Population Genetics Analyses Of Xenocypris Microlepis Based On Mitochondrial COI And Microsatellite Markers

Xenocypris microlepis is a fresh water fish in Cyprinidae family mainly distributing in the main river basin and lakes of China. In this study, one thousand five hundred and fifty-one bp mitochondrial Cytochrome oxidase subunit I (COI) gene sequences of Xenocypris microlepis was sequenced, and genetic structure of seven X. microlepis populations in Qiantang River and Caoe River were analyzed using mitochondrial COI gene sequence analysis method. And then microsatellites developed are used for analyzing the genetic diversity and genetic structure of the seven populations. ISSR-suppression PCR method is involved in selecting SSR markers.1,Sequencing and analysis of mitochondrial COI gene of X. microlepisFirst of all, degenerate primers were designed for polymerase chain reaction with Genomic DNA. And then PCR products were purified and sequenced. Finally 1551 bp full-length of COI gene sequence was obtained with start codon GTG and termination codon TAA. Homology comparison ananlyzed by BLAST showed that the similarities of the mitochondrial COI gene between X. microlepis and X. argentea, X. davidi and Distoechodon tumirostris were 94%, 94% and 92% respectively, with higher homologous. Genetic variation analyzed by MEGA4.0. And according to genetic distance, phylogenetic tree is constucted by neighbor-joining method (Neighbor-Joining, NJ), with the mitochondrial COI gene sequence of black carp as foreign groups and Kimura-2-parameter model based. The result comes out that X. argentea, X. davidi together as a team and then is clustered with X. microlepis, but relations with D. tumirostris relatively distant.2,Genetic structure in seven X. microlepis populations using mitochondrial DNA COI gene maker According to obtained COI gene sequence of full length, primer was designed to amplify all samples'genome DNA for analysis of genetic structure of seven X. microlepis populations (Fenkou, Jiangjiazhen, Fuwen, Linqi, Fenshui, Shangyu, Shendu). Five hundred and sixty base pairs partial COI gene fragments of 131 samples were sequenced and compared. The results shows that haplotype diversities (h) in seven populations of Xenocypris microlepis were 0.5000, 0, 0.1425, 0.1287, 0.3556, 0.5333 and 0.1732 respectively, and the nucleotide diversities (π) were 0.0009, 0, 0.0002, 0.0002, 0.0006, 0.0010 and 0.0003 respectively. Genetic structure analysis showed a higher level genetic diversity of Fenshui population than other three populations. AMOVA analysis disclosed that variation among populations accounting for 13% of total variation while within populations accounting for 87%, suggesting that within population variations are the main sources of total variation. This result suggests that the genetic divergence among the seven populations at a low level.3,Isolation and characterization of microsatellites for Xenocypris microlepisISSR-supression PCR is a simple method to isolate microsatellites markers compared with traditional labor-intensive, time-consuming and expensive method. The method only involves few simple steps to develop microsatellite markers without constructing and screening genomic libraries. In this study, five polymorphic microsatellite DNA markers were developed for Xenocypris microlepis using inter-simple sequence repeat cloning without constructing an enriched genomic library. And their amplification characteristics were described. The number of alleles ranged from 2 to 4. The expected and observed heterozygosities ranged from 0.1671 to 0.7409 and 0.1679 to 0.7252, respectively. No loci that deviated significantly from Hardy–Weinberg equilibrium (P>0.05) were found. These microsatellite markers could be useful in the population genetic study of Xenocypris microlepis. Population genetics analysis shows that every genetic identity between the two groups are close to 1, and genetic distances are between 0.0500 to 0.1910. According to genetic distance, phylogenetic trees were constructed by unweighted pair-group method with arithmetic means (UPGMA) method. The results came out that FK and JJZ, LQ and FW four groups are clustered together as a team, and SY and SD two groups are clustered together as a team. The result is partly in line with the geographical distance relationship.