Optimizing Hybrid de Novo Transcriptome Assembly and Extending Genomic Resources for Giant Freshwater Prawns (Macrobrachium rosenbergii): The Identification of Genes and Markers Associated with Reproduction

Abstract

The giant freshwater prawn, Macrobrachium rosenbergii, a sexually dimorphic decapod crustacean is currently the world’s most economically important cultured freshwater crustacean species. Despite its economic importance, there is currently a lack of genomic resources available for this species, and this has limited exploration of the molecular mechanisms that control the M. rosenbergii sex-differentiation system more widely in freshwater prawns. Here, we present the first hybrid transcriptome from M. rosenbergii applying RNA-Seq technologies directed at identifying genes that have potential functional roles in reproductive-related traits. A total of 13,733,210 combined raw reads (1720 Mbp) were obtained from Ion-Torrent PGM and 454 FLX. Bioinformatic analyses based on three state-of-the-art assemblers, the CLC Genomic Workbench, Trans-ABySS, and Trinity, that use single and multiple k-mer methods respectively, were used to analyse the data. The influence of multiple k-mers on assembly performance was assessed to gain insight into transcriptome assembly from short reads. After optimisation, de novo assembly resulted in 44,407 contigs with a mean length of 437 bp, and the assembled transcripts were further functionally annotated to detect single nucleotide polymorphisms and simple sequence repeat motifs. Gene expression analysis was also used to compare expression patterns from ovary and testis tissue libraries to identify genes with potential roles in reproduction and sex differentiation. The large transcript set assembled here represents the most comprehensive set of transcriptomic resources ever developed for reproduction traits in M. rosenbergii, and the large number of genetic markers predicted should constitute an invaluable resource for future genetic research studies on M. rosenbergii and can be applied more widely on other freshwater prawn species in the genus Macrobrachium.

Comparative Transcriptome Analysis of Gonads for the Identification of Sex-Related Genes in Giant Freshwater Prawns (Macrobrachium Rosenbergii) Using RNA Sequencing

“…In M. rosenbergii, See et al have developed microsatellite markers to evaluate genetic diversity [72]. Using the high throughput sequencing, it is feasible to detect and discover the large numbers of SSRs [37,73]. Correspondingly, the SSRs identified in the present study could serve as genetic markers for further QTL mapping and marker-assisted selection (MAS) in M. rosenbergii.…”

“…Mating of sex-reversed females (neo-males) with normal females (ZW) could increase a higher ratio of females, while mating of sex-reversed males (neo-females) with normal males could produce all-male (ZZ × ZZ) progeny [1,6,7]. Recently, along with the emergence of high-throughput sequencing, RNA-seq as a most powerful tool is available for illustrating the mechanisms of immune response [35], determining the expression pattern of reproduction, growth, and pheromone communication in hepatopancreas, gill, muscle, and antennal gland [36][37][38]. In the present study, we profiled to detect the transcriptomes of gonads in females, super females, and the males using RNA-seq, with the aim of interpreting the molecular mechanism involved in the sex determination, and identifying sex-related candidate genes.…”

Enrichment of genomic resources and identification of simple sequence repeats from medicinally important Clausena excavata

“…For annotation, the raw reads were filtered by removing the adapter sequences, empty reads, low-quality reads, and reads with less than a Phred quality score of 20 using NGS tool kits and Ion-Torrent server as described previously (Jung et al 2016). To increase gene identification efficiency, transcriptome de novo assembly was conducted using three assemblers, CLC Genomics Workbench (ver.…”

Guidelines for RNA-seq projects: applications and opportunities in non-model decapod crustacean species

“…The RNAseq platform is based on the analysis of the transcriptome -a small portion of the whole genome that is transcribed from chromosomal DNA into RNA molecules -a dynamic set of elements that change depending on developmental stages or physiological conditions. Also, by analysing the sequenced transcriptome, genetic polymorphisms including SNPs and SSRs can be mined and analysed with ease (Jaramillo et al 2016;Jin et al 2013;Jung et al 2011;Jung et al 2016;Lv et al 2014;Meng et al 2015;Nguyen et al 2016).…”