Development and Validation of Single Nucleotide Polymorphisms (SNPs) Markers from Two Transcriptome 454-Runs of Turbot (Scophthalmus maximus) Using High-Throughput Genotyping.
ABSTRACT: The turbot (Scophthalmus maximus) is a commercially valuable flatfish and one of the most promising aquaculture species in Europe. Two transcriptome 454-pyrosequencing runs were used in order to detect Single Nucleotide Polymorphisms (SNPs) in genes related to immune response and gonad differentiation. A total of 866 true SNPs were detected in 140 different contigs representing 262,093 bp as a whole. Only one true SNP was analyzed in each contig. One hundred and thirteen SNPs out of the 140 analyzed were feasible (genotyped), while ? were polymorphic in a wild population. Transition/transversion ratio (1.354) was similar to that observed in other fish studies. Unbiased gene diversity (He) estimates ranged from 0.060 to 0.510 (mean = 0.351), minimum allele frequency (MAF) from 0.030 to 0.500 (mean = 0.259) and all loci were in Hardy-Weinberg equilibrium after Bonferroni correction. A large number of SNPs (49) were located in the coding region, 33 representing synonymous and 16 non-synonymous changes. Most SNP-containing genes were related to immune response and gonad differentiation processes, and could be candidates for functional changes leading to phenotypic changes. These markers will be useful for population screening to look for adaptive variation in wild and domestic turbot.
Project description:BACKGROUND: The turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish, very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the turbot industry. Little is known about the molecular mechanisms for disease resistance and host-pathogen interactions in this species. In this work, thousands of ESTs for functional genomic studies and potential markers linked to ESTs for mapping (microsatellites and single nucleotide polymorphisms (SNPs)) are provided. This information enabled us to obtain a preliminary view of regulated genes in response to these pathogens and it constitutes the basis for subsequent and more accurate microarray analysis. RESULTS: A total of 12584 cDNAs partially sequenced from three different cDNA libraries of turbot (Scophthalmus maximus) infected with Aeromonas salmonicida, Philasterides dicentrarchi and from healthy fish were analyzed. Three immune-relevant tissues (liver, spleen and head kidney) were sampled at several time points in the infection process for library construction. The sequences were processed into 9256 high-quality sequences, which constituted the source for the turbot EST database. Clustering and assembly of these sequences, revealed 3482 different putative transcripts, 1073 contigs and 2409 singletons. BLAST searches with public databases detected significant similarity (e-value < or = 1e-5) in 1766 (50.7%) sequences and 816 of them (23.4%) could be functionally annotated. Two hundred three of these genes (24.9%), encoding for defence/immune-related proteins, were mostly identified for the first time in turbot. Some ESTs showed significant differences in the number of transcripts when comparing the three libraries, suggesting regulation in response to these pathogens. A total of 191 microsatellites, with 104 having sufficient flanking sequences for primer design, and 1158 putative SNPs were identified from these EST resources in turbot. CONCLUSION: A collection of 9256 high-quality ESTs was generated representing 3482 unique turbot sequences. A large proportion of defence/immune-related genes were identified, many of them regulated in response to specific pathogens. Putative microsatellites and SNPs were identified. These genome resources constitute the basis to develop a microarray for functional genomics studies and marker validation for genetic linkage and QTL analysis in turbot.
Project description:Fish sex determination (SD) systems are varied, suggesting evolutionary changes including either multiple evolution origins of genetic SD from nongenetic systems (such as environmental SD) and/or turnover events replacing one genetic system by another. When genetic SD is found, cytological differentiation between the two members of the sex chromosome pair is often minor or undetectable. The turbot (Scophthalmus maximus), a valuable commercial flatfish, has a ZZ/ZW system and a major SD region on linkage group 5 (LG5), but there are also other minor genetic and environmental influences. We here report refined mapping of the turbot SD region, supported by comparative mapping with model fish species, to identify the turbot master SD gene. Six genes were located to the SD region, two of them associated with gonad development (sox2 and dnajc19). All showed a high association with sex within families (P = 0), but not at the population level, so they are probably partially sex-linked genes, but not SD gene itself. Analysis of crossovers in LG5 using two families confirmed a ZZ/ZW system in turbot and suggested a revised map position for the master gene. Genetic diversity and differentiation for 25 LG5 genetic markers showed no differences between males and females sampled from a wild population, suggesting a recent origin of the SD region in turbot. We also analyzed associations with markers of the most relevant sex-related linkage groups in brill (S. rhombus), a closely related species to turbot; the data suggest that an ancient XX/XY system in brill changed to a ZZ/ZW mechanism in turbot.
Project description:BACKGROUND:Turbot Scophthalmus maximus is an economically important species extensively aquacultured in China. The genetic selection program is necessary and urgent for the sustainable development of this industry, requiring more and more genome background knowledge. Transcriptome sequencing is an excellent alternative way to identify transcripts involved in specific biological processes and exploit a considerable quantity of molecular makers when no genome sequences are available. In this study, a comprehensive transcript dataset for major tissues of S. maximus was produced on basis of an Illumina platform. RESULTS:Total RNA was isolated from liver, spleen, kidney, cerebrum, gonad (testis and ovary) and muscle. Equal quantities of RNA from each type of tissues were pooled to construct two cDNA libraries (male and female). Using the Illumina paired-end sequencing technology, nearly 44.22 million clean reads in length of 100 bp were generated and then assembled into 106,643 contigs, of which 71,107 were named unigenes with an average length of 892 bp after the elimination of redundancies. Of these, 24,052 unigenes (33.83% of the total) were successfully annotated. GO, KEGG pathway mapping and COG analysis were performed to predict potential genes and their functions. Based on our sequence analysis and published documents, many candidate genes with fundamental roles in sex determination and gonad differentiation (dmrt1), growth (ghrh, myf5, prl/prlr) and immune response (TLR1/TLR21/TLR22, IL-15/IL-34), were identified for the first time in this species. In addition, a large number of credible genetic markers, including 21,192 SSRs and 8,642 SNPs, were identified in the present dataset. CONCLUSION:This informative transcriptome provides valuable new data to increase genomic resources of Scophthalmus maximus. The future studies of corresponding gene functions will be very useful for the management of reproduction, growth and disease control in turbot aquaculture breeding programs. The molecular markers identified in this database will aid in genetic linkage analyses, mapping of quantitative trait loci, and acceleration of marker assisted selection programs.
Project description:BACKGROUND: Gene 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. RESULTS: We 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. CONCLUSION: Our 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.
Project description:Turbot (Scophthalmus maximus) is a commercially important flatfish species in aquaculture. It has a drastic sexual dimorphism, with females growing faster than males. In the present study, we sequenced and de novo assembled female and male turbot genomes. The assembled female genome was 568?Mb (scaffold N50, 6.2?Mb, BUSCO 97.4%), and the male genome was 584?Mb (scaffold N50, 5.9?Mb, BUSCO 96.6%). Using two genetic maps, we anchored female scaffolds representing 535?Mb onto 22 chromosomes. Annotation of the female anchored genome identified 87.8?Mb transposon elements and 20,134 genes. We identified 17,936 gene families, of which 369 gene families were flatfish specific. Phylogenetic analysis showed that the turbot, Japanese flounder and Chinese tongue sole form a clade that diverged from other teleosts approximately 78 Mya. This report of female and male turbot draft genomes and annotated genes provides a new resource for identifying sex determination genes, elucidating the evolution of adaptive traits in flatfish and developing genetic techniques to increase the sustainability of turbot aquaculture.
Project description:Chemokine receptor 4 (CXCR4) belongs to the large superfamily of G protein-coupled receptors. The EST sequence of CXCR4 from turbot (Scophthalmus maximus L.) was obtained from a subtractive cDNA library. In the present study, the full-length cDNA sequence of turbot CXCR4 was obtained, and sequence analysis indicated that its primary structure was highly similar to CXCR4 from other vertebrates. Quantitative real-time PCR demonstrated that the highest expression level of turbot CXCR4 was in the spleen following injection with physiological saline (PS). After turbot were challenged with Vibrio harveyi, the lowest expression level of CXCR4 was detected at 8 hours in the spleen and 12 hours in the head kidney, and then increased gradually to 36 hours. These findings suggested that CXCR4 may play a significant role in the immune response of turbot.
Project description:Ranaviruses have been isolated from Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus) in Denmark. Phylogenomic analyses revealed that these two ranaviruses are nearly identical and form a distinct clade at the base of the ranavirus tree branching off near other fish ranaviruses.
Project description:This paper describes the development of a high density consensus genetic linkage map of a turbot (Scophthalmus maximus L.) family composed of 149 mapping individuals using Single Nucleotide Polymorphisms (SNP) developed using the restriction-site associated DNA (RAD) sequencing technique with the restriction enzyme, PstI. A total of 6,647 SNPs were assigned to 22 linkage groups, which is equal to the number of chromosome pairs in turbot. For the first time, the average marker interval reached 0.3958 cM, which is equal to approximately 0.1203 Mb of the turbot genome. The observed 99.34% genome coverage indicates that the linkage map was genome-wide. A total of 220 Quantitative Traits Locus (QTLs) associated with two body length traits, two body weight traits in different growth periods and sex determination were detected with an LOD > 5.0 in 12 linkage groups (LGs), which explained the corresponding phenotypic variance (R2), ranging from 14.4-100%. Among them, 175 overlapped with linked SNPs, and the remaining 45 were located in regions between contiguous SNPs. According to the QTLs related to growth trait distribution and the changing of LGs during different growth periods, the growth traits are likely controlled by multi-SNPs distributed on several LGs; the effect of these SNPs changed during different growth periods. Most sex-related QTLs were detected at LG 21 with a linkage span of 70.882 cM. Additionally, a small number of QTLs with high feasibility and a narrow R2 distribution were also observed on LG7 and LG14, suggesting that multi LGs or chromosomes might be involved in sex determination. High homology was recorded between LG21 in Cynoglossus semilaevis and turbot. This high-saturated turbot RAD-Seq linkage map is undoubtedly a promising platform for marker assisted selection (MAS) and flatfish genomics research.
Project description:Sex determination in fish is a labile character in evolutionary terms. The sex-determining (SD) master gene can differ even between closely related fish species. This group is an interesting model for studying the evolution of the SD region and the gonadal differentiation pathway. The turbot (Scophthalmus maximus) is a flatfish of great commercial value, where a strong sexual dimorphism exists for growth rate. Following a QTL and marker association approach in five families and a natural population, we identified the main SD region of turbot at the proximal end of linkage group (LG) 5, close to the SmaUSC-E30 marker. The refined map of this region suggested that this marker would be 2.6 cM and 1.4 Mb from the putative SD gene. This region appeared mostly undifferentiated between males and females, and no relevant recombination frequency differences were detected between sexes. Comparative genomics of LG5 marker sequences against five model species showed no similarity of this chromosome to the sex chromosomes of medaka, stickleback, and fugu, but suggested a similarity to a sex-associated QTL from Oreochromis spp. The segregation analysis of the closest markers to the SD region demonstrated a ZW/ZZ model of sex determination in turbot. A small proportion of families did not fit perfectly with this model, which suggests that other minor genetic and/or environmental factors are involved in sex determination in this species.
Project description:The valorization of wastes generated in the processing of farmed fish is currently an issue of extreme relevance for the industry, aiming to accomplish the objectives of circular bioeconomy. In the present report, turbot (Scophthalmus maximus) by-products were subjected to Alcalase hydrolysis under the optimal conditions initially defined by response surface methodology. All the fish protein hydrolysates (FPHs) showed a high yield of digestion (>83%), very remarkable degrees of hydrolysis (30-37%), high content of soluble protein (>62 g/L), an excellent profile of amino acids, and almost total in vitro digestibility (higher than 92%). Antioxidant and antihypertensive activities were analyzed in all cases, viscera hydrolysates being the most active. The range of average molecular weights (Mw) of turbot hydrolysates varied from 1200 to 1669 Da, and peptide size distribution showed that the hydrolysate of viscera had the highest content of peptides above 1000 Da and below 200 Da.