CN102146382A - Microsatellite DNA (Deoxyribonucleic Acid) markers of Chinese three-keeled pond turtle - Google Patents
Microsatellite DNA (Deoxyribonucleic Acid) markers of Chinese three-keeled pond turtle Download PDFInfo
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Abstract
The invention discloses a microsatellite DNA (Deoxyribonucleic Acid) markers of a Chinese three-keeled pond turtle, which are determined by comprising the steps of constructing a library rich in tortoise microsatellites (CA)n (Carbonic Anhydrase)n and (GATA)n, screening and sequencing positive clones containing microsatellite sequences to obtain 59 clones containing microsatellite repetitive sequences, and determining 10 microsatellite markers with rich polymorphisms, wherein the 10 microstaellite markers are Mclw01, Mclw02, Mclw03, Mclw04, Mclw05, Mclw06, Mclw07, Mclw08, Mclw09 and Mclw10. The invention provides 10 new microsatellite loci of the Chinese three-keeled pond turtle and primer sequences and amplification methods for amplifying the 10 microsatellite loci, can be applied to a study on genetic diversity of different geographical populations of the Chinese three-keeled pond turtles and a study on paternity identification, and the molecular markers are reliable and effective with good repeatability.
Description
Technical field
The present invention relates to a kind of molecular marking technique, be specifically related to a kind of tortoise microsatellite DNA genetic marker.
Background technology
Microsatellite DNA is called short polyphone tumor-necrosis factor glycoproteins (STR), simple repeated sequence (SSR), SSLP (SSLP) again, is meant in the genome with 1-6 Nucleotide to be the nucleotide sequence of unit series connection repeating groups one-tenth; Wherein, modal is that dinucleotide repeats, as ((AC/TG) n reaches (AG/TC) n.Different bionts demonstrates polymorphism owing to the multiplicity that repeats motif is different.Studies show that, may be " chain sliding " (strand slippage) phenomenon in the dna replication dna process cause microsatellite DNA polymorphism information capacity (polymorphic information content, PIC) higher.The high mutagenicity of microsatellite DNA, codominance are expressed and the ubiquity in the eukaryotic gene group, make the superior molecule marker of evaluation, genomic mapping and the genetic breeding of its analysis that becomes population genetic diversity, sibship, with allozyme, the RAPD method is compared that certain superiority is also arranged.Owing to little satellite have quantity many, in genome, be evenly distributed, polymorphism information enriches, be easy to detect etc., and advantage is used as good genetic marker (genetic marker) is used widely.According to the formation and the distribution of repeating unit, the microsatellite DNA sequence is divided into three types: single type (pure), compound (compound), discontinuous form (interrupted), for example:
Single type: CACACACACACACACACACA
Compound: CACACACACACAGAGAGAGA
Discontinuous form: CACATTCACACATTCATTCA
Tortoise, claim grass tortoise, fragrant tortoise, mud tortoise, smelly tortoise, cockchafer again, English name Chinese Three-keeledPond Turtle, Latin literary fame Chinemys reevesii, China is not except that Qinghai, Tibet, Ningxia, Jilin, Shanxi, Liaoning, Xinjiang, Heilungkiang, the Inner Mongol are found, all there is distribution all the other various places.Be distributed in Japan, Korea abroad.Tortoise is owing to its pharmaceutical use and nutritive value, for a long time by mass consumption.And overfishing in recent years, Habitat destruction, chemical pollution etc. have caused tortoise wild stocks quantity sharply to descend.Therefore, tortoise has been used as in endangered species listed 2004 in " Chinese animals on the brink of extinction Red Data Book " and the red register of IUCN (World Conservation Union) endangered species in 2000.As seen extremely urgent to the protection of wild stocks, but to formulate effective protection plan, just must understand the information of aspects such as wild stocks structure and genetic diversity.There has been the scholar to adopt different molecular marking technique (comprising mitochondrial cytochrome b genes sequence, plastosome whole genome sequence etc.) that the genetic diversity of tortoise is studied, but above-mentioned genetic marker can not provide enough genetic information, central authorities etc. how, the segmental preliminary study of tortoise mitochondrial cytochrome b genes; Nie Liuwang etc., tortoise plastosome whole genome sequence and structural analysis.In order further to understand the information of aspects such as tortoise wild stocks structure and genetic diversity, need the new molecular genetic marker of exploitation.
Summary of the invention
Technical problem to be solved by this invention is: separate and evaluation tortoise microsatellite DNA mark, set up the technical system of tortoise microsatellite DNA and utilize these molecule markers to carry out the classification of tortoise, the analysis of paternity identification and genetic diversity.
The technical scheme of technical solution problem of the present invention: tortoise microsatellite DNA mark, comprise the little satellite of tortoise (CA) n and (GATA) structure of n enriched library, contain the screening and the order-checking of the positive colony of microsatellite sequence, obtain 59 clones that contain little satellite tumor-necrosis factor glycoproteins, determined microsatellite marker Mclw01, Mclw02, Mclw03, Mclw04, Mclw05, Mclw06, Mclw07, Mclw08, Mclw09, the Mclw10 of 10 rich polymorphism; Mclw01 is that 518 Nucleotide, Mclw02 are that 542 Nucleotide, Mclw03 are that 511 Nucleotide, Mclw04 are that 505 Nucleotide, Mclw05 are that 384 Nucleotide, Mclw06 are that 544 Nucleotide, Mclw07 are that 322 Nucleotide, Mclw08 are that 556 Nucleotide, Mclw09 are that 255 Nucleotide, Mclw10 are 330 Nucleotide.
Description of drawings
Fig. 1: the STR somatotype figure of 21 tortoise genomic dnas of primer amplification in Mclw01 site
Fig. 2: the STR somatotype figure of 21 tortoise genomic dnas of primer amplification in Mclw02 site
Fig. 3: the STR somatotype figure of 21 tortoise genomic dnas of primer amplification in Mclw03 site
Fig. 4: the STR somatotype figure of 21 tortoise genomic dnas of primer amplification in Mclw04 site
Fig. 5: the STR somatotype figure of 21 tortoise genomic dnas of primer amplification in Mclw05 site
Fig. 6: the STR somatotype figure of 21 tortoise genomic dnas of primer amplification in Mclw06 site
Fig. 7: the STR somatotype figure of 21 tortoise genomic dnas of primer amplification in Mclw07 site
Fig. 8: the STR somatotype figure of 21 tortoise genomic dnas of primer amplification in Mclw08 site
Fig. 9: the STR somatotype figure of 21 tortoise genomic dnas of primer amplification in Mclw09 site
Figure 10: the STR somatotype figure of 21 tortoise genomic dnas of primer amplification in Mclw10 site
In Fig. 1-Figure 10,1-21 represents 1-21 tortoise respectively.
Embodiment
Be described in further detail the present invention below in conjunction with accompanying drawing.
1, the structure of tortoise microsatellite DNA enriched library:
1.1 genomic extraction and enzyme are cut: with reference to Sambrook etc. (2002, Molecular Cloning, method 3nd463-471) is extracted the tortoise genomic dna, cut genome with restriction enzyme Sau3AI (available from TaKaRa company) enzyme, enzyme is cut product electrophoresis on 1% sepharose, downcuts the enzyme of dna fragmentation blended rubber recovery test kit (giving birth to the worker available from Shanghai) the purifying recovery of 400-1000bp size under the UV-light and cuts product.
1.2 add joint:
The two ends of the enzyme of purifying recovery being cut product add joint:
SauL-F(5’-GGCCAGAGACCCCAAGCTTCG-3’)
SauL-R(5’-PO
4-GATCCGAAGCTTGGGGTCTCTGGCC-3’)，
16 ℃ of water-baths connections are spent the night under T4 ligase enzyme (available from Promega company) effect.
1.3PCR detection tabs connects:
With SauL-F is primer, is that template is carried out PCR with the connection product of 1.2 operation systems, and 50 μ L reaction systems are as follows: 5 μ L, 10 * exTaq damping fluid, 4 μ L MgCl
2, 4 μ L dNTP, 0.25 μ L exTaq enzyme (available from TaKaRa company), 2 μ L primer SauL-F, 5 μ L connect product, add distilled water (ddH
2O) mend to 50 μ L.The PCR response procedures is as follows: 95 ℃ of pre-sex change 5 minutes, and 94 ℃ of sex change 45 seconds, 60 ℃ of annealing 45 seconds, 72 ℃ were extended 1 minute, and sex change to three steps of annealing repeat 35 times, and 72 ℃ were fully extended 10 minutes.The PCR product carries out purifying with purification kit (purchasing the company in Axygen).
1.4 enrichment with magnetic bead:
With 1.3 made purified product and biotin labeled oligonucleotide probe hybridization probes (CA)
12(GATA)
6Hybridized 7 hours down at 72 ℃, 55 ℃ respectively.Hybridization solution is added the good magnetic bead of balance, and room temperature was placed 30 minutes.Centrifuge tube is used lavation buffer solution (6 * SSC, 0.1%SDS successively; 2 * SSC, 0.1%SDS; 1 * SSC 0.1%SDS) washes twice in 45 ℃, washes once with the distilled water room temperature again.Add 60 μ L distilled waters at last, 95 ℃ of sex change are 15 minutes on the PCR instrument, collect supernatant.
1.5PCR enrichment:
With the enrichment with magnetic bead product is template, and SauL-F is that primer carries out pcr amplification, and 50 μ L reaction systems are as follows:
DNA30 μ L after the enrichment, 5 μ L, 10 * exTaq damping fluid, 4 μ L MgCl
2, 4 μ L dNTP, 0.25 μ LexTaq polysaccharase, 2 μ L primer SauL-F add distilled water and mend to 50 μ L.The PCR response procedures is as follows: 95 ℃ of pre-sex change 5 minutes, and 94 ℃ of sex change 40 seconds, 60 ℃ of annealing 45 seconds, 72 ℃ were extended 1 minute, and sex change to three steps of annealing repeat 35 times, and 72 ℃ were fully extended 10 minutes.The PCR product carries out purifying with purification kit.
1.6 the preparation of competent cell:
The E.coli DH5 α nutrient solution of 1mL incubated overnight is connected in the 50mL liquid LB substratum, and 37 ℃ of shaking culture 2 hours are to logarithmic phase (OD
600Reaching 0.3-0.5) time takes out.Bacterium liquid is centrifugal 10 minutes in 4 ℃.Abandon supernatant, add the CaCl of 1mL 0.1mol/L precooling
2Again suspension cell is beaten even with the rifle head gently.4 ℃ centrifugal 10 minutes.Abandon supernatant, add the glycerine/CaCl of precooling
2The mixture thalline that suspends again, beat with the rifle head even ,-80 ℃ of preservations.
1.7 fall dull and stereotyped:
The LB solid medium is melted fully, be cooled to about 50 ℃, (ratio is: LB solid medium 100mL to be made into the LB/Amp/X-Gal/IPTG plate culture medium; Penbritin (Amp) 100 μ L; 20% isopropyl-(IPTG), 7 μ L; 2%5-bromo-4-chloro-3-indoles-β-D-galactoside (X-Gal) 40 μ L), pours into after mixing in the culture dish, treat to put into 37 ℃ of baking ovens after the culture medium solidifying, dry water vapour.
Transform 1.8 connect:
The purified product of getting after the made PCR enrichment of 1.5 operations adds pMD18-T carrier (available from TaKaRa company) after 16 ℃ of water-baths connect 2 hours, is put in 4 ℃ of refrigerator overnight, makes the connection product.
Take out the competent cell that 1.6 operations are made, recovery on ice adds above-mentioned connection product, mixing to liquid.Ice bath is put into 42 ℃ of water-baths after 30 minutes, heat shock 90 seconds was put into the ice ice bath 2 minutes immediately, added fresh LB substratum, 37 ℃ of shaking culture 1 hour.Get 100 μ L nutrient solutions and coat on the LB/Amp/X-gal/IPTG substratum, be inverted overnight incubation for 37 ℃.
2, contain the design of screening, order-checking and the micro-satellite primers of the positive colony of microsatellite sequence:
2.1 bacterium colony PCR:
White single bacterium colony on the flat board is put into the EP pipe that the LB/Amp substratum is housed in advance respectively.37 ℃ of shaking culture are to bacterium liquid muddiness.12.5 μ L PCR reaction system comprises: Taq enzyme 0.25U (available from TaKaRa company), 10 * Buffer, 1.25 μ L, dNTP 0.2 μ M, MgCl
22 μ M, primer 0.4 μ M (SauL-F, primer-(CA)
12(GATA)
6), bacterium liquid 0.5 μ L adds distilled water and supplies volume.Response procedures: 95 ℃ of pre-sex change 5 minutes, 94 ℃ of sex change 40 seconds, 60 ℃ of annealing 45 seconds, 72 ℃ were extended 1 minute, and sex change to three steps of annealing repeat 35 times, and 72 ℃ were fully extended 10 minutes, and 1.5% agarose gel electrophoresis detects.
2.2 positive colony order-checking and design of primers:
Positive colony send biotech firm's order-checking, obtains 59 sequences that contain microsatellite DNA altogether.According to the microsatellite DNA flanking sequence, utilize 59 pairs of micro-satellite primers of software primerpremier5 design.And these 59 pairs of primers are carried out pcr amplification detect, finally there are 17 pairs of primers to stablize and amplify the purpose band.Upstream primer 5 ' end to these 17 pairs of primers is used FAM respectively, HEX, and TAMRA carries out fluorescent mark.
3, the screening and the interpretation of result of tortoise polymorphic micro-satellite site primer
3.1 screening has the primer of polymorphism:
Extract 21 tortoise genomes with genomic methods of extraction such as Sambrook in 1.1 operations.17 pairs of primer amplification genomes that get with 2.2 operation designing institutes.Response procedures: 95 ℃ of pre-sex change 5 minutes, 94 ℃ of sex change 40 seconds, 60 ℃ of annealing 45 seconds, 72 ℃ were extended 1 minute, and sex change to three steps of annealing repeat 35 times, and 72 ℃ were fully extended 10 minutes, and 1.5% agarose gel electrophoresis detects.The PCR product carries out gene type (Genotyping) through ABI PRISM3730 genetic analyzer (Applied Biosystem company), uses the segmental concrete numerical value of Genemarker1.6 software (Applied Biosystem company) interpretation allelotrope.Obtain 10 microsatellite markers (seeing Table 1) altogether: Mclw01, Mclw02, Mclw03, Mclw04, Mclw05, Mclw06, Mclw07, Mclw08, Mclw09, Mclw10 with polymorphism; Mclw01 is that 518 Nucleotide, Mclw02 are that 542 Nucleotide, Mclw03 are that 511 Nucleotide, Mclw04 are that 505 Nucleotide, Mclw05 are that 384 Nucleotide, Mclw06 are that 544 Nucleotide, Mclw07 are that 322 Nucleotide, Mclw08 are that 556 Nucleotide, Mclw09 are that 255 Nucleotide, Mclw10 are 330 Nucleotide.
3.2 interpretation of result:
Use Cervus3.0 computed in software expectation heterozygosity and observe heterozygosity.Use the value of Genepop4.0 computed in software Hardy one Weinberg and linkage disequilibrium, describe the feature of 10 tortoise microsatellite DNA polymorphic sites with this.
Can find out that by accompanying drawing 1-10 the length of 10 microsatellite sequences of the present invention diversity all occurred in 21 tortoises for examination, this shows that these 10 microsatellite markers of the present invention can be used to study assortment and the genetic affinity analysis of tortoise.
The invention provides the primer sequence and the amplification method of the new microsatellite locus of 10 tortoises and these 10 microsatellite locus that increase, can be applicable to the research of different geographical population genetic Study on Diversity of tortoise and paternity identification, good reproducibility is a kind of reliable and effective molecule marker.
Table 1 primer property list: the site, repeat motif, primer sequence, annealing temperature
Sequence table
＜110〉Anhui Normal University
＜120〉tortoise microsatellite DNA mark
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<212>DNA
<213>Chinemys?reevesii
<400>1
gattagccac?tcattgtgta?ctgaaaggga?aaccctcccc?gggagagaat?cagattcatg 60
aaggctgcag?gcatgccagg?cctgtaaacg?gctggtgaaa?gggaatccta?tacccagtgg 120
gacatgttca?tggtgttcca?gtgaaaatgt?cagtgaccgt?accccttgct?cagactgtgt 180
gtgtgtgtgt?gtgtgttgat?tggggctcaa?cctctgcaca?atccaaatga?agtgttacct 240
tttatcagtt?acatggctga?tggatgggtg?aaacttaccc?cagggagaag?gggtgtactc 300
agaaaatact?gctaaacaaa?aatcctccaa?ggtaactggg?caagtgtctg?tgggcctcct 360
ccttttgttg?gagcacactc?cccatcttgc?ttccaggaga?cattcacacc?cactgagtgt 420
tcttccacag?ctgtcttgct?tacagtgtct?gtgcagttat?tgattatctg?tgtcagtaat 480
accaaagcta?taagggattg?attcttaagg?agaaggtt 518
<210>2
<211>542
<212>DNA
<213>Chinemys?reevesii
<400>2
gatccggcac?atctgccgtc?actgcccaac?cccgtgtggg?ggcaccagca?ctgtgccggg 60
tcgtgatggg?aaattgtcca?agctggccca?ggagagacgt?gtgtgtccct?gctgcccctg 120
ctggtggggc?cgagccctac?tctgtgtgtg?tgtgcatctg?tgacactgtt?tgtatcagtg 180
tgttgctggg?ctaaccacta?ggaaatggct?ttacaggatt?ttgtattctg?cagcaagtca 240
cacacacaca?cacacacaca?cacactctga?cgcacaaaga?gactcacaca?atcgcaagaa 300
cactcagaca?ccacacaccc?agagggacat?gctagcccaa?cccccaaaga?gagcaacaat 360
caccgccccc?agccacactc?acaatcaccc?ccacacacac?accataatat?tcacaatcgt 420
gctcagagat?acaactgcac?gcagttcact?cccacagctc?ccaatacaag?gacctcctgc 480
cccacacagc?atgtgccaag?gcctgcagaa?ctcactgcca?ctggatggct?accaatgaga 540
at 542
<210>3
<211>511
<212>DNA
<213>Chinemys?reevesii
<400>3
gatcagatgg?aaggccgttt?aggaatgaga?gcagattact?ggggcgaggg?ggttggtgga 60
gctgactggg?gaatgtggtg?gcacagaggt?agttgaggaa?tgtgtgtgtg?tgtgtgtgtg 120
tgagaaaaga?ggggactgag?gcgaaggagt?gtgggttgga?tgtactgatg?tgtgttgctt 180
ataacaatgg?caggtttaaa?agaatcagac?ctaggtgtga?tttttttatg?ctaatagtgt 240
ccctttaaaa?gtagtgaaat?tatgacacct?gaaaaacaat?aaccaaactg?aaaatgtgag 300
atttgatttc?tctgagtcac?cagggtggtc?aaactttgga?acacaaaaca?atgagatagg 360
cataaaccag?tgttcctttt?agcaatacta?acaatttggt?ctctgggcat?gacaaatgaa 420
atctgataaa?gagccccact?ggcagagtac?ttgaattaaa?aaaatatgag?gctatttact 480
gaactgtatt?gtttacatac?agaacattgt?t 511
<210>4
<211>505
<212>DNA
<213>Chinemys?reevesii
<400>4
atcattcctt?cccctctccc?ctgtagctgg?aagcagctca?tgtcccttcc?ttcccacaca 60
gtgccgaaag?gctgctgctc?accacgttct?ggtgtgaaac?ctggcagaaa?ttgtgtgtgt 120
gtgtgtgtgt?gtgaccctgc?atgccctccc?cccatgtgtt?gcctcaggaa?aatgtggtgc 180
caggtaccag?gagaggtggg?tccgtcttgg?gggcccaacc?aggcatggag?ggcagaaagc 240
accaagcagg?gagggttggg?tcagttggtt?acccccatgt?gtgagagata?tatatgtgtg 300
tgtgtcacct?ctccccatgt?gtgtgggggg?ggggagggaa?aggtgtgtgt?gtcacccctc 360
cccttgtgaa?ccctaaaacc?ttaaagataa?gatggtaaat?aaaaagaatc?caactatgca 420
gtatttcttt?ttaagagggg?ctcagtcaac?ttgatgttaa?tttgaacatt?tgtactgcat 480
aattctgatt?gattgctgtt?gatcc 505
<210>5
<211>384
<212>DNA
<213>Chinemys?reevesii
<400>5
gatctctgtg?cagctgggtg?tggccctgcc?tgtgtgcttg?gctggggagc?ctaactcagc 60
aaggccaggt?aaaggggacc?caggctggga?gaatagtctg?actcagtggc?gtcccagcac 120
accaggtgac?acactcaggg?tgtcccaaat?agtcactttg?gtataaaact?aggaagctga 180
gaccagtgag?ggctgcagtg?tggaagtgtg?tagttactct?caagctttag?acctgttcta 240
cattaggaga?ttaggatggt?cgatctgtgt?gtgtgtgtgt?gtgtgtgtgt?gtgtgagaga 300
gagagagaga?gaaatccaca?cccctgagca?atgcaattaa?attgacctaa?ccctcagcgc 360
agatggtgct?gtgttgacca?gatc 384
<210>6
<211>544
<212>DNA
<213>Chinemys?reevesii
<400>6
gatcatggcc?taacttcact?cctaagaatg?tgctgcccac?agaaaagaaa?aacctgcagc 60
tggcctgtgc?tcacgggact?tgggctgcag?gggctatttc?attgctgtgt?agactactag 120
actagggctt?gagcccaggc?tctaggaccc?tgcaggggag?tccactcact?tcacgggagg 180
taggtagggg?tgtgtgtgtg?tgtgtgtgtg?tgtgtgtgtg?tgtaggcagt?gcaggaggat 240
ggggaaataa?tgtgtacaca?catgcagggg?tgggcaggag?agagggaaaa?caggtctgtg 300
catacagggg?aatgtagaga?atgcaagaag?ggaaacttgg?tgggatgcac?cagggaatac 360
agagaggtgc?aagaagcagg?tggcatttga?aggacggggt?ggaggatgga?aaaaacttgg 420
gttttaagct?gctctagcaa?aacaaagcag?ttgtaaaccc?aatttaactg?gccaatatgt 480
tccggcttct?tgtgctgctt?cagagtggca?ggaagcagcc?agcgcacatt?ggtgcaactg 540
atcc 544
<210>7
<211>322
<212>DNA
<213>Chinemys?reevesii
<400>7
gatcactgcc?gcggatcgcc?ttcgaccgaa?gagccgaccg?tcaacctgaa?tcaccatttg 60
tcccccctcc?ccttctcaca?atgtgtgtgt?gtgtgtgtga?gagagagaga?gagagagaga 120
gagagagaga?gagaggggtg?tgcaaaacta?aatgcaatgg?cgtaacactg?ggtagtaggg 180
agtagcattt?gcatctcgtg?tgagcatagt?ttgcagcgtt?gcgggacttc?gctgacgagg 240
gaggagcggc?tcgagggcaa?atcgtggttt?gaaaggctac?gggacctcac?tgaagccgag 300
tgggcgttgg?tcgacatgat?cg 322
<210>8
<211>556
<212>DNA
<213>Chinemys?reevesii
<400>8
gatcattact?ccttgttctg?tcatctgcta?ctactgagaa?caagcaaagg?gatggtgctg 60
gggcctcagt?gacaagctag?gtaccagcag?tgaagagggc?agcctcccgg?taggtggtaa 120
ctgtgacaga?tattgcaatc?acatgcaata?tctttgggga?ccatattgtg?ataagtttat 180
ggatggtttg?ttatcactgt?gggccaggga?ttgtatgcag?gaggaatgga?ggaagggtta 240
ccatagctcc?tccaggaact?aaaaactgtg?tgtgtgtgtg?tgtgtgtgtg?tgggggggtg 300
atttaagatg?aatcactctt?gtttgtaaac?tcctctggag?aggtaccact?ccctggggag 360
gcttgcatat?actggttcag?actgaatttt?ccagagtaaa?ctgacacaga?aagggctttt 420
ggcataaaga?ctgagtttag?actggcacag?gatttttttt?tgattcagca?aatggatggg 480
accttctctc?caaggggagc?cgaggggagg?gggagggcac?tgttgtggaa?gggttggaaa 540
gactttggtt?tatcct 556
<210>9
<211>255
<212>DNA
<213>Chinemys?reevesii
<400>9
cagcagaagc?atcagctggg?caggctgggg?gcaaggtcca?gaggcaggcg?gacagtgccc 60
cagggagtat?ataggtatgg?agccaccacc?atcccatttc?atagaacagt?cacgtggtca 120
gggctcctgg?ggatgtcact?ttgtcatcct?gcaggaaatt?tattacatat?acacacacac 180
acacacacac?acacacacac?acaccgggaa?tggtagcaca?caggcacgct?gcctggatgc 240
cttccccgct?gatcc 255
<210>10
<211>330
<212>DNA
<213>Chinemys?reevesii
<400>10
gatcctgaga?aatcctggac?acaacccttg?tgtggtggga?gagatagaga?gatggttgtg 60
tataggaata?gatagataga?tagatagata?gatagataga?tagatagata?gggtgtgttg 120
ggctagatag?atagatagat?agaggggggt?gtatggggat?agatagatag?atagatagat 180
agatagatag?catttagaca?aacggagatc?atgaaaaaat?gaagctaatg?tctcaatgcg 240
catggtttcc?aaagtccata?ggagtttcat?accattgaag?tcatctggaa?tggtttactc 300
cagtacaagc?attaagacat?catgataaat 330
Claims (2)
1. tortoise microsatellite DNA mark is characterized in that: described microsatellite marker numbering is respectively: Mclw01, Mclw02, Mclw03, Mclw04, Mclw05, Mclw06, Mclw07, Mclw08, Mclw09, Mclw10;
Its nucleotides sequence is classified as:
Mclw01：
gattagccac?tcattgtgta?ctgaaaggga?aaccctcccc?gggagagaat?cagattcatg 60
aaggctgcag?gcatgccagg?cctgtaaacg?gctggtgaaa?gggaatccta?tacccagtgg 120
gacatgttca?tggtgttcca?gtgaaaatgt?cagtgaccgt?accccttgct?cagactgtgt 180
gtgtgtgtgt?gtgtgttgat?tggggctcaa?cctctgcaca?atccaaatga?agtgttacct 240
tttatcagtt?acatggctga?tggatgggtg?aaacttaccc?cagggagaag?gggtgtactc 300
agaaaatact?gctaaacaaa?aatcctccaa?ggtaactggg?caagtgtctg?tgggcctcct 360
ccttttgttg?gagcacactc?cccatcttgc?ttccaggaga?cattcacacc?cactgagtgt 420
tcttccacag?ctgtcttgct?tacagtgtct?gtgcagttat?tgattatctg?tgtcagtaat 480
accaaagcta?taagggattg?attcttaagg?agaaggtt 518
Mclw02：
gatccggcac?atctgccgtc?actgcccaac?cccgtgtggg?ggcaccagca?ctgtgccggg 60
tcgtgatggg?aaattgtcca?agctggccca?ggagagacgt?gtgtgtccct?gctgcccctg 120
ctggtggggc?cgagccctac?tctgtgtgtg?tgtgcatctg?tgacactgtt?tgtatcagtg 180
tgttgctggg?ctaaccacta?ggaaatggct?ttacaggatt?ttgtattctg?cagcaagtca 240
cacacacaca?cacacacaca?cacactctga?cgcacaaaga?gactcacaca?atcgcaagaa 300
cactcagaca?ccacacaccc?agagggacat?gctagcccaa?cccccaaaga?gagcaacaat 360
caccgccccc?agccacactc?acaatcaccc?ccacacacac?accataatat?tcacaatcgt 420
gctcagagat?acaactgcac?gcagttcact?cccacagctc?ccaatacaag?gacctcctgc 480
cccacacagc?atgtgccaag?gcctgcagaa?ctcactgcca?ctggatggct?accaatgaga 540
at 542
Mclw03：
gatcagatgg?aaggccgttt?aggaatgaga?gcagattact?ggggcgaggg?ggttggtgga 60
gctgactggg?gaatgtggtg?gcacagaggt?agttgaggaa?tgtgtgtgtg?tgtgtgtgtg 120
tgagaaaaga?ggggactgag?gcgaaggagt?gtgggttgga?tgtactgatg?tgtgttgctt 180
ataacaatgg?caggtttaaa?agaatcagac?ctaggtgtga?tttttttatg?ctaatagtgt 240
ccctttaaaa?gtagtgaaat?tatgacacct?gaaaaacaat?aaccaaactg?aaaatgtgag 300
atttgatttc?tctgagtcac?cagggtggtc?aaactttgga?acacaaaaca?atgagatagg 360
cataaaccag?tgttcctttt?agcaatacta?acaatttggt?ctctgggcat?gacaaatgaa 420
atctgataaa?gagccccact?ggcagagtac?ttgaattaaa?aaaatatgag?gctatttact 480
gaactgtatt?gtttacatac?agaacattgt?t 511
Mclw04：
atcattcctt?cccctctccc?ctgtagctgg?aagcagctca?tgtcccttcc?ttcccacaca 60
gtgccgaaag?gctgctgctc?accacgttct?ggtgtgaaac?ctggcagaaa?ttgtgtgtgt 120
gtgtgtgtgt?gtgaccctgc?atgccctccc?cccatgtgtt?gcctcaggaa?aatgtggtgc 180
caggtaccag?gagaggtggg?tccgtcttgg?gggcccaacc?aggcatggag?ggcagaaagc 240
accaagcagg?gagggttggg?tcagttggtt?acccccatgt?gtgagagata?tatatgtgtg 300
tgtgtcacct?ctccccatgt?gtgtgggggg?ggggagggaa?aggtgtgtgt?gtcacccctc 360
cccttgtgaa?ccctaaaacc?ttaaagataa?gatggtaaat?aaaaagaatc?caactatgca 420
gtatttcttt?ttaagagggg?ctcagtcaac?ttgatgttaa?tttgaacatt?tgtactgcat 480
aattctgatt?gattgctgtt?gatcc 505
Mclw05：
gatctctgtg?cagctgggtg?tggccctgcc?tgtgtgcttg?gctggggagc?ctaactcagc 60
aaggccaggt?aaaggggacc?caggctggga?gaatagtctg?actcagtggc?gtcccagcac 120
accaggtgac?acactcaggg?tgtcccaaat?agtcactttg?gtataaaact?aggaagctga 180
gaccagtgag?ggctgcagtg?tggaagtgtg?tagttactct?caagctttag?acctgttcta 240
cattaggaga?ttaggatggt?cgatctgtgt?gtgtgtgtgt?gtgtgtgtgt?gtgtgagaga 300
gagagagaga?gaaatccaca?cccctgagca?atgcaattaa?attgacctaa?ccctcagcgc 360
agatggtgct?gtgttgacca?gatc 384
Mclw06：
gatcatggcc?taacttcact?cctaagaatg?tgctgcccac?agaaaagaaa?aacctgcagc 60
tggcctgtgc?tcacgggact?tgggctgcag?gggctatttc?attgctgtgt?agactactag 120
actagggctt?gagcccaggc?tctaggaccc?tgcaggggag?tccactcact?tcacgggagg 180
taggtagggg?tgtgtgtgtg?tgtgtgtgtg?tgtgtgtgtg?tgtaggcagt?gcaggaggat 240
ggggaaataa?tgtgtacaca?catgcagggg?tgggcaggag?agagggaaaa?caggtctgtg 300
catacagggg?aatgtagaga?atgcaagaag?ggaaacttgg?tgggatgcac?cagggaatac 360
agagaggtgc?aagaagcagg?tggcatttga?aggacggggt?ggaggatgga?aaaaacttgg 420
gttttaagct?gctctagcaa?aacaaagcag?ttgtaaaccc?aatttaactg?gccaatatgt 480
tccggcttct?tgtgctgctt?cagagtggca?ggaagcagcc?agcgcacatt?ggtgcaactg 540
atcc 544
Mclw07：
gatcactgcc?gcggatcgcc?ttcgaccgaa?gagccgaccg?tcaacctgaa?tcaccatttg 60
tcccccctcc?ccttctcaca?atgtgtgtgt?gtgtgtgtga?gagagagaga?gagagagaga 120
gagagagaga?gagaggggtg?tgcaaaacta?aatgcaatgg?cgtaacactg?ggtagtaggg 180
agtagcattt?gcatctcgtg?tgagcatagt?ttgcagcgtt?gcgggacttc?gctgacgagg 240
gaggagcggc?tcgagggcaa?atcgtggttt?gaaaggctac?gggacctcac?tgaagccgag 300
tgggcgttgg?tcgacatgat?cg 322
Mclw08：
gatcattact?ccttgttctg?tcatctgcta?ctactgagaa?caagcaaagg?gatggtgctg 60
gggcctcagt?gacaagctag?gtaccagcag?tgaagagggc?agcctcccgg?taggtggtaa 120
ctgtgacaga?tattgcaatc?acatgcaata?tctttgggga?ccatattgtg?ataagtttat 180
ggatggtttg?ttatcactgt?gggccaggga?ttgtatgcag?gaggaatgga?ggaagggtta 240
ccatagctcc?tccaggaact?aaaaactgtg?tgtgtgtgtg?tgtgtgtgtg?tgggggggtg 300
atttaagatg?aatcactctt?gtttgtaaac?tcctctggag?aggtaccact?ccctggggag 360
gcttgcatat?actggttcag?actgaatttt?ccagagtaaa?ctgacacaga?aagggctttt 420
ggcataaaga?ctgagtttag?actggcacag?gatttttttt?tgattcagca?aatggatggg 480
accttctctc?caaggggagc?cgaggggagg?gggagggcac?tgttgtggaa?gggttggaaa 540
gactttggtt?tatcct 556
Mclw09：
cagcagaagc?atcagctggg?caggctgggg?gcaaggtcca?gaggcaggcg?gacagtgccc 60
cagggagtat?ataggtatgg?agccaccacc?atcccatttc?atagaacagt?cacgtggtca 120
gggctcctgg?ggatgtcact?ttgtcatcct?gcaggaaatt?tattacatat?acacacacac 180
acacacacac?acacacacac?acaccgggaa?tggtagcaca?caggcacgct?gcctggatgc 240
cttccccgct?gatcc 255
Mclw10：
gatcctgaga?aatcctggac?acaacccttg?tgtggtggga?gagatagaga?gatggttgtg 60
tataggaata?gatagataga?tagatagata?gatagataga?tagatagata?gggtgtgttg 120
ggctagatag?atagatagat?agaggggggt?gtatggggat?agatagatag?atagatagat 180
agatagatag?catttagaca?aacggagatc?atgaaaaaat?gaagctaatg?tctcaatgcg 240
catggtttcc?aaagtccata?ggagtttcat?accattgaag?tcatctggaa?tggtttactc 300
cagtacaagc?attaagacat?catgataaat 330。
2. tortoise microsatellite marker according to claim 1 is characterized in that: described Mclw01, Mclw02, Mclw03, Mclw04, Mclw05, Mclw06, Mclw07, Mclw08, Mclw09, Mclw10; The primer sequence in ten sites is:
Mclw01F?TCCTATACCCAGTGGGACATG
Mclw01S?AAGTTTCACCCATCCATCAGC
Mclw02F?ACTGTGCCGGGTCGTGATGGG
Mclw02S?GTGGTGTCTGAGTGTTCTTGC
Mclw03F?GTGGTGGCACAGAGGTAGTTG
Mclw03S?CTCACATTTTCAGTTTGGTTA
Mclw04F?GAAAGGCTGCTGCTCACCACG
Mclw04S?ACTGACCCAACCCTCCCTGCT
Mclw05F?AGCAAGGCCAGGTAAAGG
Mclw05S?CATCTGCGCTGAGGGTTA
Mclw06F?GGGCTATTTCATTGCTGT
Mclw06S?CGTCCTTCAAATGCCACC
Mclw07F?GAAGAGCCGACCGTCAACCT
Mclw07S?CCCGTAGCCTTTCAAACCAC
Mclw08F?AGCTCCTCCAGGAACTAAAA
Mclw08S?AAACCAAAGTCTTTCCAACC
Mclw09F?GAGCCACCACCATCCCATTT
Mclw09S?AAGGCATCCAGGCAGCGT
Mclw10F?CTGAGAAATCCTGGACAC
Mclw10S?CTATGGACTTTGGAAACC。
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| CN103276097A (en) * | 2013-06-07 | 2013-09-04 | 浙江省水产技术推广总站 | PCR detection method for identifying germplasms of four populations of Pelodiscus sinensis, primer group and kit |
| CN103614374A (en) * | 2013-12-04 | 2014-03-05 | 广东省昆虫研究所 | Platysternon megacephalum microsatellite marked specific primer and detection method thereof |
| 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 |
| CN110317883A (en) * | 2019-07-29 | 2019-10-11 | 湖北省药品监督检验研究院 | One group for identifying the SNP marker of tortoise, flower tortoise and its cenospecies |
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| CN103276097A (en) * | 2013-06-07 | 2013-09-04 | 浙江省水产技术推广总站 | PCR detection method for identifying germplasms of four populations of Pelodiscus sinensis, primer group and kit |
| CN103614374A (en) * | 2013-12-04 | 2014-03-05 | 广东省昆虫研究所 | Platysternon megacephalum microsatellite marked specific primer and detection method thereof |
| 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 |
| CN110317883A (en) * | 2019-07-29 | 2019-10-11 | 湖北省药品监督检验研究院 | One group for identifying the SNP marker of tortoise, flower tortoise and its cenospecies |
| CN110317883B (en) * | 2019-07-29 | 2022-10-21 | 湖北省药品监督检验研究院 | SNP markers for identifying tortoise, pond turtle and hybrid species thereof |
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