Development Of The Enrichment And Typing-by-Sequencing Approach For Large-scale Microsatellite Analysis

Microsatellite is widely used molecular genetic marker.Genotyping of microsatellites includes locus selection,PCR amplification,genotyping by capillary electrophoresis or by massive parallel sequencing.This approach requires screening and validation of loci from genome resources,setting up of PCR system and cycling program,polymorphism analysis and genotyping error quantification,as well as population genetic characterization for each locus before application to genetic analysis.This approach is costly of both fund and labor but often obtains a small number of qualified loci.In order to break these limits,we proposed a novel technical resolution based on high throughput sequencing platform to achieve the integration of loci capture and typing-by-sequencing.This resolution includes blunt-ended restriction digestion of DNA,A-tailing and adaptor ligation,microsatellite capture,parallel amplification,massive parallel sequencing and bioinformatic genotyping.Among all these steps,microsatellite capture was performed using a 5’-end biotinated primer made up of GA and CA motifs via one cycle of denaturation,annealing,strand extension and slow cooling to create triplex of the double strands of microsatellite fragment and the newly primed strand.The triplexes were captured using silica bead.New strand was separated and removed from the triplex by heating,leaving double strands of microsatellite fragment.This approach was tested using four vertebrate species namely black spotted frog(Pelophylax nigromaculatus,muscle),King rat snake(Elaphe carinata,muscle),hazel grouse(Bonasa bonasia,muscle)and sika deer(Cervus nippon,muscle and feces).Results showed that 5729±2124 to 614±254 microsatellite loci were obtained from five samples.Loci could be identified via flanking sequence alignment and genotyped based on top one or two of identical reads.It was observed that microsatellites could be enriched by PCR after with positive correlation with cycling number(P=0.695).Microsatellites we obtained include 23.67±5.43～67.67±4.99 types of motifs besides perfect GA and AC.These results suggest our resolution could directly analyze massive microsatellite loci at low cost and without needs of species-specific screening,selection and validation.It has great potential to play important role in the analysis of genome variation after further optimization.