Isolation Of Microsatellite Markers And 16S RRNA Sequences Analysis In Rana Catesbeiana

The American Bullfrog(Rana catesbeiana) is a important commercially cultured species in our country. To isolate microsatellite markers from bullfrog is useful for the characterization of genetic stocks, broodstock selection, investigate population structure, mapping economically important quantitative traits, identifying genes responsible for these traits and application to marker-assisted breeding programmes.The protocol is an enrichment protocol based on the ability to recover microsatellite DNA by PCR amplification, after selectivhybridisation. High quality genomic DNA is fragmented using a restriction enzyme (TaqI) and than ligated to a adaptor (TaqI adaptor). Following the fragmentation-ligation step the DNA is then hybridised to specific selected 5' biotinylated probes and bound to streptavidin coated beads. After the hybridisation step and several washes to remove DNA that has bound non-specifically, the DNA eluted and recovered by PCR. Enriched DNA fragments are then cloned into a plasmid vector. The resultant recombinant clones are in turn screene for microsatellite repeats by directly sequencing them using primers specific for the vector. Following the identification of clones containing repeats, primers are designed for mark optimisation.This protocol was optimised for Rana catesbeiana and four out of nine potential loci were identified as being polymorphic with two being monomorphic and the others exhibiting unstable amplification reactions.Mitochondrial 16S rRNA gene fragment was amplified by polymerase chain reaction(PCR) in Rana catesbeiana.The 530 base-pair nuckeotide sequences of 16S rRNA were examined and analyzed for genetic polymorphism in three culture populations.The results showed that there were no differences in sequence among the tested samples.