Genetic Diversity Of Oratosqilla Oratoria Based On The Mitochondrial COI Gene Sequences Analysis And Isolation Of Microsatellite

Oratosquilla oratoria (Crustacea: Squillidae) is the most dominant species in the indo-pacific ocean,which has been regarded as the the commercially valuable species for its good taste and rich nutrition infishery.Four populations of O.oratoria were collected from Pikou, Suizhong, Qingdao and Guangzhou sea watersof China. This study analyzed the genetic diversity and phylogenetic relationship by amplifying andsequencing the mitochondrial cytochrome oxidase subunit I (CO I) gene of O.oratoria. A total number of54variable sites (9.2%) and35haplotypes were detected in585bp sites for100CO I sequences. This paper madean analysis of CO I sequences and indicated that the A+T content(64.8%) was higher than that of C+G(35.2%),which was corresponding with the characteristics of studies on Arthropods’s mitochondrial CO I gene. Theratio of TV and TS was7.84which indicated that the base substitution of CO I gene was not satured in O.oratoria. The average values of haplotype and nucleotide diversity were0.9616and0.02027. The analysis ofhaplotype and nucleotide diversity in four different populations of O.oratoria showed a middle geneticdiversity, but the genetic diversity level of Guangzhou is the highest. AMOVA analysis showed that thegenetic difference among populations (84.53%) was significantly higher than that within populations (15.47%).The fix index (Fst) across all populations revealed that there were no genetic differentiations among Pikou,Suizhong and Qingdao sea waters(Fst＜0), but Guangzhou has a high level.19homologous sequences ofO.oratoria of the eastern coast of Guangdong province was download from GenBank, together with thesequences obtained in this study, reconstructed the Neighbor-joining tree. The result revealed that fivepopulations could be divided into two groups, one group included Pikou, Suizhong and Qingdao populationsand the other group included Guangzhou and the east coast of Guangdong province. This study had made thehaplotype phylogenetic tree and haplotype network of35haplotypes which all revealed the big geneticdifferentiation of O.oratoria in Guangzhou seawaters.This study analyze the genetic variation and the geneticstructure of four O.oratoria populations and the results obtained in this study could provide a molecularbiology basis for protection and reasonable utilization of germplasm resources of O.oratoria in the coastalareas of China.To study the genetic diversity of O.oratoria without information of the whole genome, the SSR primerswere designed based on the transcriptome sequencing data (unpublished) through high-throughput sequencingof O.oratoria. The SSR loci were analyzed98818unigene sequences, and we screened out19050SSR lociwhich distributed in13309unigene sequences, accounting for2.9%of transcriptome unigene sequences,among which has3563unigene sequences contain2or more than2SSR loci. This study selected di-nucleotidewhich replicated more than14times, tri-nucleotide replicated more than7times, tetra-nucleotide replicatedmore than6times, penta-nucleotide and hexa-nucleotide replicated more than5times as well as compoundrepeat motifs to design the primer by Primer-Blast on NCBI according to the conservative sequence at theends of microsatellite sequences. The primers were identified with PCR, polyacrylamide gel electrophoresisusing the genomic DNA extraction of O.oratoria which were collected from Pikou, Suizhong, Qingdao andGuangzhou sea waters, only10primers were amplified clear and polymorphism bands at last. This study hadan important application value to analyze genetic diversity, phylogeny and maker assisted selection.