RESEARCH ARTICLE Construction, De-Novo Assembly and Analysis of Transcriptome for Identification of Reproduction-Related Genes and Pathways from Rohu, Labeo rohita (original) (raw)
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PLOS ONE, 2015
Rohu is a leading candidate species for freshwater aquaculture in SouthEast Asia. Unlike common carp the monsoon breeding habit of rohu restricts its seed production beyond season indicating strong genetic control over spawning. Genetic information is limited in this regard. The problem is exacerbated by the lack of genomic-resources. We identified 182 reproduction-related genes previously by Sanger-sequencing which were less to address the issue of seasonal spawning behaviour of this important carp. Therefore, the present work was taken up to generate transcriptome profile by mRNAseq. 16GB, 72bp paired end (PE) data was generated from the pooled-RNA of twelve-tissues from pre-spawning rohu using IlluminaGA-II-platform. There were 64.97 million high-quality reads producing 62,283 contigs and 88,612 numbers of transcripts using velvet and oases programs, respectively. Gene ontology annotation identified 940 reproduction-related genes consisting of 184 mainly associated with reproduction, 223 related to hormone-activity and receptor-binding, 178 receptoractivity and 355 embryonic-development related-proteins. The important reproduction-relevant pathways found in KEGG analysis were GnRH-signaling, oocyte-meiosis, steroid-biosynthesis, steroid-hormone biosynthesis, progesterone-mediated oocyte-maturation, retinolmetabolism, neuroactive-ligand-receptor interaction, neurotrophin-signaling and phototransduction. Twenty nine simple sequence repeat containing sequences were also found out of which 12 repeat loci were polymorphic with mean expected-&-observed heterozygosity of 0.471 and 0.983 respectively. Quantitative RT-PCR analyses of 13-known and 6unknown transcripts revealed differences in expression level between preparatory and postspawning phase. These transcriptomic sequences have significantly increased the genetic-&-genomic resources for reproduction-research in Labeo rohita.
Gene, 2013
Labeo rohita (Ham.) also called rohu is the most important freshwater aquaculture species on the Indian sub continent. Monsoon dependent breeding restricts its seed production beyond season indicating a strong genetic control about which very limited information is available. Additionally, few genomic resources are publicly available for this species. Here we sought to identify reproduction-relevant genes from normalized cDNA libraries of the brain-pituitary-gonad-liver (BPGL-axis) tissues of adult L. rohita collected during post preparatory phase. 6161 random clones sequenced (Sanger-based) from these libraries produced 4642 (75.34%) high-quality sequences. They were assembled into 3631 (78.22%) unique sequences composed of 709 contigs and 2922 singletons. A total of 182 unique sequences were found to be associated with reproduction-related genes, mainly under the GO term categories of reproduction, neuro-peptide hormone activity, hormone and receptor binding, receptor activity, signal transduction, embryonic development, cell-cell signaling, cell death and anti-apoptosis process. Several important reproduction-related genes reported here for the first time in L. rohita are zona pellucida sperm-binding protein 3, aquaporin-12, spermine oxidase, sperm associated antigen 7, testis expressed 261, progesterone receptor membrane component, Neuropeptide Y and Pro-opiomelanocortin. Quantitative RT-PCR-based analyses of 8 known and 8 unknown transcripts during preparatory and post-spawning phase showed increased expression level of most of the transcripts during preparatory phase (except Neuropeptide Y) in comparison to post-spawning phase indicating possible roles in initiation of gonad maturation. Expression of unknown transcripts was also found in prolific breeder common carp and tilapia, but levels of expression were much higher in seasonal breeder rohu. 3631 unique sequences contained 236 (6.49%) putative microsatellites with the AG (28.16%) repeat as the most frequent motif. Twenty loci showed polymorphism in 36 unrelated individuals with allele frequency ranging from 2 to 7 per locus. The observed heterozygosity ranged from 0.096 to 0.774 whereas the expected heterozygosity ranged from 0.109 to 0.801. Identification of 182 important reproduction-related genes and expression pattern of 16 transcripts in preparatory and post-spawning phase along with 20 polymorphic EST-SSRs should be highly useful for the future reproductive molecular studies and selection program in Labeo rohita.
Computational analysis of transcriptome of Indian major carp, Labeo rohita (Hamilton-Buchanan, 1822)
2012
A total of 1671 ESTs of Labeo rohita were retrieved from dbEST database and analysed for functional annotation using various computational approaches. The result indicated 1387 non-redundant (184 contigs and 1203 singletons) putative transcripts with an average length of 542 bp. These 1387 transcript sequences were matched with Refseq_RNA, UniGene and Swiss-Prot on high threshold cutoff for functional annotation along with help of gene ontology and SSRs markers. We developed extensive Perl programming based modules for processing all alignment files, comparing and extracting common hits from all files on a threshold, evaluating statistics for alignment results and assigning gene ontology terms. In this study, 92 putative transcripts predicted as orthologous genes and among those, 44 putative transcripts were annotated with gene ontology terms. The annotated orthologous gene of our result associated with some very important proteins of L. rohita involved in biotic and abiotic stresses and glucose metabolism of spermatogenic cells etc. The unidentified transcripts, if found important in expression profiling can be vital resource after re-sequencing. The predicted genes can further be used for enhancing productivity and controlling disease of L. rohita.
Bioinformation, 2012
A total of 1671 ESTs of Labeo rohita were retrieved from dbEST database and analysed for functional annotation using various computational approaches. The result indicated 1387 non-redundant (184 contigs and 1203 singletons) putative transcripts with an average length of 542 bp. These 1387 transcript sequences were matched with Refseq_RNA, UniGene and Swiss-Prot on high threshold cut-off for functional annotation along with help of gene ontology and SSRs markers. We developed extensive Perl programming based modules for processing all alignment files, comparing and extracting common hits from all files on a threshold, evaluating statistics for alignment results and assigning gene ontology terms. In this study, 92 putative transcripts predicted as orthologous genes and among those, 44 putative transcripts were annotated with gene ontology terms. The annotated orthologous gene of our result associated with some very important proteins of L. rohita involved in biotic and abiotic stresses and glucose metabolism of spermatogenic cells etc. The unidentified transcripts, if found important in expression profiling can be vital resource after re-sequencing. The predicted genes can further be used for enhancing productivity and controlling disease of L. rohita.
Reviews in Aquaculture, 2020
Farmed rohu, Labeo rohita, is an economically important freshwater aquaculture species in south Asia. Consumer preference for this species has driven researchers to develop genetically improved rohu (Jayanti) for important traits such as growth and disease resistance. The feeding and breeding biology of rohu is wellstudied, but the physiological responses to stress or climate change at the molecular level are less understood. We summarized the genetic and advanced molecular tools used for biological insights into farmed rohu. Recently, next-generation sequencing technology (NGS) and genome informatics have helped to identify biological questions in fish using genome, transcriptome, metagenome, proteome and epigenome sequencing. We briefly reviewed past rohu research using traditional selective breeding methods for genetic improvement, as well as the other molecular tools that have been utilized to understand the fish's physiology, health, nutrition, stress and reproduction. We discuss the molecular techniques used in rohu, such as gene cloning and expression profiling, DNA markers, NGS, metagenomics, epigenomics, proteomics, cell culture/cell line development and transgenics. Finally, we address the future perspectives of rohu research, which may be driven by NGS and other advanced techniques like genome editing to improve rohu aquaculture production. The comprehensive information presented here will provide insights for the carp aquaculture research community.
General and Comparative Endocrinology, 2018
Common carp (Cyprinus carpio) is a worldwide freshwater fish of eutrophic waters. C. carpio, have various reproductive traits, including early sexual maturity, that may make them excellent, large, realistic, aquaculture model species. In the present work, de novo assembly of gonadal (testicular and ovarian) transcriptomes from juvenile common carp was performed to identify genes involved in gonadal development. A total of 81,757 and 43,257 transcripts with average lengths of 769 and 856 bp, were obtained from the immature testicular and ovarian transcriptomes, respectively. About 84,367 unigenes were constructed after removing redundancy involving representation of transcripts in both gonadal transcriptomes. Gene ontology (39,171 unigenes), clusters of orthologous group's analysis (6,651 unigenes) and Kyoto encyclopedia of genes, and genomes automatic annotation server analysis (4,783 unigenes) were performed to identify potential genes along with their functions. Furthermore, 18,342 (testis) and 8,693 (ovary) simple sequence repeats were identified. About 298 differentially expressed genes were identified, of which 171 and 127 genes were up-regulated in testis and ovary, respectively. Quantitative realtime reverse transcription PCR was performed to validate differential expression of selected genes in testis and ovary. Nearly 809 genes related to reproduction were identified, sex-wise expression pattern of genes related to steroid synthesis, endocrine regulation, germ cell maintenance and others factors related to gonadal differentiation was observed, and expression analysis of nanos, ad4bp/sf-1, and gdf9 was performed. The present study identified certain important genes/factors involved in the gonadal development of C. carpio which may provide insights into the understanding of sex-differentiation and gonadal development processes.
Expressed Sequences and Polymorphisms in Rohu Carp (Labeo rohita, Hamilton) Revealed by mRNA-seq
Marine Biotechnology, 2012
Expressed genes and polymorphisms were identified in lines of rohu Labeo rohita selected for resistance or susceptibility to Aeromonas hydrophila, an important bacterial pathogen causing aeromoniasis. All animals were grown in a common environment and RNA from ten individuals from each line pooled for Illumina mRNA-seq. De novo transcriptome assembly produced 137,629 contigs with 40× average coverage. Forty-four percent of the assembled sequences were annotated with gene names and ontology terms. Of these, 3,419 were assigned biological process terms related to "stress response" and 1,939 "immune system". Twenty-six contigs containing 38 single nucleotide polymorphisms (SNPs) were found to map to the Cyprinus carpio mitochondrial genome and over 26,000 putative SNPs and 1,700 microsatellite loci were detected. Seventeen percent of the 100 transcripts with coverage data most indicative of higher-fold expression (>5.6 fold) in the resistant line pool showed homology to major histocompatibility (MH), heat shock proteins (HSP) 30, 70 and 90, glycoproteins or serum lectin genes with putative functions affecting immune response. Forty-one percent of these 100 transcripts showed no or low homology to known genes. Of the SNPs identified, 96 showing the highest allele frequency differences between susceptible and Electronic supplementary material The online version of this article
BMC Genomics
Background Carp fish, rohu (Labeo rohita Ham.) is important freshwater aquaculture species of South-East Asia having seasonal reproductive rhythm. There is no holistic study at transcriptome level revealing key candidate genes involved in such circannual rhythm regulated by biological clock genes (BCGs). Seasonality manifestation has two contrasting phases of reproduction, i.e., post-spawning resting and initiation of gonadal activity appropriate for revealing the associated candidate genes. It can be deciphered by RNA sequencing of tissues involved in BPGL (Brain-Pituitary-Gonad-Liver) axis controlling seasonality. How far such BCGs of this fish are evolutionarily conserved across different phyla is unknown. Such study can be of further use to enhance fish productivity as seasonality restricts seed production beyond monsoon season. Result A total of ~ 150 Gb of transcriptomic data of four tissues viz., BPGL were generated using Illumina TruSeq. De-novo assembled BPGL tissues reveal...
Frontiers in Genetics, 2023
In most of the aquaculture selection programs, harvest body weight has been a preferred performance trait for improvement. Molecular interplay of genes linked to higher body weight is not elucidated in major carp species. The genetically improved rohu carp with 18% average genetic gain per generation with respect to harvest body weight is a promising candidate for studying genes' underlying performance traits. In the present study, muscle transcriptome sequencing of two groups of individuals, with significant difference in breeding value, belonging to the tenth generation of rohu carp was performed using the Illumina HiSeq 2000 platform. A total of 178 million paired-end raw reads were generated to give rise to 173 million reads after quality control and trimming. The genomeguided transcriptome assembly and differential gene expression produced 11,86,119 transcripts and 451 upregulated and 181 downregulated differentially expressed genes (DEGs) between high-breeding value and low-breeding value (HB & LB) groups, respectively. Similarly, 39,158 high-quality coding SNPs were identified with the Ts/Tv ratio of 1.23. Out of a total of 17 qPCR-validated transcripts, eight were associated with cellular growth and proliferation and harbored 13 SNPs. The gene expression pattern was observed to be positively correlated with RNA-seq data for genes such as myogenic factor 6, titin isoform X11, IGF-1 like, acetyl-CoA, and thyroid receptor hormone beta. A total of 26 miRNA target interactions were also identified to be associated with significant DETs (p-value < 0.05). Genes such as Myo6, IGF-1-like, and acetyl-CoA linked to higher harvest body weight may serve as candidate genes in markerassisted breeding and SNP array construction for genome-wide association studies and genomic selection.