Isolation And Characterization Of Polymorphic Microsatellite Loci Of Anopheles Sinensis And Anopheles Lesteri (Diptera: Culicidae)

Anopheles sinensis and An. lesteri (synonym as An. anthropophagus) are distributed widely in China. Their population are high density and malaria vectors in distribution regions. The efficient vector control strategy is an important element for malaria prevention. It is necessary to clarity the vector's biology in developing the control measures. In this study, the microsatellite DNA library of An. sinensis and An. lesteri will be constructed, the polymorphic microsatellite loci of An. sinensis will be isolated and characterized. It is certainly that the results will provide invaluable information for genomic. The polymorphic microsatellite loci will be new molecular markers for reseach on gene location, population genetic structure and ecology of An. sinensis and An. lesteri.Genomic DNA fragments which was digested by Sau3AI were hybridizied with biotinylated oligonucleotide probes, as (AAT)17,(GA)25,(CCT)17,(AC)25,(CAG)17,(CA)18,(CAC)5,(TC)10,(GT)8 and (TG)18. The hybridization fragments were captured with Avidin D and concentrated by ultrization using ultra-4 column. The enrichment were proceeded twice. The fragments which were contained microsatellite DNA were amplified three times,and ligated with T-vector, transformed to E.coli strain DH5α. After the positive clones picked and sequenced, the microsatellite geomic library of An. sinensis and An. lesteri was constructed. The suitable microsatellite loci were chosen in An. sinensis's library, and their amplified primers were designed and synthesized. The PCR assay of amplified microsatellite loci was established. The polymorphic microsatellite loci screening was used PAGE gel electrophoresis and Genescan assay by field populations of An. sinensis idetified by molecular marker. The results were as follow:1. Microsatllite genomic library of An. sinensis contained 282 sequences. There were 18 sequences which was the same in the library. So, a total of 273 unique microsatellite DNA sequences were obtained.2. The dinucleotide microsatellite loci were the most in An. sinensis's library. The microsatellite sequences contained (CA)n and (GT)n were abundance. The range of repeate number per loci was from 2 to 54. There were 89 perfect microsatellite sequences (32.6%), 82 imperfect sequences (30.1%) and 102 compund sequences (37.3%).3. The 22 suitable microsatellite loci were chosen and their primers were designed and synthesized. The PCR assay was established. The amplification products of 15 loci were specific.4. After molecular identification, the 40 field samples from 4 An. sinensis populations were used to amplification and screen microsatellite loci. There were 10 polymorphic microsatellite loci were identified, as AS5, AS6, AS9, AS13, AS14, AS15, AS16, AS22, AS31 and AS43.5. A total of 61 microsatellite sequences of An. lesteri were isolated, the 60 unique sequences were obtained.