Development Of Microsatellite DNA Markers And Population Genetics Assessment For Theblack Rockfish, Sebastes Schlegelii

Sebastes schlegelii has a high value of economy and is an important fish ofcultivation and exploitation. Commercial exploitation and coastal ecologicalenvironment changes has caused a gradually decrease of the wild S. schlegeliiresources, the genetic diversity of wild population is threatened. Reasonable stockenhancement efforts by releasing artificial propagation seeds to natural waters on thebasis of a sufficient understanding of its germplasm resources in wild populations, willbenefit its resource quantity and biodiversity conservation. To select and breedhigh-quality varieties of fast growth and strong disease resistance from the wildpopulations for artificial cultivation also have important economic and social benefits.The widely use of DNA molecular markers especially the microsatellite markers on thestudy of marine fishes has provided a convenient and effective tool for these studys.Here we developed fifty-one polymorphic microsatellite loci from S. schlegelii with anenriched partial genomic library by magnetic beads. Then13primers were selectedalong with4primers from other papers to evaluate the structure of five wildpopulations of S. schlegelii collected from Muping, Changdao, Rizhao, Rongcheng,Qingdao, respectively.1The isolation and characterization of fifty-one microsatellite lociGenomic DNA was digested with restriction enzyme Sau3AI and DNA fragmentsranging from400to1000bp were collected. The fragments were hybridized tobiotinylated (CA)12and (GA)12probes，then captured by streptavidin-coated magneticbeads. The enriched DNA was then ligated to vectors and transformed into competentEscherichia coli DH5α cells to construct an enriched microsatellite sequence library.213positive clones were isolated from353colonies for sequencing by three primersPCR technique,188(88.26%) of them revealed to be have microsatellite sequences, and most of the sequences were based on CA/GT or GA/CT repeat motif. Among thesequences,83were identified to be perfect microsatellites,47imperfect and58compound microsatellites, the percentage is44.15%,25.00%and30.85%, respectively.169primer pairs were designed and synthesized using software, of which144amplified a clear objective stripe.51pairs of primers showed polymorphism afterscreening by a wild population of32individuals. The numbers of alleles of all loci was375, ranged from2to21, averaging7.5294alleles per locus. The mean value of Ho,He and PIC was0.5106,0.5794and0.5443respectively.13and27loci showed a highor medium polymorphic level, with a total ratio of78.43%. Most of the loci conformedto Hardy-Weinberg equilibrium after Bonferroni correction except five loci. The resultshowed that most of the microsatellite markers we isolated had a high level ofpolymorphism, and will facilitate studies of genetic analysis of S. schlegelii.2The genetic structure evaluation of five wild populationsWe selected17primers to analysis the genetic structure of five wild populationsof S. schlegelii. Alleles were separated by high-resolution capillary electrophoresistechnique. The loci yielded an average of14.353to14.8242alleles per locus in onepopulation, the mean value of observed, expected heterozygosity and polymorphicinformation content ranged from0.785to0.793,0.824to0.841and0.791to0.808,respectively, and no significant difference observed between the populations. Theresult indicates that these populations have a high level of genetic diversity andselective breeding potential. The inbreeding coefficient (Fis) showed that Muping,Changdao and Rongcheng population had a varying degrees of inbreeding in one lociand Qingdao population in three loci (P<0.05), the mean Fis ranged from0.025to0.065in one population, Rongcheng population showed the mimimum value andQingdao the largest. Genetic differentiation index (Fst) ranged from-0.0027to0.0064,the differentiation of Rizhao and Rongcheng population was minimum. There’s a verysignificant differentiation between Qingdao and Changdao population and a significantdifferentiation between Qingdao and the other three populations, but the differentiationlevel revealed to be low because the index was less than0.05. The genetic similarityindex ranges from0.8844to0.9350and the genetic distance ranges from0.0672to 0.1229, the genetic distance between Qingdao and the other four populations revealedlarger than the value between the four populations, and the genetic similarity indexshowed an opposite trend. Rongcheng and Rizhao populations possessed the largestgenetic similarity index and the mimimum genetic distance value. UPGMA clusteranalysis tree based on Nei’s genetic distance indicated that Rizhao and Rongchengpopulations were first clustered, then clustered with Changdao population followed byMuping population, Qingdao population clustered last. The result indicated that S.schlegelii populations of Shandong peninsula had no significant differentiationgenetically, and could be regarded as a pangamy population. Mild differentiation andinbreeding appeared in Qingdao population might be caused by relative closing of thesampling site.These results indicated that these polymorphic microsatellite markers would beuseful for the studies of population structure analysis, genetic linkage mapping, QTLanalysis and other relevant research in S. schlegelii. The information of populationstructure analysis would provide basic data for resources conservation and selectivebreeding of S. schlegelii.