Isolation Of Microsatellite Markers And Population Genetic Diversity Analysis In Solenaia Oleivora

Solenaia oleivora Heude (Bivalvia: Unionidae) is an endemic rare freshwater mussel in China. It is delicious and nutritious because of its large axe foot. But it has suffered from over-fishing and excessive exploitation. In recent years, the natural resources of Solenaia oleivora were dramatically declined, it has become a rare threatened or endangered unionid mussel in China, and it is urgent to find the major threats and protect natural resources of this species. In this study, we used the magnetic beads enriched method to isolate microsatellite loci from the genome of Solenaia oleivora, and evaluated population genetic diversity of three wild Solenaia oleivora populations by using the isolated microsatellite loci. In addition, morphology methods were used to compare the morphological variation among three populations. Our purpose is to investigate the population genetic structure of this species through phenotypic traits and molecular characters. The results will contribute to effective conservation and rational utilization of natural resources of this species.1. The morphological analysis of Solenaia oleivoraTotally,244individuals were collected from Tianmen River and Jingzhou in Hubei Province, and from Funan in Anhui Province. According to the principal component analysis (PCA) of the characters, three principal components were extracted and the related contribution ratios were45.97%,25.62%and10.32%, respectively, with the cumulative contribution ratio of81.92%. Discriminant analysis showed the discriminant accuracy in three populations (Tianmen, Funan and Jingzhou) was78.95%,93.02%, and74.60%, with the comprehensive discrimination rate was82.79%.The cluster analysis indicated that the Tianmen population and Jingzhou population get together first, then they cluster with Funan population.2. The development and evaluation of microsatellite markers of Solenaia oleivoraFifteen pairs of dinucleotide micro satellites and17pairs of tri-and tetranucleotide microsatellites were developed using magnetic beads enriched method. Firstly, using repeat-enrichment method with biotin-labeled oligo (CA)12,(AAC)8and (GATA)4streptavidin magnetic beads, then selecting475clones screened by PCR randomly. Sequencing analysis found107clones containing microsatellite repeat units. The repeats within the microsatellite core sequence are7to27times, and most of them are more than10times. Thirteen two pairs of designed primers were effective for PCR amplification and showed polymorphism as tested in a population (n=46). The PIC values for the microsatellite loci ranged from0.250to0.892with an average of0.646. The number of alleles ranged from2to15with an average of7. The observed heterozygosity ranged from0.357to0.929with an average of0.625, and the expected heterozygosity ranged from0.293to0.880with an average of0.726.3. Population genetic diversity and genetic structure as revealed by microsatellite DNAFifteen microsatellite loci were selected to analyze genetic diversity and genetic structure of the three wild populations. The genotypic linkage disequilibrium and conformation to Hardy-Weinberg equilibrium were tested, and some genetic diversity indices (number of alleles, effective number of alleles, heterozygosity, polymorphism information content) were calculated. The results showed that the average number of alleles was from5.20to7.47in each population, the average effective number of alleles was between3.04and4.93. The average observed heterozygosity and expected heterozygosity were from0.501to0.620and from0.598to0.701in each population. The overall polymorphism of the three populations was high with the PIC value ranging from0.547to0.604(PIC>0.5). AMOVA analysis showed that there was significant genetic differentiation among three populations (Fst=0.094), and pairwise fixed index (Fst) between populations was from0.1733to0.2240(P<0.01). The genetic distances was from0.1149to0.1965among three populations. The results showed that the genetic diversity was the highest in the Funan populatin, and the lowest in the Jingzhou population. There were significant genetic differentiation among the three populations, the Tianmen and Jingzhou populations have closer genetic relationship.