized in that, described pcr amplification program is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, annealing 30s, 72 ℃ are extended 30s, 30 circulations; 72 ℃ are extended 5min; 16 ℃ of preservations; The annealing temperature of the Auele Specific Primer of Sacalis bealei microsatellite marker is respectively: microsatellite locus tSb-SSR3:55 ℃, microsatellite locus tSb-SSR12:55 ℃, microsatellite locus tSb-SSR15:57 ℃, microsatellite locus tSb-SSR19:57 ℃, microsatellite locus tSb-SSR30:53 ℃, microsatellite locus tSb-SSR33:56 ℃, microsatellite locus tSb-SSR36:53 ℃, microsatellite locus tSb-SSR48:57 ℃.
5. the detection method of Sacalis bealei microsatellite marker according to claim 2, is characterized in that, described native polyacrylamide gel electrophoresis is used 10% gel strength.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106967805A (en) * | 2017-04-01 | 2017-07-21 | 安徽师范大学 | The tortoise microsatellite DNA mark screened based on high-flux sequence |
| CN112011601A (en) * | 2020-08-19 | 2020-12-01 | 广东省生物资源应用研究所 | Identification primer, kit and identification method for sex-related microsatellite markers of scarab beetles |
| 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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| 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 |
| 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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| 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 |
| 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 |
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Cited By (4)
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| CN106967805A (en) * | 2017-04-01 | 2017-07-21 | 安徽师范大学 | The tortoise microsatellite DNA mark screened based on high-flux sequence |
| CN106967805B (en) * | 2017-04-01 | 2021-03-19 | 安徽师范大学 | Tortoise microsatellite DNA marker based on high-throughput sequencing screening |
| CN112011601A (en) * | 2020-08-19 | 2020-12-01 | 广东省生物资源应用研究所 | Identification primer, kit and identification method for sex-related microsatellite markers of scarab beetles |
| 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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| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150520 Termination date: 20211206 |