Isolation of microsatellite markers from Paralichthys olivaceus and its application in genetic structure analysis

Magnetic bead enrich-method and plasmid detection-method were used to isolate microsatellite DNA from Paralichthys olivaceus genome.The microsatellite markers were applied to analyze natural population structure of four populations,which were collected from Dalian,Beidaihe,Dandong,and Qingdao.Two thousand eight hundred and five microsatellite sequence clones were isolated and 3120 microsatellite loci were obtained.From these sequences,57.97% were perfect,7.25% were imperfect and 34.78% were compound.This allowed to design 135 pairs of primers with the software Primer Premier 3.0.Seventy-eight pairs were synthesized and 30 pairs were polymorphic.The effective number of alleles ranged from 3.93 to 9.94(6.95 in average) with observed and expected heterozygosities ranging from 0.532 to 0.895(0.753 in average) and 0.635 to 0.902(0.820 in average),respectively.Hardy-Weinberg equilibrium analysis in 30 microsatellite loci by genetic deviation index(d) ranged from-0.247 to 0.512,among which 7 loci had heterozygote excess(d0) and the rest showed heterozygote deficit(d0).A dendrogram was constructed based on NJ methods using MEGA3.0 software package.Four populations were divided into two clusters.Dalian population and Dandong population were grouped in one cluster,while Beidaihe population and Qingdao population were classified into another cluster.