Diego Robledo

Analysis of qPCR reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (Scophthalmus maximus) gonad dataset

BackgroundGene expression analysis by reverse transcription quantitative PCR (qPCR) is the most widely used method for analyzing the expression of a moderate number of genes and also for the validation of microarray results. Several issues are crucial for a successful qPCR study, particularly the selection of internal reference genes for normalization and efficiency determination. There is no agreement on which method is the best to detect the most stable genes neither on how to perform efficiency determination. In this study we offer a comprehensive evaluation of the characteristics of reference gene selection methods and how to decide which one is more reliable when they show discordant outcomes. Also, we analyze the current efficiency calculation controversy. Our dataset is composed by gonad samples of turbot at different development times reared at different temperatures. Turbot (Scophthalmus maximus) is a relevant marine aquaculture European species with increasing production in the incoming years. Since females largely outgrow males, identification of genes related to sex determination, gonad development and reproductive behavior, and analysis of their expression profiles are of primary importance for turbot industry.ResultsWe analyzed gene stability of six reference genes: RPS4, RPL17, GAPDH, ACTB, UBQ and B2M using the comparative delta-CT method, Bestkeeper, NormFinder and GeNorm approaches in gonad samples of turbot. Supported by descriptive statistics, we found NormFinder to be the best method, while on the other side, GeNorm results proved to be unreliable. According to our analysis, UBQ and RPS4 were the most stable genes, while B2M was the least stable gene. We also analyzed the efficiency calculation softwares LinRegPCR, LREanalyzer, DART and PCR-Miner and we recommend LinRegPCR for research purposes since it does not systematically overestimate efficiency.ConclusionOur results indicate that NormFinder and LinRegPCR are the best approaches for reference gene selection and efficiency determination, respectively. We also recommend the use of UBQ and RPS4 for normalization of gonad development samples in turbot.

Whole Genome Sequencing of Turbot (Scophthalmus maximus; Pleuronectiformes): A Fish Adapted to Demersal Life

The turbot is a flatfish (Pleuronectiformes) with increasing commercial value, which has prompted active genomic research aimed at more efficient selection. Here we present the sequence and annotation of the turbot genome, which represents a milestone for both boosting breeding programmes and ascertaining the origin and diversification of flatfish. We compare the turbot genome with model fish genomes to investigate teleost chromosome evolution. We observe a conserved macrosyntenic pattern within Percomorpha and identify large syntenic blocks within the turbot genome related to the teleost genome duplication. We identify gene family expansions and positive selection of genes associated with vision and metabolism of membrane lipids, which suggests adaptation to demersal lifestyle and to cold temperatures, respectively. Our data indicate a quick evolution and diversification of flatfish to adapt to benthic life and provide clues for understanding their controversial origin. Moreover, we investigate the genomic architecture of growth, sex determination and disease resistance, key traits for understanding local adaptation and boosting turbot production, by mapping candidate genes and previously reported quantitative trait loci. The genomic architecture of these productive traits has allowed the identification of candidate genes and enriched pathways that may represent useful information for future marker-assisted selection in turbot.

RNA-seq analysis reveals significant transcriptome changes in turbot (Scophthalmus maximus) suffering severe enteromyxosis

BackgroundEnteromyxosis caused by the intestinal myxozoan parasite Enteromyxum scophthalmi is a serious threat for turbot (Scophthalmus maximus, L.) aquaculture, causing severe catarrhal enteritis leading to a cachectic syndrome, with no therapeutic options available. There are still many aspects of host-parasite interaction and disease pathogenesis that are yet to be elucidated, and to date, no analysis of the transcriptomic changes induced by E. scophthalmi in turbot organs has been conducted. In this study, RNA-seq technology was applied to head kidney, spleen and pyloric caeca of severely infected turbot with the aim of furthering our understanding of the pathogenetic mechanisms and turbot immune response against enteromyxosis.ResultsA huge amount of information was generated with more than 23,000 identified genes in the three organs, amongst which 4,762 were differently expressed (DE) between infected and control fish. Associate gene functions were studied based on gene ontology terms and available literature, and the most interesting DE genes were classified into five categories: 1) immune and defence response; 2) apoptosis and cell proliferation; 3) iron metabolism and erythropoiesis; 4) cytoskeleton and extracellular matrix and 5) metabolism and digestive function. The analysis of down-regulated genes of the first category revealed evidences of a connexion failure between innate and adaptive immune response, especially represented by a high number of DE interferon-related genes in the three organs. Furthermore, we found an intense activation of local immune response at intestinal level that appeared exacerbated, whereas in kidney and spleen genes involved in adaptive immune response were mainly down-regulated. The apoptotic machinery was only clearly activated in pyloric caeca, while kidney and spleen showed a marked depression of genes related to erythropoiesis, probably related to disorders in iron homeostasis. The genetic signature of the causes and consequences of cachexia was also demonstrated by the down-regulation of the genes encoding structural proteins and those involved in the digestive metabolism.ConclusionsThis transcriptomic study has enabled us to gain a better understanding of the pathogenesis of enteromyxosis and identify a large number of DE target genes that bring us closer to the development of strategies designed to effectively combat this pathogen.

Applications of genotyping by sequencing in aquaculture breeding and genetics

Abstract Selective breeding is increasingly recognized as a key component of sustainable production of aquaculture species. The uptake of genomic technology in aquaculture breeding has traditionally lagged behind terrestrial farmed animals. However, the rapid development and application of sequencing technologies has allowed aquaculture to narrow the gap, leading to substantial genomic resources for all major aquaculture species. While high‐density single‐nucleotide polymorphism (SNP) arrays for some species have been developed recently, direct genotyping by sequencing (GBS) techniques have underpinned many of the advances in aquaculture genetics and breeding to date. In particular, restriction‐site associated DNA sequencing (RAD‐Seq) and subsequent variations have been extensively applied to generate population‐level SNP genotype data. These GBS techniques are not dependent on prior genomic information such as a reference genome assembly for the species of interest. As such, they have been widely utilized by researchers and companies focussing on nonmodel aquaculture species with relatively small research communities. Applications of RAD‐Seq techniques have included generation of genetic linkage maps, performing genome‐wide association studies, improvements of reference genome assemblies and, more recently, genomic selection for traits of interest to aquaculture like growth, sex determination or disease resistance. In this review, we briefly discuss the history of GBS, the nuances of the various GBS techniques, bioinformatics approaches and application of these techniques to various aquaculture species.

Comparative expression analysis in mature gonads, liver and brain of turbot (Scophthalmus maximus) by cDNA-AFLPS

Turbot is one of the most important farmed fish in Europe. This species exhibits a considerable sexual dimorphism in growth and sexual maturity that makes the all-female production recommended for turbot farming. Our knowledge about the genetic basis of sex determination and the molecular regulation of gonad differentiation in this species is still limited. Our goal was to identify and compare gene expression and functions between testes and ovaries in adults in order to ascertain the relationship between the genes that could be involved in the gonad differentiation or related to the sex determination system. The identification of differentially expressed sex related genes is an initial step towards understanding the molecular mechanisms of gonad differentiation. For this, we carried out a transcriptome analysis based on cDNA-AFLP technique which allowed us to obtain an initial frame on sex-specific gene expression that will facilitate further analysis especially along the critical gonad differentiating period. With the aim of widening the study on sex-biased gene expression we reproduced the same experiments in two somatic tissues: liver and brain. We have selected the liver because it is the most analyzed one regarding sexual dimorphic gene expression and due to its importance in steroid hormones metabolism and the brain because the functional relationship between brain and gonad is documented. We found slight but important differences between sexes which deserve further investigation.

Integrative Transcriptome, Genome and Quantitative Trait Loci Resources Identify Single Nucleotide Polymorphisms in Candidate Genes for Growth Traits in Turbot

Growth traits represent a main goal in aquaculture breeding programs and may be related to adaptive variation in wild fisheries. Integrating quantitative trait loci (QTL) mapping and next generation sequencing can greatly help to identify variation in candidate genes, which can result in marker-assisted selection and better genetic structure information. Turbot is a commercially important flatfish in Europe and China, with available genomic information on QTLs and genome mapping. Muscle and liver RNA-seq from 18 individuals was carried out to obtain gene sequences and markers functionally related to growth, resulting in a total of 20,447 genes and 85,344 single nucleotide polymorphisms (SNPs). Many growth-related genes and SNPs were identified and placed in the turbot genome and genetic map to explore their co-localization with growth-QTL markers. Forty-five SNPs on growth-related genes were selected based on QTL co-localization and relevant function for growth traits. Forty-three SNPs were technically feasible and validated in a wild Atlantic population, where 91% were polymorphic. The integration of functional and structural genomic resources in turbot provides a practical approach for QTL mining in this species. Validated SNPs represent a useful set of growth-related gene markers for future association, functional and population studies in this flatfish species.

Construction and Annotation of a High Density SNP Linkage Map of the Atlantic Salmon (Salmo salar) Genome

High density linkage maps are useful tools for fine-scale mapping of quantitative trait loci, and characterization of the recombination landscape of a species’ genome. Genomic resources for Atlantic salmon (Salmo salar) include a well-assembled reference genome, and high density single nucleotide polymorphism (SNP) arrays. Our aim was to create a high density linkage map, and to align it with the reference genome assembly. Over 96,000 SNPs were mapped and ordered on the 29 salmon linkage groups using a pedigreed population comprising 622 fish from 60 nuclear families, all genotyped with the ‘ssalar01’ high density SNP array. The number of SNPs per group showed a high positive correlation with physical chromosome length (r = 0.95). While the order of markers on the genetic and physical maps was generally consistent, areas of discrepancy were identified. Approximately 6.5% of the previously unmapped reference genome sequence was assigned to chromosomes using the linkage map. Male recombination rate was lower than females across the vast majority of the genome, but with a notable peak in subtelomeric regions. Finally, using RNA-Seq data to annotate the reference genome, the mapped SNPs were categorized according to their predicted function, including annotation of ∼2500 putative nonsynonymous variants. The highest density SNP linkage map for any salmonid species has been created, annotated, and integrated with the Atlantic salmon reference genome assembly. This map highlights the marked heterochiasmy of salmon, and provides a useful resource for salmonid genetics and genomics research.

Comprehensive transcriptomic analysis of the process of gonadal sex differentiation in the turbot (Scophthalmus maximus)

The turbot is a flatfish with a ZW/ZZ sex determination system but with a still unknown sex determining gene(s), and with a marked sexual growth dimorphism in favor of females. To better understand sexual development in turbot we sampled young turbot encompassing the whole process of gonadal differentiation and conducted a comprehensive transcriptomic study on its sex differentiation using a validated custom oligomicroarray. Also, the expression profiles of 18 canonical reproduction-related genes were studied along gonad development. The expression levels of gonadal aromatase cyp19a1a alone at three months of age allowed the accurate and early identification of sex before the first signs of histological differentiation. A total of 56 differentially expressed genes (DEG) that had not previously been related to sex differentiation in fish were identified within the first three months of age, of which 44 were associated with ovarian differentiation (e.g., cd98, gpd1 and cry2), and 12 with testicular differentiation (e.g., ace, capn8 and nxph1). To identify putative sex determining genes, ∼4.000 DEG in juvenile gonads were mapped and their positions compared with that of previously identified sex- and growth-related quantitative trait loci (QTL). Although no genes mapped to the previously identified sex-related QTLs, two genes (foxl2 and 17βhsd) of the canonical reproduction-related genes mapped to growth-QTLs in linkage group (LG) 15 and LG6, respectively, suggesting that these genes are related to the growth dimorphism in this species.

RNA-seq analysis of early enteromyxosis in turbot (Scophthalmus maximus): new insights into parasite invasion and immune evasion strategies

Enteromyxum scophthalmi, an intestinal myxozoan parasite, is the causative agent of a threatening disease for turbot (Scophthalmus maximus, L.) aquaculture. The colonisation of the digestive tract by this parasite leads to a cachectic syndrome associated with high morbidity and mortality rates. This myxosporidiosis has a long pre-patent period and the first detectable clinical and histopathological changes are subtle. The pathogenic mechanisms acting in the early stages of infection are still far from being fully understood. Further information on the host-parasite interaction is needed to assist in finding efficient preventive and therapeutic measures. Here, a RNA-seq-based transcriptome analysis of head kidney, spleen and pyloric caeca from experimentally-infected and control turbot was performed. Only infected fish with early signs of infection, determined by histopathology and immunohistochemical detection of E. scophthalmi, were selected. The RNA-seq analysis revealed, as expected, less intense transcriptomic changes than those previously found during later stages of the disease. Several genes involved in IFN-related pathways were up-regulated in the three organs, suggesting that the IFN-mediated immune response plays a main role in this phase of the disease. Interestingly, an opposite expression pattern had been found in a previous study on severely infected turbot. In addition, possible strategies for immune system evasion were suggested by the down-regulation of different genes encoding complement components and acute phase proteins. At the site of infection (pyloric caeca), modulation of genes related to different structural proteins was detected and the expression profile indicated the inhibition of cell proliferation and differentiation. These transcriptomic changes provide indications regarding the mechanisms of parasite attachment to and invasion of the host. The current results contribute to a better knowledge of the events that characterise the early stages of turbot enteromyxosis and provide valuable information to identify molecular markers for early detection and control of this important parasitosis.

De novo transcriptome assembly of Perkinsus olseni trophozoite stimulated in vitro with Manila clam (Ruditapes philippinarum) plasma.

The protistan parasite Perkinsus olseni is a deadly causative agent of perkinsosis, a molluscan disease affecting Manila clam (Ruditapes philippinarum), having a significant impact on world mollusc production. Deciphering the underlying molecular mechanisms in R. philippinarum-P. olseni interaction is crucial for controlling this parasitosis. The present study investigated the transcriptional expression in the parasite trophozoite using RNA-seq. Control and treatment (in vitro challenged with Manila clam-plasma) P. olseni trophozoite RNA were extracted and sequenced on the Illumina HiSeq 2000 instrument using a 100-bp paired-end sequencing strategy. Paired reads (64.7 million) were de novo assembled using Trinity, and the resultant transcripts were further clustered using CAP3. The re-constructed P. olseni transcriptome contains 47,590 unique transcripts of which 23,505 were annotated to 9764 unique proteins. A large number of genes were associated with Gene Ontology terms such as stress and immune-response, cell homeostasis, antioxidation, cell communication, signal transduction, signalling and proteolysis. Among annotated transcripts, a preliminary gene expression analysis detected 679 up-regulated and 478 down-regulated genes, linked to virulence factors, anti-oxidants, adhesion and immune-response molecules. Genes of several metabolic pathways such as DOXP/MEP, FAS II or folate biosynthesis, which are potential therapeutic targets, were identified. This study is the first description of the P. olseni transcriptome, and provides a substantial genomic resource for studying the molecular mechanisms of the host-parasite interaction in perkinsosis. In this sense, it is also the first evaluation of the parasite gene expression after challenge with clam extracellular products.