Isolation Of Microsatellite Markers And Population Genetic Diversity Analysis In Mastacembelus Armatus

The Zig-zag eel Mastacembelus armatus is an important freshwater fish that widely distributes in south China.M.armatus belongs to the family of Mastacembelidae,order of Symbranchiformes.As M.armatus is an economic fish that faced with the challenges resulting from overfishing,water pollution,and habitat destruction,the research of population genetics for M.armatus is imperative.Study on genetic diversity of fish species is the basis for the adaptation to the environment and the maintenance of potential for evolution.In this study,we aimed to characterize genetic diversity and population genetic structure of M.armatus populations using polymorphic microsatellite loci and EPIC marker.Results from this research can guide management of germplasm resource and conservation strategies for this species.The main studies are as follows::1.We developed and characterized 66 novel polymorphic microsatellite markers for M.armatus through the PCR isolation of microsatellite arrays(PIMA)and restriction-site associated DNA tags sequencing(RAD-seq).Six loci developed by PIMA method had an average polymorphic information content(PIC)value of 0.234.The average number of alleles was 2.333,the average observed and expected heterozygosity were 0.139 and 0.0.272,respectively.Four loci was found to deviate from HWE(P<0.05)and no linkage disequilibrium was found between loci.Number of alleles of 60 pairs of primers developed by RAD-seq method was ranged from 1 to23 per locus,with 6.383 alleles per locus on average.The average expected heterozygosity per locus were 0.602 and 0.760.Twenty six loci deviated significantly from Hardy-Weinberg equilibrium after the correction for Bonferroni correction.2.We used 10 microsatellite markers and represented a population genetic analysis of 10 M.armatus wild populations collected from south China.Totally 253 alleles were obtained from the 10 microsatellite loci in 10 M.armatus populations.The observed and expected heterozygosity were ranged from 0.536 to 0.913 and from0.452 to 0.799,respectively.Mean allele numbers at the 10 loci ranged from3.3(Xishuangbanna)to 11.1(Lechang).The polymorphism information content(PIC)ranged from 0.383(Xishuangbanna)to 0.691(Enping).Deviations from Hardy-Weinberg equilibrium were detected in some loci after the Bonferroni correction.The Fst values ranged from 0.060 to 0.393 which estimated in AMOVA indicated significant genetic differentiation among populations,the genetic variation mainly from individuals within populations(75 %).Structure analysis showed that the10 populations were grouped into five clusters.The UPGMA tree showed that the 10 populations clustered into five groups,Xishuangbanna population showed clear divergence to the other 9 populations.Bottleneck effect test indicated that Chaozhou,Wuhua and Baise had suffer bottleneck effect in recent time.3.We invested the genetic diversity of M.armatus using EPIC marker,We obtained 266 variable sites of 1090 base pairs,including 189 singleton variable sites and 77 parsimony informative sites.The nucleotide diversity was highest in Hechi(0.00579),Lechang(0.00544)and Enping(0.00548)and lowest in Xishuangbanna(0.00265)and Baise(0.00243).One hundred and forty haplotypes were identified,with 12 haplotypes shared among populations.The pattern of genetic differentiation among populations was highly significant with an overall Fst mean of0.210.The average genetic distance of 10 populations was 0.0013.Xishuangbanna significantly differentiated with the other 9 populations.The results of haplotype network diagram indicated that some haplotypes from the same or neighboring locations grouped together.The phylogenetic tree showed that Hechi,Baise and Lechang were allocated into one cluster,then grouped together with Enping.Xishuangbanna and Baisha diverged and could form another relatively independent branch,respectively.Neutral test and mismatch analysis indicated that Longyan,Zengcheng and Wuhua had undergone a recent population expansion.4.The universal applicability of 60 microsatellite markers of M.armatus were tested in seven species,15 loci were found to be applicable in Monopterus albus,and13 loci were applicable in Macrognathus aculeatus.These results indicated that the universal rate of M.armatus microsatellite markers in Synbranchiformes was higherthan in Cypriniformes.Loci Mat01 were applicable in seven fishes,and Mad22,Mad27,Mat09 were be applicable in five fishes.The amplification rate in dinucleotide was significantly higher than trinucleotide and tetranucleotide.