High-throughput Transcriptome Library Sequencing And Molecular Marker Development In The Clam Saxidomus Purpuratus

Saxidomus purpuratus,a marine bivalve mollusk,mainly distributes relatively restricted areas around China,Japan and Korea.Due to its high nutritional and economic value,it makes it an important economic seafood.In recent years,the natural resources of Saxidomus purpuratus have been declining due to overfishing and environmental degradation.In order to protect the wild population,it is necessary to develop and utilize molecular markers for genetic analysis of the wild population of Saxidomus purpuratus.In this study,we used the Illumina Hiseq 2500 sequencing platform to carry out transcriptome library sequencing of the gills of Saxidomus purpuratus,aiming at obtaining transcriptome data for the development of molecular markers,which laid the foundation for genetic diversity analysis and molecular marker assisted breeding of this species.In the sequencing report,we obtained 100,480,000 raw reads.After removing inferior sequences,adaptor sequences and rRNA,68,080,636 clean reads were obtained,5,115,494 assembled contigs were generated from these clean reads.These contigs were assembled into 120,479 transcripts and 66,388 unigenes.A total of 26,781 unigenes were annotated by hitting against the NCBI database.A total of 414 microsatellite sequences were obtained from the 66,388 unigenes of the transcriptome database using bioinformatics software.Amongtheserepeat motifs,trinucleotide repeatsequences were 217,followed by 135 dinucleotide and 58 tetranucleotide repeatsequences,accounting for 52.4%,32.6% and 14% of the total number of microsatellite sequences.35 primers were randomly selected,and of which 26 were used to amplify the target product.Polymorphisms of microsatellites were validated among 30 individuals,of which 13 were polymorphic.The alleles ranged from 3 to 6 with an average of 4.69 alleles per locus,and the observed and expected heterozygosity ranged from 0.4667 to 0.6667 and from 0.5847 to 0.8322,respectively.No significant linkage disequilibrium(LD)between pairs of loci was found and 2 loci significantly deviated from the Hardy-Weinberg equilibrium(HWE).The transcriptome sequencing platform demonstrated the ability to rapidly develop molecular resources in this species.These new polymorphic microsatellite markers provide basic information for further population studies and conservation genetics.