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[Isolation of microsatellite DNA and the polymorphic locus screening from Phlebotomus chinensis
Objective:
To isolate the microsatellite DNA of Phlebotomus chinensis and screen the polymorphic loci.
Methods:
Genomic DNA fragments were hybridized with biotinylated oligonucleotide probes (AAT)17, (GA)25, (CCT)17, and (TG)18. The hybridized fragments were captured with Vectren Avidin D and enriched by centrifugal ultrafiltration with ultra-4-column ultrafiltrate. The target fragments were amplified, cloned and sequenced. The suitable microsatellite loci were chosen in Ph. chinensis's library to establish PCR amplification assay. The polymorphism screening was conducted by PAGE with Ph. chinensis field specimens.
Results:
The enrichment protocol used in this study was efficient, with a percentage of recombinant clone as 78.6%. There were 118 microsatellite sequences in library, the GenBank accession numbers were from FJ919812 to FJ919932 (except GenBank accession numbers FJ919833, FJ919836, and FJ919869). There were 72 typical microsatellite sequences occupying 61.0% and the rest were 46 nontypical microsatellite sequences in the library. Twenty-two loci were chosen to polymorphism screening and PAGE showed that 14 loci were polymorphic. The loci of dinucleotide repeat were more polymorphic than those of trinucleotide and polynucleotide repeat.
Conclusion:
The microsatellite-containing library of Ph. chinensis has been constructed with 118 sequences, and 14 new polymorphic microsatellite loci are reported.
