CN104328118A - Microsatellite loci of Rana chensinensis, primers and application thereof - Google Patents
Microsatellite loci of Rana chensinensis, primers and application thereof Download PDFInfo
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- CN104328118A CN104328118A CN201410641083.2A CN201410641083A CN104328118A CN 104328118 A CN104328118 A CN 104328118A CN 201410641083 A CN201410641083 A CN 201410641083A CN 104328118 A CN104328118 A CN 104328118A
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- 108091092878 Microsatellite Proteins 0.000 title claims abstract description 33
- 241001336827 Rana chensinensis Species 0.000 title 1
- 241001285221 Breviceps Species 0.000 claims description 17
- 238000004458 analytical method Methods 0.000 claims description 5
- 238000009395 breeding Methods 0.000 claims description 3
- 230000001488 breeding effect Effects 0.000 claims description 3
- 238000011835 investigation Methods 0.000 claims description 3
- 238000011160 research Methods 0.000 claims description 3
- 108700028369 Alleles Proteins 0.000 abstract description 3
- 241001560470 Quasipaa spinosa Species 0.000 abstract 2
- 238000003205 genotyping method Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 8
- 208000005652 acute fatty liver of pregnancy Diseases 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000270878 Hyla Species 0.000 description 1
- 241000601854 Hyla tsinlingensis Species 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 239000012459 cleaning agent Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a Qinling mountain rana spinosa microsatellite locus, a primer and application thereof, wherein the Qinling mountain rana spinosa microsatellite locus comprises 12 stable polymorphic microsatellite loci, and the sequences of the polymorphic microsatellite loci are respectively shown as SEQ ID NO: 1-SEQ ID NO: shown at 12. By utilizing the Qinling frog microsatellite locus, 20 Qinling frog individuals are analyzed for genotyping data, the allele factor is 3-12, the expected heterozygosity He is 0.145-0.903, and the observed heterozygosity Ho is 0-0.579.
Description
Technical field
The present invention relates to biological technical field, in particular Qinling rainfrog microsatellite locus, primer and uses thereof.
Background technology
Qinling rainfrog (Hyla tsinlingensis) is under the jurisdiction of Hylidae Hyla, is Chinese unique wheat, and distribution range is comparatively extensive, is distributed in Yuexi County, Anhui Province and Huoshan County and Southern Shaanxi, SOUTH OF GANSU and Chongqing.Under the background that global Amphibians is forced, strengthen the protection of Amphibians very urgent.Molecular biosciences information is set up to change species conservation unit and provide certain theoretical foundation.And the research of this aspect is in blank.
Summary of the invention
The invention discloses 12 microsatellite sequences and 12 pairs of micro-satellite primers of Qinling rainfrog.These sites play an important role studying the conservative genetics of Qinling rainfrog, in Germplasm Resource Investigation and assistant breeding.
Technical scheme of the present invention is as follows:
Qinling rainfrog microsatellite locus, comprise 12 stable polymorphic micro-satellite sites, its sequence is respectively as shown in SEQ ID NO:1-SEQ ID NO:12.
The primer of described Qinling rainfrog microsatellite locus, its sequence is respectively as shown in SEQ ID NO:13-SEQ ID NO:36.
Described Qinling rainfrog microsatellite locus or the purposes of described primer, comprise molecular ecology research, pedigree analysis, Germplasm Resource Investigation, assistant breeding and individual recognition, described 12 stable polymorphic micro-satellite Sites Combination use, or the combination of primers of its correspondence uses.By analyzing the genotype data of 20 Qinling rainfrog individualities, number of alleles is 3-12, expects that heterozygosity He is 0.145-0.903, and observation heterozygosity Ho is that 0-0.579. has more much higher state property, reaches the requirement of individual recognition.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
1, DNA extracting
Utilize muscle or blood tissues, extract the genomic dna of Qinling rainfrog.The extracting of genomic dna adopts the cracking of SDS/ Proteinase K, phenol-chloroform extracting approach (Sambrook et al., 1999).
2, the structure of Qinling rainfrog enriched microsatellite library
Utilize and extract genomic dna, adopt AFLP Rapid Isolation (Fast Isolation by AFLP of Sequences Containing repeats, FIASCO) enriched microsatellite library is built, detailed process is as follows: get genomic dna about 250ng, use MseI digestion, be connected with the AFLP acceptor joint (5 '-TAC TCA GGA CTC AT-3 '/5 '-GAC GAT GAG TCC TGA G-3 ') of MseI simultaneously.Digestion product is diluted after ten times, do pcr amplification with the primer (5 '-GAT GAG TCC TGA GTA AN-3 ' is called for short MseI-N) of AFLP receptor-specific.Amplified production 1% agarose gel electrophoresis after 30 minutes for being greater than the dispersion plating of 200bp.
Use probe (AC)
12or (AG)
12cut after connecting fluid pcr amplified fragment hybridizes with enzyme, use magnetic bead to carry out absorb-elute, the micro-satellite repetitive sequence of required strand can be obtained.With the DNA caught for template, be that primer carries out double-stranded DNA recovery with MseI-N.PCR reaction volume is 50 μ L, and reaction system consists of: dNTP 5 μ L, Buffer 5 μ L, r Taq 0.5 μ L, MseI-N Primer 2 μ L, magnetic bead elutriant 5 μ L.PCR is circularly set as 95 DEG C of denaturation 5min; 95 DEG C of sex change 30sec; 60 DEG C of annealing 30sec; 72 DEG C extend 45sec, carry out 5 circulations; Carry out other 92 DEG C of sex change 30sec; 60 DEG C of annealing 30sec; 30 circulations of 72 DEG C of extension 45sec, 72 DEG C extend 10min.
PCR primer is through PCR cleaning agents box (Axygen) purifying, and by 2% agarose-EB gel detection, result is that the dispersion plating being greater than 200bp is then considered as successfully.Double-strand after purifying is recovered transform in DH5-α competent cell (Takara) after product is connected with pMD19-T carrier (Takara).The competent cell transformed is coated on the Amp+LB flat board of interpolation 0.5mMIPTG and 0.5 μ g/mL X-Gal, at 37 DEG C, cultivate 13h, obtain the micro-satellite library comprising AC, AG two kinds repetition.
3, the screening of positive colony, order-checking and design of primers
The full bacterium colony of picking white is placed in Amp+LB liquid nutrient medium enlarged culturing (37 DEG C, 5h).Tri-Primer-PCR (Zane et al., 2002) is adopted to detect the positive recombinant containing micro-satellite fragment.The probe (AC) of the lifeless matter element mark of M13 carrier universal primer (M13-47) and clone's correspondence is selected in experiment
12/ (AG)
12react.If product occurs that two or more band can think that this mono-clonal contains aim sequence, otherwise then thinks without aim sequence.Successfully checked out the positive recombinant 135 of aim sequence by three-primer method, these recons are checked order.
The preliminary screening of 4, sequential analysis, design of primers and microsatellite locus
Check sequencing result, after removing the carrier sequence of M13, use TRF software (Tandem Repeats Finder, Version3.2.1) to find the sequence (Benson, 1999) wherein containing micro-satellite repeating unit.Use PRIMER5 software (Lalitha, 2000; Singh et al., 1998) in the flanking sequence of repeating unit sequence upstream and downstream, design primer, object fragment is set between 100-200bp.24 micro-satellites are selected to repeat site as alternate labels altogether.
5, the reading of genotype data and the acquisition of microsatellite locus
Fluorescent mark (FAM/HEX/TAMRA) is carried out to the one-sided primer 5 ' end of 24 microsatellite locus just selected, then the DNA of 50 Qinling rainfrog individualities is used to carry out amplification screening to above-mentioned microsatellite locus, process is as follows: the pcr amplification first treating bit selecting point, is circularly set as 95 DEG C of denaturation 5min; 95 DEG C of sex change 30sec, anneal at Tm temperature 60sec, and 72 DEG C extend 60sec, react 30 circulations; 72 DEG C extend 10min.Amplified production uses Tamara350 fluorescence molecule amount standard to use polyacrylamide gel to carry out gene type in ABI 377DNA sequenator, and the result of genetic analysis uses Genescan 2.0 software to read.The analysis of genotypic results, removes following alternative site: without the site of genotypic results; At arbitrary individual allelic number in plural site; In 20 sites of Qinling rainfrog allelic below two.After screening, the microsatellite locus that final acquisition 12 is stable, microsatellite sequence is as follows:
＞Hts35
TAACTAATCTAGTGGAAGAGACTCGAGTGCTAAAATCCACGGGTTAGGGGGCGCAAATTACTTG
CCTTGCCCCGGGTGCTGACAACCCACGCTACGCCACTGGGAGTGCCATAGTGTTTGTTCATTCA
CTTGTAACAGCTACACTGGAGCCAACACCCCTTATAAAAACAGCATGCAATATACACACATATCT
ATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCACACACACTAAAGA
AAGCTGTAGGACATAGTGATAGCTACAGATAGCTGTAGGACATAGTGAAAGCCACAACTGTGAC
T
＞Hts99
TAAGGGTGCCAGTAATTTTGTCTAGCCCATTTTTGTCCAGTTTGCTTTTTTCCTCCCTTTTTTAGT
TTAGTTCCAATACACACAAAGGGAATAAACATGTGTATAGCAAAACATGTGTTACTGCAATCCTT
TTCTGTGAGAAATACTAGAAAAATGTCAGGGGTGCCAACATTTACGGCCATGACTGTATGTAGT
GAGCTCTCCCCAGAGTAGTGTTTCCCAACGGCTGTCTGGGCATGCTGGGAGTTGTAGTTTTGCA
ACAGCTGGAGGCACCCTGGTTGAGAAACACTGCTAGAGGCATGAATCTACTGATACTAATGATA
AATCTCTCCTATGTAGTGGTGGTTATGACGGTGAAATAGTGTGAGGTCTTTCTATCTTCTGCAAT
CTGCAAAAACCTATTCCGATATTTGTCATCAGATTTCCTAATAAGTAGGTATCTAGGATAAGAAA
AACATAGCTGCTTTCTTCCAGAAACAGCGTCACTCTTGTTCTCAGTTTGGGTGTGGTATTGCAG
CTTAGCTCCATTGAAGTGAATTGAGAAACGTTGTACTACCACACACAACCGGAGGACAGGAGA
GCTGTTTATAGAAAGCAAATACTGTTCATTTATTCCTGGACTTTTTGAT
＞Hts195
TCCTGAGTAACCCTTTTACTGTCTTGGACCACCAGTCATGTAACCATAAACCCCTCTCCT
GCCCTAGACCAACAAAGATGTAACCATAAAGCCCTTTGTTGCATTAGGCCACTAGTTATG
TAACCATAGATTCCTTTACTGTCCTAAACCACCAGGTATGTACACCCTTCTGAATTCTGT
AATAGCCAGAGTGGGGAGATCCACTACAAAGGGCAATCATGTTGATGTAAGTTCTTAGTA
TGCAGGCAAAATGGGGCCAACATGCTATTCTTGCACAGGGGCCCAAAGTTATCTAGATAG
ATAGATAGATAGATAGATAGATAGATAGATAGATAGATATTTCATGTTTTACATTTCATG
TTTTACATATATCATTGTGCTGGCAGTGTGCTGCTGTATTCTCCTGTTTATGTATATTCA
GCCTAGCAGCGTGCACCTGTATGCCAGGTCCATGCAATAGTTTGTATCAGTATGTGCTGG
CCAACTTATTCTTTTTCTGTGTTACACATATGGGGGTGTGGCTATCCTCCATGTAGGTTC
TTGCACCCCACCCTGCTCATAAGGAGCTTATATAAGGTGCTGGATGTTCTAGACCTTGCA
GTGCCTGTTGTCTGATTACACAGAGGCACATTCACAGTGTAGGGTCCGTCCCAATGAGGT
CACCTTATCGCGGTGGGATGGTCCAAGCTATTTGGCTGCCAAATATTTTGCTAAGTACCT
CGCTTTTTCATTTTTTCATTGTAGCAATATAGCATTTCATGTTTTACATATATCATTGTG
CTGGCAGTGTGCTGCTGTATTCTCCTGTTTATAGATAGATAGATAGATAGATAGATAGAT
AGATAGACAGATAGATAGACAGACATCATCTAAAGGTCGTGGCTATTTTCTTTTGCCAGA
AAGTCAACAATTCACGGGTGACAATAATCATCTTTAATGTACAGT
＞Hts92
TAAGGGGTTGTCCATCTTTTCTCTGTTGATTTCTTCAATAAACCGTGAGTAATTTTCCATTTTTCT
TCACCAGATTATACTACATTGTTAACTTGGCCATGATCGTCTGATTTATCTCTGGTGTCGTTCTAC
TGTCCAGGATAGAAATCTTATTTACAGCATCTTCTTACTCACTCGGGGGGATCGGGTGTTGGCTG
AGGAGTTCTCTAGAACAGGGTATTACAGGGAAGCATGAATGATACCAGTCAGGAGATATTCTAT
CTAGATATATATAAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATTTTTGACTGG
TTGGTTTCATGCTTGCTTGTATGAGATT
＞Hts87
TAACATGCTTGAAAAAGGCAGTGAGAGACTGCCGAAACGTTGCACTGGTTTTGCTGTGAAGAT
GCAATAATTTTATACACTTTTTGAAGTTTACTCCTGTGTGCTGCGGTCCTTTGCTATGTGGATTTA
TCATGGTTACTCTGACCAGGAACCTGATGACGGCCTGCACCTAATGGATTGAGGATACTCCTGC
TGGTTGTGCTGATTTTGGGGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGAT
GATAGATAGATAGATAGATAGATACACGAAAAAATAGATGCAGCACACCACAGCTTAGAAGCAA
AGTGCAAGGTTTATTCCATAGTGTATTCCAAAGGACTACGTTTCTCCAGCCTCACGCTGGCTTTT
TCAAGTACAAAAATGTGATCAGTGCTGGGCTTTATACAACGCCCGCAGGAAGTAACATGACATC
ATACAACATTTACATATATAAACAAACAAAATTTCAGTGCACAATATATAACTCCAATGCATAATA
TCATACATTTGTATATATACATACTCATATATACATATGCT
＞Hts70
TAATGATTGTAATATGGATTTATTGGACTGTTTGATTTGTTATCACACAAATATGTGGCACTTCTAC
CTTGCTGTATGCTTGAAAAAGCCGGTGTGAGACCGGTGAAACGTTGTATCACTTTTCCTGCATG
GGATAAATAAATCACCACTTTTGAGTTGATTTGAGTGCTGCGGAACTTCTTTGTAATTCTATCTCT
CTCGTATTTATCATCTCTCTTGTATCTATCTATCTCCTATCTATCTATCTCATATCTATCTATCTCCTAT
CTATCCATCCATCTCATATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTCTCTCATATCTAT
CTATCTTGTATCTATCTCATATCTATCTATCTCCTATCTATCTATCTCTCTCCTTGGTTGTCCGGTTAT
GATGGAGTGATAGTCTGAGCCGGTTTGGCGCACTCTCATATCCCCGTGCCTCCTCTTACGCTTTG
CCCGCGTCTCCTCTTCTCTGTAAAACATTTAGGTCAGTAGGT
＞Hts2
TAAGTGCCATTCCTTTTTTGTTTTAGCTGGGAAATCTTGCAATTCCAATAATCCCTGACTGAGGC
AAATAATTTGTCTCAGGACCAGGGTTGTCACTTTGTGTTTTGGACCTGATCCTTGGGAGTGCTG
CTGGACGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATGGATAGATAGATAGGTA
GGTAGGTAGGTAGGTAGGTAGGTAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAG
ATAGATAGATAGATAGATAGATAGTTCAATGCAAAAAAGAGCAGCACATCCAGCAGCGTCAAGG
AGTGGGGGTGCAAGCGGGCCCAGTCGCAGCCCTGGTCACAGGCAGCAGTGAT
＞Hts28
TAACATAGGTAAGATAATTTTACATGACCAAGAATACCAGTATAAAAGGAGGAAATATATACCAT
CACACAATAACTTGATAATACCACCATACTGTACTGAATAAAACCACTATACACAGACCAGTATT
CCCCCATACAGTGCCCATATAGTGAAAGTTGTCAGCTGTACACAGGATATATATACCATATAAGTG
ATTATATACATGAGCAGGGCTGCCATCAGAGATAGATAGATAAATAGATAGATAGATAGATATGAG
ATAGATAAATAGATATGAGATAGATAGATAGATAGATATGTGGTAGATAGATATGAGATAGATAGA
TATGAGATATAGATATGAGATAGATAGATTGATAAATAGATAGATAAAAAAAAGTGTAGAAAAGC
AGCACATCCACAGGTAGACAGCAGAT
＞Hts233
TAACTGATTACACAGAGGCACATTCACAGTGTAGGGTCCGTCCCAATGAGGTCACCTTATCGCG
GTGGGATGGTCCAAGCTATTTGGCCGCCAAATAATTAGCTAAGTACCTCACTTTTTCATTTTTTC
ACTGTAGCAATATAGCATTTCATGTTTTACATATATCATTGTGCTAGCAGTGTGCTGCTGTATTCTC
CTGTTTATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGAAAAAGACCCAGACT
GCACAACATCCATGAATGTGTCCTGTGGTGTCTGCACCAAGACTTCAGCAGCAGATCCTGTAAG
TCCTGTAAGTTGTGAGGTGTGACCTCTATAGATCAGACTTGTTCTTTTACTAGATCTTTTAGGAG
CTGATCTAGATTTAGATGTTCCTCTCATCATCACTATGAAGGGTTGTAATAAGTGGCCCATATCAA
AAAACATTCTTTATATCCTCTTTCACTGCAGTTCCTTCTCTGACCAGTAGTTGTTGCCTTGCT
＞Hts39
TAACACGTGTCATATTCTAGGTTCTTCAAAGTAGCCACCTTTTGCTTTGATTACTGCTTTGCACA
CTCTTGGCATTCTCTTGATGAGCTTCAAGAGGTAGTCACCTGAAATGGTCTTCCAACAGTCTTG
AAGGAGTTCCCAGAGATGCTTAGCACTTGTTGGCCCTTTTGCCTTCACTCTGCGGTCCAGCTCA
CCCCAAACCATCTGGATTGGGTTCAGGTCCGGTGACTGTGGAGGCCAGGTCATCTGGCGCAGC
ACCCCATCACTCTCCTTCTTGGTCAAATAGTCCTTACACAGCCTGGAGGTGTGTTTGGGGTCATT
GTCCTGTTGGAAAATAAATGATGGTCCAACTAAACGCAAACCGGATGGAATAGCATGTCGCTGA
AGAAAACTCTTTGAATGAGAAGGTGTGTCCAAACTTTTGGTCTGTACTGTAGATAGATATGATAT
AGATAGATAGATAGATAGATAGATAGATAGATAGATAATATAGATAGATAGATAGATAGATAGATAT
GAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGTGAATTC
AGGTAGAGAAAAAACAGACATGGTCGCTAGTGTTGCTCACGAATATTCGCATTTCGAATATTCG
CATCGAATATTGCATATTCGTATATTCGCGAATATCACGAAATTTCGCCATT
＞Hts85
TAATCAAATAGGAATTTTGTTATCTATCTATCATCTATCTATCTATCATATCTCATATCTATCTATCTA
TCTCCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTCACCTTATCATGGTG
GGATGGTCCAAACTATTTGGCCGCCAAATGGTGAGCAAAGTACCTCACTTTTTCATTGTAGCAAT
ATAGCATTTCATGTTTTATCTGTATCTTTGTGCTAGCAGTGTGCTGCTGTATTCTCCTGTTTATCTA
TCTATCTATCTATCTATCTATCTATCTATCTATCTATCTAATCTCCAGACCCCATCTGAATATGTGAC
CTTCTCTCTCCCATCCCATAGTGTAATGTTCTTGTCTTGCCCATGTCTTGTAGGTGCCAATCACTC
CCATGTGAGACCCCCACCTCTCTGCTCGGGGTCAGGGGTGTTATTCTTTCAGTGCGGGGCTCTG
TTTAGGTTGAGTGCAGAATAAATATATCAGGGGAGAGGAGTCCAAGTCTGGTAAATGATTCCCG
CTGTGCAACTTTCCAAGGAGTAGGAAATGGGCAGGAAATGAATAGCAGGCCTGGAGTGGACCT
GGATGTGTCTCTATTTGTGTTTATCCTGCCAGTGCTGTGCCACACGTGAATGACAACTCCTGCCA
GTGTTCTCCCCGCCATGTTTGTATCTCCTGGACAATCTGAGCACTAGTACAGGTTGTTCTGCATT
GCTCTGCCTTCTGACATACTGTATG
＞Hts53
TAACGAATTCAGATTCGTCAGATTCGATGCGCTCATCCCTAATTATCTATCTATCTATCATCTCATA
TCTATCTCATATCTATCTATCTCATATCTATCTATCTATCTATCTCTTATCTATCTATCTATCTCATATC
TATCTCATATCTATCTATCTCATATCTATCTATCTATCTATCTCTTATCTATCTATCTATCTCATATCTAT
CTCATATCTATCTATCTATCTCATATCTATCTATCTCATATTTATCTATCTCATATCTATCTATCTCATA
TCTATATATCTATCTAGCATGTAGAAAAAGCTGCAGCACTCCCAATGAAGGTGAAATAAAAAAG
TGGTTTATTCCATCCAAACGGATACAACGTTTCGGTCGCCCAATGCGGCCATTTTCAAGCATGTA
TATGGATATATATCTATCTATCTCATATCTATCTATCTCATATCTATCTATCTATCTATCTCCTATCTAT
CTCATATCTATCTATCTCATATCTATCTCATATCTATCTATCTCATATCTATCTATCTCATATCTATCTC
ATATCTATCTATCTATCTCATATCTATCTATCTATCTCATATCTATCTCATATCTATCTATCTATATATCT
ATCTCATATCTATCTATCTCATATCTATCTATCTATTTATCTATCTCATATCTAT
The primer sequence in table 1 12 sites
The genotype data of table 2 20 Qinling rainfrog individualities
Table 3 typing data result by analysis
Typing data by analysis after, number of alleles is 3-12, expects that heterozygosity He is 0.145-0.903, and observation heterozygosity Ho is that 0-0.579. has more much higher state property, reaches the requirement of individual recognition.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.
Claims (3)
1. Qinling rainfrog microsatellite locus, comprise 12 stable polymorphic micro-satellite sites, its sequence is respectively as shown in SEQ ID NO:1-SEQ ID NO:12.
2. the primer of Qinling rainfrog microsatellite locus according to claim 1, its sequence is respectively as shown in SEQ ID NO:13-SEQID NO:36.
3. the purposes of Qinling rainfrog microsatellite locus according to claim 1 or primer according to claim 2, comprise molecular ecology research, pedigree analysis, Germplasm Resource Investigation, assistant breeding and individual recognition, described 12 stable polymorphic micro-satellite Sites Combination use, or the combination of primers of its correspondence uses.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003189885A (en) * | 2001-10-05 | 2003-07-08 | Japan Science & Technology Corp | Centromere-localized repeated sequence derived from wheat |
| CN104087584A (en) * | 2014-07-21 | 2014-10-08 | 张洁 | Dasyatis zugei microsatellite sites, primers and application thereof |
| CN104087585A (en) * | 2014-07-21 | 2014-10-08 | 王慧 | New salangid microsatellite locus, primer and application thereof |
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- 2014-11-14 CN CN201410641083.2A patent/CN104328118A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003189885A (en) * | 2001-10-05 | 2003-07-08 | Japan Science & Technology Corp | Centromere-localized repeated sequence derived from wheat |
| CN104087584A (en) * | 2014-07-21 | 2014-10-08 | 张洁 | Dasyatis zugei microsatellite sites, primers and application thereof |
| CN104087585A (en) * | 2014-07-21 | 2014-10-08 | 王慧 | New salangid microsatellite locus, primer and application thereof |
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