Tianlong FU， Shan SUN， Chaonan ZHU and Yanfu QU

Jiangsu Key Laboratory for Biodiversity and Biotechnology， College of Life Sciences， Nanjing Normal University，Nanjing 210023， Jiangsu， China

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Isolation and Characterization of Nine Microsatellite Markers for Red-backed Ratsnake， Elaphe rufodorsata

Tianlong FU， Shan SUN， Chaonan ZHU and Yanfu QU*

Jiangsu Key Laboratory for Biodiversity and Biotechnology， College of Life Sciences， Nanjing Normal University，Nanjing 210023， Jiangsu， China

The red-backed ratsnake （Elaphe rufodorsata） is widely distributed in East Asia， especially China. This species is a common snake in plain river network region. In the past several decades， E. rufodorsata has dramatically declined due to the effect of human activities and over hunting for traditional Chinese medicine. We developed nine species-specific microsatellite loci in 190 individuals collected from Huzhou， Zhejiang province in China. These markers revealed a high degree of genetic diversity （13-41 alleles per locus） and heterozygosity （HOranged from 0.266 to 0.941， and HEranged from 0.851 to 0.937）. No locus exhibited significant deviations from Hardy-Weinberg equilibrium. There was no evidence of linkage disequilibrium among pairs of loci. These microsatellite markers were described in our study will be valuable tools for the long term management and population-level studies （e.g. the population structure， genetic diversity and variation， individual paternity and evolutionary history） of the species.

Colubridae， Elaphe rufodorsata， Microsatellite markers， Polymorphism， PCR primers

The red-backed ratsnake （Elaphe rufodorsata） is a kind of small-sized， viviparous， nontoxic snake can be found in semiaquatic areas， widely distributes in most parts of China， eastern Russia and Korean Peninsula （Zhao 1998）. This snake has been declined because of its medical value for traditional Chinese medicine and habitat destruction. As a result， this snake is listed as the state’s ‘‘three are’’（state protection beneficial or of important economic，scientific value of the list of terrestrial wildlife） animal in China by State Forestry Administration of the People’s Republic of China. To better address conservation plan and comprehend population genetic information of this species， we developed nine species-specific microsatellite loci that can be used to study the population genetics of E. rufodorsata.

In this study， the tissues were took from the tail tip of three unrelated snakes and preserved in 95 % ethanol before brought to Nanjing Normal University from Huzhou， Zhejiang province. Total genomic DNA fromthree unrelated individuals was extracted with dBIOZOL Genomic DNA Extraction Reagent （Bioer Technology）and dissolved in TE buffer （10 mM Tris-HCl， pH 8.0，0.1 mM EDTA）. We used the total genetic DNA to construct the library enriched for AC/AG/GATA/TATC motif microsatellite sequences according to previous study （Hua et al.， 2012）. The genomic DNA was digested with Mse I restriction enzyme and ligated to Mse I AFLP adaptor. After diluted the digestion-ligation mixture with ten times water， we amplified it with AFLP adaptorspecific primers Mse I-N （5'-GAT GAG TCC TGA GTA AN-3'） for 26 cycles and hybridized the DNA product with （AC）10， （AG）10， （GATA）6， and （TATC）10probes，separately. The microsatellite-enriched DNA fragments were obtained following the protocol （biotin capture method）. These DNA fragments were amplified with Mse I-N primers， then ligated the PCR fragments into PMD19-T vector （Promega）， and transformed into Escherichia coli dH5α competent cell （TAKARA Biotechnology）. The transformed cells were plated on Luria-Bertani （LB） agar plate containing X-Gal and IPTG for blue-white selection at 37 °C for 16h.

White colonies were transferred into the liquid mediumcontaining ampicillin and kept oscillation culture for 6-8 h at 37 °C. The recombinants were screened by PCR in according with Hua et al. （2012）. We separated PCR products using an ABI PRISM 3500 XL genetic analyzer with a GS350 size standard， and analyzed genotypes by using GENEMARKER V1.85. We tested 190 individuals to analyze microsatellite data using GENETIX V4.0.

These nine sequences containing microsatellite locus were deposited in GenBank （KM597056-KM597064）. Polymorphic information of nine microsatellite loci was presented in Table 1. All the nine loci were polymorphic exhibiting 13-41 （mean of 21.667） alleles per locus（Table 1）. HO（observed heterozygosities） ranged from 0.266 to 0.941 （mean of 0.658）， and HE（expected heterozygosities） ranged from 0.851 to 0.937 （mean of 0.896）. The PIC （polymorph information content） value ranged from 0.833 to 0.961 （mean of 0.890）. MICROCHECKER detected no null alleles. No locus exhibited significant deviations from Hardy-Weinberg equilibrium after sequential Bonferroni correction （Rice， 1989）. There was no evidence of linkage disequilibrium among pairs of loci in the samples. These microsatellite loci were described in our study would be used for assessing the genetic diversity， population structure and individual paternity. In addition， these microsatellite loci may be helpful to set up effective conservation measures for E.rufodorsata.

Acknowledgements This study was carried out in compliance with the current laws of China， and was supported by grants from Natural Science Foundation of China （31200283， 31272294 and 31270571）， the Priority Academic Program Development of Jiangsu Higher Education Institutions， Teaching Reform Research of Nanjing Normal University （2014-61） and Postgraduate Research Innovation Projects （KYLX_0716） of Nanjing Normal University. The authors would like to thank Yan-Qing Wu for help in sample collection and experimental procedures during the research.

References

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Table 1 Characteristics of nine Elaphe rufodorsata microsatellites DNA loci： locus designation， primer sequences， repeat motif， allele sizes，annealing temperature （Ta）， number of alleles （Na）， observed （HO） and expected （HE） heterozygosities， polymorph information content （PIC）and GenBank Accession No.

The 9 microsatellite sequences are as following：

＞KM597056

AGTAACGGAGCAAACACAATGCTGCTGCAAATTCATCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTC TCTCTCTCTCTCTCTCTCTCTCTCTCACTCACTCACTTTGGAT

＞KM597057

TTCCTGGTGACTGACATTGTGTAGGTGTGTATATCTCTATCTCTATCATCTCTGTCTATCTATCTATCTATCTAT CTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCGGAAAAGAAGGAAGGCAAATGAGGTCCAGGCA GAAGAAGA

＞KM597058

CATTCCAGATCACCGATCACTCTGCCATTTTTTTTACTGCTACTTTTGTTCTCTGTACACACACACACACACA CACACACACACACACACACACACACAAACACACAAAACCAAAAACATTATGGAGAGAAAGATGGAAGAG CCACATAAGGAATTCGAGCAGGTAAGGACATCAAGC

＞KM597059

CCAATGCTATGAGTCTGCTGAGCCACCAGGCCCTTGTGTGTAGGTTACCTACACACACACACACACACACA CACACACACACACACACACACACACACAAAAACATGGAGCTGACATTTGGATTATCAGATGTCACTTCTAG AATAAATGAAATCACAGCATCTGAGACGTATCCTGAGAGTAGATAAAAAGACTCCAGCTTTTGCCCTACGG AATGAA

＞KM597060

ACCACCAGTCATCTCTCTTTCCTACACTCAGGGCAGCTCACTCTCTAATACAAAAATAAAATAAAATTCAAA GCAGTGCAAAACACACACACACACACACATGCTATGTGCTATATATGCTATGTAGCATATATAGCATGCATCT GTCTGTCTGTCTGTCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTAACTA GACAACTTGAATCTCCAGCATA

＞KM597061

CTGGATGTGGTTCTCAGTGGGCCCTTGGCTGAACATAGTAAGAATTGCACACATGTGACAATTTTGAGATAT GGAAATATAGATAGATAGATGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGA TAGATAGACAGACAGATTGATCTAGGTCTTCAAATATAGACAATCTTTATAATTCGTGGGTTCTATATTTGCA AATTTGCCTACACGCTACAATTTCTTT

＞KM597062

TGAATTGGTAGAGTTTGGTGTGCCGGTACCGACATTTGGAGTTTGCAGGACTGCTTGCTTCTTCTTTATTTG TGGACTCAATACCTTGTTCCTGACTTTGTGGACTCATTGTTTGTTCCTTGGTTCATGGACACATTTCCCTGTT CCTTTGTTTTGCTGGACTGGGCGCAGCCAGAGGCTGCTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG TGTGTGTGTGTGCCTTGCTCTGGGACTTGC

＞KM597063

GGCTTGGCTTGACATGCTAGGTCTTATTTTTGGGATAGGTTTTATTTTCAGGGAAACATCAGATAGATAGATA GAAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGAT AGATAGATAGATATGGTTCAAGACCACACAGGCATTCCAAAACACAACCTACTCATTGTTGGAAGAGAAGA AAGTCTAGGCCCAGAGACCCCCCAGAGATACGGAGGATTCCAGA

＞KM597064

CCATCTAGCAGCAGAGTTCACCACATGGCACCTGGGGATATTACCTCACTCAGCACAGTTCAATATGACTTA TCTCAGACAAACTCCTAGGATTTGCACATGAGCCCTACAAAACAACCAATAGGATACATTTATTGCACAGGA ATAGGAAGCTCTAGGGCAAGGCCCAAGAGGGTATATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTA TCTATCTATCTATCTATCTATCTATCATCTCCACTCCACTCCACTCCACTC

10.16373/j.cnki.ahr.150065

Appendix Comparative mtDNA sequences examined.

ROM， Royal Ontario Museum， Toronto， Canada； FMNH， Field Museum of Natural History， Chicago， USA； CAS， California Academy of Sciences， San Francisco， USA； KIZ， Kunming Institute of Zoology， the Chinese Academy of Sciences， Kunming， China； ZFMK，Zoologisches Forschungsinstitut und Museum A. Koenig， Bonn， Germany； NHMG， Natural History Museum of Guangxi， Nanning， China；CIB， Chengdu Institute of Biology， the Chinese Academy of Sciences， Chengdu， China； SCUM， Sichuan University Museum， Chengdu，China.

Dr. Yanfu QU， Nanjing Normal University，Jiangsu， China. with his research mainly focusing on evolutionary biology.

E-mail： imwx-q@163.com

4 November 2015 Accepted： 6 June 2016