Transcriptome Characterization And Genetic Map Construction For Apostichopus Japonicus

1. Determination of genome size of Apostichopus japonicusThe C-values of four different tissues (body wall, muscle, intestines and respiratorytrees) of A. japonicus were measured in a flow cytometer using Chlamys farreri as aninternal standard. It was noted that the C-values of different tissues were closebetween one another. The results indicate that the C-value of A. japonicus is0.878±0.02pg with the genome size of859Mb. The results of this study will be helpful infuture genomic study of the sea cucumber.2. Transcriptome sequencing and characterization for the sea cucumber A.japonicusWe performed the large-scale transcriptome profiling and characterization bypyrosequencing diverse cDNA libraries from A. japonicus. In total,1,061,078readswith an average length of344bp were obtained by454sequencing of eight cDNAlibraries representing different developmental stages and adult tissues of A. japonicus.Of all these reads,92%(974,004reads) passed through our quality filters andrepresented high-quality (HQ) reads. The HQ reads were assembled into33,835contigs and199,011singletons. Contigs were then assembled into29,666isotigs,which were further clustered into21,071isogroups. About47%of the isogroupsshowed significant matches to known proteins or nucleotide based on sequencesimilarity. As determined by Gene ontology (GO) annotation and Kyoto Encyclopediaof Genes and Genomes (KEGG) pathway mapping, functional annotation of theunigenes recovered diverse biological functions and processes. Candidate genes that were potentially involved in aestivation were identified. Twelve candidate DGEs wererandomly selected for Q-PCR validation. Q-PCR results verified the differentialexpression of these genes between the active and aestivating sea cucumbers.Transcriptome comparison with the sea urchin Strongylocentrotus purpuratusrevealed similar patterns of GO term representation. In addition,4,882putativeorthologous genes were identified, of which202were not present in thenon-echinoderm organisms. More than700simple sequence repeats (SSRs) and54,000single nucleotide polymorphisms (SNPs) were detected in the A. japonicustranscriptome.This transcriptome resource would lay an important foundation forfuture genetic or genomic studies on this species.3. Construction of SNP-based linkage map for A. japonicusLinkage mapping was performed using an F1family of90A. japonicus individuals,and a genetic linkage map was constructed using a2b-RAD method. Two thousandsand ten SNP markers were genotyped and1,484markers that conformed to theexpected Mendelian ratios (P≥0.05) were included in the linkage analysis. The geneticlinkage map contained917markers ordered on22linkage groups and spanned1,467cM, with an average intermarker spacing of1.64cM and94.89%of genome coverage.The size of the linkage groups ranged from13.5cM to113.8cM, and the number ofmarkers per linkage group varied from16to98, with an average number of42. Thisis the first SNP-based linkage map of A. japonicus and will be useful for furthergenetic analyses such as gene mapping and molecular marker-assisted selection(MAS).