Identification of male gametogenesis expressed genes from the scallop Nodipecten subnodosus by suppressive subtraction hybridization and pyrosequencing.

Despite the great advances in sequencing technologies, genomic and transcriptomic information for marine non-model species with ecological, evolutionary, and economical interest is still scarce. In this work we aimed to identify genes expressed during spermatogenesis in the functional hermaphrodite scallop Nodipecten subnodosus (Mollusca: Bivalvia: Pectinidae), with the purpose of obtaining a panel of genes that would allow for the study of differentially transcribed genes between diploid and triploid scallops in the context of meiotic arrest and reproductive sterility. Because our aim was to isolate genes involved in meiosis and other testis maturation-related processes, we generated suppressive subtractive hybridization libraries of testis vs. inactive gonad. We obtained 352 and 177 ESTs by clone sequencing, and using pyrosequencing (454-Roche) we maximized the identified ESTs to 34,276 reads. A total of 1,153 genes from the testis library had a blastx hit and GO annotation, including genes specific for meiosis, spermatogenesis, sex-differentiation, and transposable elements. Some of the identified meiosis genes function in chromosome pairing (scp2, scp3), recombination and DNA repair (dmc1, rad51, ccnb1ip1/hei10), and meiotic checkpoints (rad1, hormad1, dtl/cdt2). Gene expression analyses in different gametogenic stages in both sexual regions of the gonad of meiosis genes confirmed that the expression was specific or increased towards the maturing testis. Spermatogenesis genes included known testis-specific ones (kelch-10, shippo1, adad1), with some of these known to be associated to sterility. Sex differentiation genes included one of the most conserved genes at the bottom of the sex-determination cascade (dmrt1). Transcript from transposable elements, reverse transcriptase, and transposases in this library evidenced that transposition is an active process during spermatogenesis in N. subnodosus. In relation to the inactive library, we identified 833 transcripts with functional annotation related to activation of the transcription and translation machinery, as well as to germline control and maintenance.

Identification of a tubulin-α gene specifically expressed in testis and adductor muscle during stable reference gene selection in the hermaphrodite gonad of the lion's paw scallop Nodipecten subnodosus.

For non-model species, as many used for aquaculture, with minimal or no genomic information, relative quantification of gene expression studies requires preliminary research including the isolation of potential reference genes and the identification of those stably expressed under the biological conditions of interest. Here we report on the isolation of five partial gene sequences from gonad tissue cDNA in the functional hermaphrodite scallop Nodipecten subnodosus to be evaluated as reference genes: 18S-rRNA, riboprotein l8 (rp-l8), actin-ß (act-ß), elongation factor 1α (ef-1α) and alpha-tubulin-α (tub-α). We found that 18S-rRNA was stably expressed independently of the priming method used to reverse transcribe RNA to cDNA, oligo-dT or random hexamer. Stability analysis for the five putative reference genes with geNorm and NormFinder indicated that 18S together with rp-l8 were the most stable genes for normalization of gene expression during gonad development in both, male and female sexual regions of the hermaphrodite N. subnodosus. The least stable gene was tub-α, showing a biased expression profile between sexual regions of the gonad, therefore this gene was analyzed thereafter as a target gene together with vitellogenin (vit) and a DEAD-box RNA helicase (dbx) gene. Relative expression, estimated by normalization with the combination of 18S and rp-l8 as reference genes, indicated that as gonad development advanced two of the target genes were up-regulated, tub-α in the male region and vit in the female region. Whereas an increased expression was expected during development for vit for its known role in vitellogenesis, the increased expression of tub-α in the male sexual region was unexpected, and pointed toward this gene being a testis-specific α-tubulin isotype. Further analyses of gene expression among tissues indicated that tub-α is specifically and highly expressed in the male gonad, although expression in adductor muscle was also observed at significantly lower levels. The existence of testis specific α- and ß-tubulins has been previously reported in other taxa, relating their function to sperm axoneme formation. Tissue-specific tubulin genes, particularly their promoters, have recently found an application as native promoters for transgene tissue-specific expression in research and reproductive control of insect plagues. The third target gene, a putative member of the DEAD-box RNA helicase family (dbx), showed no changes in expression during gonad development or between sexual regions, therefore it was chosen to discuss the different statistical inferences resulting from the arbitrary use of 'randomly chosen' reference genes when normalizing gene expression.