High-throughput Transcriptome Library Sequencing And Molecular Marker Development In The Scallop Crassadoma Gigantea

Rock scallop(Crassadoma gigantea),which belongs to Mollusca,Lamellibranchia,Pterioida,was native to North America Pacific coast.There are several valuable characteristics making C.gigantea favored in the international market,including fast growth,strong resistance and delicious meat.The C.gigantea was introduced to China to solve the problem of lacking varieties,low production and High mortality in scallop aquaculture.To date,the studies on C.gigantea are mainly focused on physiology,ecology,production and aquaculture,while the genetics background was rarely reported.In this study,high-throughput transcriptome sequencing method and RNA-Seq technique were used to sequence and analyze the DNA mixture of the mantle,viscera,gills and the adductor muscle of scallop,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.1.We used the Illumina to obtained 80,832,518 raw reads,after removing inferior sequences,adaptor sequences and rRNA,77,306,198(95.64%)clean reads were obtained.These contigs were assembled into 158,855 transcripts and 96007 unigenes.A total of 24795 microsatellite sequences were obtained from the 19182 unigenes of the transcriptome database using bioinformatics software.The number of sequences containing more than 1 SSR is 4148,21.6%.Among these repeat motifs,trinucleotide repeat sequences were 5602,followed by 1924 dinucleotide and 186 tetranucleotide repeat sequences,accounting for 22.6%,7.8% and 0.8% of the total number of microsatellite sequences.97 primers were randomly selected,and of which 42 were used to amplify the target product.Polymorphisms of microsatellites were validated among 30 individuals,of which 12 were polymorphic.The alleles ranged from 3 to 9 with an average of 3.25 alleles per locus,and the observed and expected heterozygosity ranged from 0.033 to 0.923 and from 0.254 to 0.820,respectively.Two loci significantly deviated from the Hardy-Weinberg equilibrium(HWE)(P < 0.01).2.In this paper,the mitochondrial DNA 16 s rRNA and COI gene fragments of scallop were preliminarily studied by PCR amplification and sequencing techniques.The size of 16 s rRNA gene fragment was found to be between 613bp-634 bp.The average content of A,T,G,C was 26.5%,29.7%,24.5% and 19.3%,respectively.The size of COI gene fragment was 660 bp.The average content of A,T,G,C was 17%,40%,27% and 15%.16 s in rock scallop,respectively.The codon with U or G end was amplified in rock scallop,and the total usage was 12.76 and 8.02,respectively.The synonymous codon usage of GUU is more than 2,which is the most frequently used line of 16 s in rock scallop.The phylogenetic tree shows that the closest phylogenetic relationship with the rock scallop is Azumapecten farreri and the next is the Chlamys islandica based on COI analysis.The phylogenetic analysis revealed the more close relationship of Chlamys andica is Patinopecten caurinusus,Mizuhopecten yessoensis,Chlamys andica from 16 s sequence.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.