Isolation and characterization of polymorphic microsatellite DNA markers of Anopheles sinensis(Diptera:Culicidae)

AIM:To construct microsatellite-containing library of Anopheles sinensis and to isolate and characterize the polymorphic microsatellite loci.METHODS:Genomic DNA fragments were hybridized with biotinylated oligonucleotide probes.The hybridized fragments were captured with Vectrex Avidin D and enriched by centrifugalization with ultra-4-column ultrafiltrate.The target fragments were amplified,cloned and sequenced.The suitable microsatellite loci were chosen in An.sinensis's library to establish PCR amplification assay.The polymorphism screening was conducted by PAGE gel electrophoresis with An.sinensis field populations.RESULTS:An enrichment protocol yielded 252 microsatellite sequences,their GenBank Accession Numbers were from EF620047 to EF620298.There were the most percentage of dinucleotide repeat,the more of tri-nucleotide repeat and fewer of multi-nucleotide repeat.The(CA)n and(GT)n were higher abundance in the microsatellite DNA libary.The number of motif repeate was from 2 to 54.The percentage of perfect microsatellite DNA sequence was 35.3%,imperfect sequence was 20.2% and compound sequence was 18.7%,and the rest was non-typical microsatellite sequence.We designed primers to amplify 22 unique microsatellites,and 20 of which amplified successfully.A survey of 30 individuals showed that 15 loci were highly polymorphic.CONCLUSION:A total of 15 polymorphic microsatellite loci of An.sinensis are first reported.These markers will be useful for population genetic studies and genome mapping in An.sinensis.