Development and characterization of fourteen novel microsatellite loci in Chinese muntjac ( Muntiacus reevesi )
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TECHNICAL NOTE
Development and characterization of fourteen novel microsatellite loci in Chinese muntjac (Muntiacus reevesi) Hui Wang • Xia Luo • Wenbo Shi • Baowei Zhang
Received: 15 April 2013 / Accepted: 21 June 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Fourteen novel polymorphic microsatellite markers were isolated and characterized for Muntiacus reevesi, a small deer species. An (AG)n enriched library was created from two individuals following the FIASCO protocol (Fast Isolation by AFLP of Sequences COtaining repeats). 14 primers were designed from 98 microsatellite sequences and tested in 32 samples. The number of alleles per locus ranged from 2 to 13 and the expected heterozygosities from 0.123 to 0.916. The loci had an average polymorphic information content value of 0.676. Five loci showed significant deviations from Hardy–Weinberg equilibrium and no linkage disequilibrium was detected. These markers should be a useful tool for further population genetic studies of Muntiacus reevesi. Keywords Chinese muntjac Muntiacus reevesi Microsatellite Polymorphic Population genetics
Chinese muntjac (Muntiacus reevesi) is a small deer species with a native distribution centred in South China and parts of Southeast Asia (Zhang et al. 2004). It was introduced to England in the nineteenth century and it has expanded its range greatly during the last 30 years (Harris et al. 1995). Density of M. reevesi was so high in some areas that negative impacts on the biodiversity and conservation interest of forests and other animals such as roe deer were suspected (Capreolus capreolus) (Fuller and Gill 2001; Joys et al. 2004; Chapman et al. 1993; Staines et al. 1998; Hemami et al. 2005). By contrast, the population of
Hui Wang and Xia Luo have contributed equally to this article. H. Wang X. Luo W. Shi B. Zhang (&) School of Life Science, Anhui University, Hefei 230039, China e-mail: [email protected]
M. reevesi in China has declined sharply due to habitat degradation and overhunting, and it is in urgent need of protection. Here we report fourteen polymorphic microsatellite primers we developed in M. reevesi. Genomic DNA was extracted and purified using EasyPure Genomic DNA kit (TransGen Biotech) following the manufacturer’s guidelines. Microsatellite library enrichment was following the FIASCO protocol (Fast Isolation by AFLP of Sequences COtaining repeats) (Zane et al. 2002). 50 -biotinylated (AG) 12 oligonucleotides were used as probes and hybridization products were captured with Streptavidin MagneSphere Paramagnetic Particles (Promega). The enriched fragments were ligated into pEASY-T1 simple cloning vector (TransGen Biotech) and transferred into Trans5a chemically competent cells (TransGen Biotech). Recombinants were screened by PCR using three primers: vector primers (M13F and M13R) and (AG)12 oligonucleotide. Lanes showing multiple bands in agarose electrophoresis were considered as positive clones. Plasmids from positive clones were sequenced and primers were designed using software
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