Publications by authors named "José Alvarez-Dios"

17 Publications

  • Page 1 of 1

New insights into the Manila clam - Perkinsus olseni interaction based on gene expression analysis of clam hemocytes and parasite trophozoites through in vitro challenges.

Int J Parasitol 2020 03 20;50(3):195-208. Epub 2020 Feb 20.

Departamento de Zooloxía, Xenética e Antropoloxía Física, Universidade de Santiago de Compostela, 27002 Lugo, Spain; Instituto de Acuicultura, Universidade de Santiago de Compostela, Campus Vida s/n, Santiago de Compostela 15782, Spain. Electronic address:

The Manila clam (Ruditapes philippinarum) is the bivalve species with the highest global production from both fisheries and aquaculture, but its production is seriously threatened by perkinsosis, a disease caused by the protozoan parasite Perkinsus olseni. To understand the molecular mechanisms underlying R. philippinarum-P. olseni interactions, we analysed the gene expression profiles of in vitro challenged clam hemocytes and P. olseni trophozoites, using two oligo-microarray platforms, one previously validated for R. philippinarum hemocytes and a new one developed and validated in this study for P. olseni. Manila clam hemocytes were in vitro challenged with trophozoites, zoospores, and extracellular products from P. olseni in vitro cultures, while P. olseni trophozoites were in vitro challenged with Manila clam plasma along the same time-series (1 h, 8 h, and 24 h). The hemocytes showed a fast activation of the innate immune response, particularly associated with hemocyte recruitment, in the three types of challenges. Nevertheless, different immune-related pathways were activated in response to the different parasite stages, suggesting specific recognition mechanisms. Furthermore, the analyses provided useful complementary data to previous in vivo challenges, and confirmed the potential of some proposed biomarkers. The combined analysis of gene expression in host and parasite identified several processes in both the clam and P. olseni, such as redox and glucose metabolism, protease activity, apoptosis and iron metabolism, whose modulation suggests cross-talk between parasite and host. This information might be critical to determine the outcome of the infection, thus highlighting potential therapeutic targets. Altogether, the results of this study aid understanding the response and interaction between R. philippinarum and P. olseni, and will contribute to developing effective control strategies for this threatening parasitosis.
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http://dx.doi.org/10.1016/j.ijpara.2019.11.008DOI Listing
March 2020

Tracking age-correlated DNA methylation markers in the young.

Forensic Sci Int Genet 2018 09 13;36:50-59. Epub 2018 Jun 13.

Faculty of Mathematics, University of Santiago de Compostela, Spain.

DNA methylation is the most extensively studied epigenetic signature, with a large number of studies reporting age-correlated CpG sites in overlapping genes. However, most of these studies lack sample coverage of individuals under 18 years old and therefore little is known about the progression of DNA methylation patterns in children and adolescents. In the present study we aimed to select candidate age-correlated DNA methylation markers based on public datasets from Illumina BeadChip arrays and previous publications, then to explore the resulting markers in 209 blood samples from donors aged between 2 to 18 years old using the EpiTYPER® DNA methylation analysis system. Results from our analyses identified six genes highly correlated with age in the young, in particular the gene KCNAB3, which indicates its potential as a highly informative and specific age biomarker for childhood and adolescence. We outline a preliminary age prediction model based on quantile regression that uses data from the six CpG sites most strongly correlated with age ranges extended to include children and adolescents.
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http://dx.doi.org/10.1016/j.fsigen.2018.06.011DOI Listing
September 2018

Gene expression analysis of Ruditapes philippinarum haemocytes after experimental Perkinsus olseni zoospore challenge and infection in the wild.

Fish Shellfish Immunol 2018 Jan 21;72:611-621. Epub 2017 Nov 21.

Departamento de Zoología, Genética y Antropología Física, Universidade de Santiago de Compostela, Lugo 27002, Spain. Electronic address:

The production of Manila clam (Ruditapes philippinarum) is seriously threatened by the protistan parasite Perkinsus olseni. We characterized and compared gene expression of Manila clam haemocytes in response to P. olseni in a time-course (10 h, 24 h, 8 d) controlled laboratory challenge (LC), representing the first step of infection, and in a more complex infection in the wild (WI), using a validated oligo-microarray containing 11,232 transcripts, mostly annotated. Several immune-genes involved in NIK/NF-kappaB signalling, Toll-like receptor signalling and apoptosis were activated at LC-10 h. However, down-regulation of genes encoding lysozyme, histones, cathepsins and heat shock proteins indicated signals of immunodepression, which persisted at LC-24 h, when only down-regulated genes were detected. A rebound of haemocyte activity occurred at LC-8 d as shown by up-regulation of genes involved in cytoskeleton organization and cell survival. The WI study showed a more complex picture, and several immune-relevant processes including cytoskeleton organization, cell survival, apoptosis, encapsulation, cell redox- and lipid-homeostasis were activated, illustrating the main mechanism of host response. Our results provide useful information, including potential biomarkers, to develop strategies for controlling Manila clam perkinsosis.
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http://dx.doi.org/10.1016/j.fsi.2017.11.033DOI Listing
January 2018

Construction of an Ostrea edulis database from genomic and expressed sequence tags (ESTs) obtained from Bonamia ostreae infected haemocytes: Development of an immune-enriched oligo-microarray.

Fish Shellfish Immunol 2016 Dec 1;59:331-344. Epub 2016 Nov 1.

Departamento de Zoología, Genética y Antropología Física, Facultad de Veterinaria, Universidade de Santiago de Compostela, Campus de Lugo, 27002 Lugo, Spain. Electronic address:

The flat oyster, Ostrea edulis, is one of the main farmed oysters, not only in Europe but also in the United States and Canada. Bonamiosis due to the parasite Bonamia ostreae has been associated with high mortality episodes in this species. This parasite is an intracellular protozoan that infects haemocytes, the main cells involved in oyster defence. Due to the economical and ecological importance of flat oyster, genomic data are badly needed for genetic improvement of the species, but they are still very scarce. The objective of this study is to develop a sequence database, OedulisDB, with new genomic and transcriptomic resources, providing new data and convenient tools to improve our knowledge of the oyster's immune mechanisms. Transcriptomic and genomic sequences were obtained using 454 pyrosequencing and compiled into an O. edulis database, OedulisDB, consisting of two sets of 10,318 and 7159 unique sequences that represent the oyster's genome (WG) and de novo haemocyte transcriptome (HT), respectively. The flat oyster transcriptome was obtained from two strains (naïve and tolerant) challenged with B. ostreae, and from their corresponding non-challenged controls. Approximately 78.5% of 5619 HT unique sequences were successfully annotated by Blast search using public databases. A total of 984 sequences were identified as being related to immune response and several key immune genes were identified for the first time in flat oyster. Additionally, transcriptome information was used to design and validate the first oligo-microarray in flat oyster enriched with immune sequences from haemocytes. Our transcriptomic and genomic sequencing and subsequent annotation have largely increased the scarce resources available for this economically important species and have enabled us to develop an OedulisDB database and accompanying tools for gene expression analysis. This study represents the first attempt to characterize in depth the O. edulis haemocyte transcriptome in response to B. ostreae through massively sequencing and has aided to improve our knowledge of the immune mechanisms of flat oyster. The validated oligo-microarray and the establishment of a reference transcriptome will be useful for large-scale gene expression studies in this species.
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http://dx.doi.org/10.1016/j.fsi.2016.10.047DOI Listing
December 2016

Whole genome sequencing of turbot (Scophthalmus maximus; Pleuronectiformes): a fish adapted to demersal life.

DNA Res 2016 Jun 6;23(3):181-92. Epub 2016 Mar 6.

Departamento de Xenética, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo 27002, Spain

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.
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http://dx.doi.org/10.1093/dnares/dsw007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4909306PMC
June 2016

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

Int J Mol Sci 2016 Feb 17;17(2):243. Epub 2016 Feb 17.

Departamento de Xenética, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo 27002, Spain.

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.
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http://dx.doi.org/10.3390/ijms17020243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4783974PMC
February 2016

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

J Invertebr Pathol 2016 Mar 25;135:22-33. Epub 2016 Jan 25.

Departamento de Xenética, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo 27002, Spain.

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.
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http://dx.doi.org/10.1016/j.jip.2016.01.009DOI Listing
March 2016

Exploration of SNP variants affecting hair colour prediction in Europeans.

Int J Legal Med 2015 Sep 11;129(5):963-75. Epub 2015 Jul 11.

Forensic Genetics Unit, Institute of Legal Medicine, University of Santiago de Compostela, A Coruña, Spain.

DNA profiling is a key tool for forensic analysis; however, current methods identify a suspect either by direct comparison or from DNA database searches. In cases with unidentified suspects, prediction of visible physical traits e.g. pigmentation or hair distribution of the DNA donors can provide important probative information. This study aimed to explore single nucleotide polymorphism (SNP) variants for their effect on hair colour prediction. A discovery panel of 63 SNPs consisting of already established hair colour markers from the HIrisPlex hair colour phenotyping assay as well as additional markers for which associations to human pigmentation traits were previously identified was used to develop multiplex assays based on SNaPshot single-base extension technology. A genotyping study was performed on a range of European populations (n = 605). Hair colour phenotyping was accomplished by matching donor's hair to a graded colour category system of reference shades and photography. Since multiple SNPs in combination contribute in varying degrees to hair colour predictability in Europeans, we aimed to compile a compact marker set that could provide a reliable hair colour inference from the fewest SNPs. The predictive approach developed uses a naïve Bayes classifier to provide hair colour assignment probabilities for the SNP profiles of the key SNPs and was embedded into the Snipper online SNP classifier ( http://mathgene.usc.es/snipper/ ). Results indicate that red, blond, brown and black hair colours are predictable with informative probabilities in a high proportion of cases. Our study resulted in the identification of 12 most strongly associated SNPs to hair pigmentation variation in six genes.
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http://dx.doi.org/10.1007/s00414-015-1226-yDOI Listing
September 2015

Development of a forensic skin colour predictive test.

Forensic Sci Int Genet 2014 Nov 10;13:34-44. Epub 2014 Jul 10.

Forensic Genetics Unit, Institute of Forensic Science "Luis Concheiro", University of Santiago de Compostela, Spain.

There is growing interest in skin colour prediction in the forensic field. However, a lack of consensus approaches for recording skin colour phenotype plus the complicating factors of epistatic effects, environmental influences such as exposure to the sun and unidentified genetic variants, present difficulties for the development of a forensic skin colour predictive test centred on the most strongly associated SNPs. Previous studies have analysed skin colour variation in single unadmixed population groups, including South Asians (Stokowski et al., 2007, Am. J. Hum. Genet, 81: 1119-32) and Europeans (Jacobs et al., 2013, Hum Genet. 132: 147-58). Nevertheless, a major challenge lies in the analysis of skin colour in admixed individuals, where co-ancestry proportions do not necessarily dictate any one person's skin colour. Our study sought to analyse genetic differences between African, European and admixed African-European subjects where direct spectrometric measurements and photographs of skin colour were made in parallel. We identified strong associations to skin colour variation in the subjects studied from a pigmentation SNP discovery panel of 59 markers and developed a forensic online classifier based on naïve Bayes analysis of the SNP profiles made. A skin colour predictive test is described using the ten most strongly associated SNPs in 8 genes linked to skin pigmentation variation.
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http://dx.doi.org/10.1016/j.fsigen.2014.06.017DOI Listing
November 2014

A combined strategy involving Sanger and 454 pyrosequencing increases genomic resources to aid in the management of reproduction, disease control and genetic selection in the turbot (Scophthalmus maximus).

BMC Genomics 2013 Mar 15;14:180. Epub 2013 Mar 15.

Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain.

Background: Genomic resources for plant and animal species that are under exploitation primarily for human consumption are increasingly important, among other things, for understanding physiological processes and for establishing adequate genetic selection programs. Current available techniques for high-throughput sequencing have been implemented in a number of species, including fish, to obtain a proper description of the transcriptome. The objective of this study was to generate a comprehensive transcriptomic database in turbot, a highly priced farmed fish species in Europe, with potential expansion to other areas of the world, for which there are unsolved production bottlenecks, to understand better reproductive- and immune-related functions. This information is essential to implement marker assisted selection programs useful for the turbot industry.

Results: Expressed sequence tags were generated by Sanger sequencing of cDNA libraries from different immune-related tissues after several parasitic challenges. The resulting database ("Turbot 2 database") was enlarged with sequences generated from a 454 sequencing run of brain-hypophysis-gonadal axis-derived RNA obtained from turbot at different development stages. The assembly of Sanger and 454 sequences generated 52,427 consensus sequences ("Turbot 3 database"), of which 23,661 were successfully annotated. A total of 1,410 sequences were confirmed to be related to reproduction and key genes involved in sex differentiation and maturation were identified for the first time in turbot (AR, AMH, SRY-related genes, CYP19A, ZPGs, STAR FSHR, etc.). Similarly, 2,241 sequences were related to the immune system and several novel key immune genes were identified (BCL, TRAF, NCK, CD28 and TOLLIP, among others). The number of genes of many relevant reproduction- and immune-related pathways present in the database was 50-90% of the total gene count of each pathway. In addition, 1,237 microsatellites and 7,362 single nucleotide polymorphisms (SNPs) were also compiled. Further, 2,976 putative natural antisense transcripts (NATs) including microRNAs were also identified.

Conclusions: The combined sequencing strategies employed here significantly increased the turbot genomic resources available, including 34,400 novel sequences. The generated database contains a larger number of genes relevant for reproduction- and immune-associated studies, with an excellent coverage of most genes present in many relevant physiological pathways. This database also allowed the identification of many microsatellites and SNP markers that will be very useful for population and genome screening and a valuable aid in marker assisted selection programs.
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http://dx.doi.org/10.1186/1471-2164-14-180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3700835PMC
March 2013

An Expressed Sequence Tag (EST)-enriched genetic map of turbot (Scophthalmus maximus): a useful framework for comparative genomics across model and farmed teleosts.

BMC Genet 2012 Jul 2;13:54. Epub 2012 Jul 2.

Departamento de Genética, Facultade de Veterinaria, Universidade de Santiago de Compostela (USC), Campus de Lugo, 27002, Lugo, Spain.

Background: The turbot (Scophthalmus maximus) is a relevant species in European aquaculture. The small turbot genome provides a source for genomics strategies to use in order to understand the genetic basis of productive traits, particularly those related to sex, growth and pathogen resistance. Genetic maps represent essential genomic screening tools allowing to localize quantitative trait loci (QTL) and to identify candidate genes through comparative mapping. This information is the backbone to develop marker-assisted selection (MAS) programs in aquaculture. Expressed sequenced tag (EST) resources have largely increased in turbot, thus supplying numerous type I markers suitable for extending the previous linkage map, which was mostly based on anonymous loci. The aim of this study was to construct a higher-resolution turbot genetic map using EST-linked markers, which will turn out to be useful for comparative mapping studies.

Results: A consensus gene-enriched genetic map of the turbot was constructed using 463 SNP and microsatellite markers in nine reference families. This map contains 438 markers, 180 EST-linked, clustered at 24 linkage groups. Linkage and comparative genomics evidences suggested additional linkage group fusions toward the consolidation of turbot map according to karyotype information. The linkage map showed a total length of 1402.7 cM with low average intermarker distance (3.7 cM; ~2 Mb). A global 1.6:1 female-to-male recombination frequency (RF) ratio was observed, although largely variable among linkage groups and chromosome regions. Comparative sequence analysis revealed large macrosyntenic patterns against model teleost genomes, significant hits decreasing from stickleback (54%) to zebrafish (20%). Comparative mapping supported particular chromosome rearrangements within Acanthopterygii and aided to assign unallocated markers to specific turbot linkage groups.

Conclusions: The new gene-enriched high-resolution turbot map represents a useful genomic tool for QTL identification, positional cloning strategies, and future genome assembling. This map showed large synteny conservation against model teleost genomes. Comparative genomics and data mining from landmarks will provide straightforward access to candidate genes, which will be the basis for genetic breeding programs and evolutionary studies in this species.
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http://dx.doi.org/10.1186/1471-2156-13-54DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3464660PMC
July 2012

Gene expression profiles of spleen, liver, and head kidney in turbot (Scophthalmus maximus) along the infection process with Philasterides dicentrarchi using an immune-enriched oligo-microarray.

Mar Biotechnol (NY) 2012 Oct 26;14(5):570-82. Epub 2012 Feb 26.

Departamento de Genética, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus de Lugo, Lugo, Spain.

We evaluated the expression profiles of turbot in spleen, liver, and head kidney across five temporal points of the Philasterides dicentrarchi infection process using an 8x15K Agilent oligo-microarray. The microarray included 2,176 different fivefold replicated gene probes designed from a turbot 3' sequenced EST database. We were able to identify 221 differentially expressed (DE) genes (8.1% of the whole microarray), 113 in spleen, 83 in liver, and 90 in head kidney, in at least 1 of the 5 temporal points sampled for each organ. Most of these genes could be annotated (83.0%) and functionally categorized using GO terms (69.1%) after the additional sequencing of DE genes from the 5' end. Many DE genes were related to innate and acquired immune functions. A high proportion of DE genes were organ-specific (70.6%), although their associated GO functions showed notable similarities in the three organs. The most striking difference in functional distribution was observed between the up- and downregulated gene groups. Upregulated genes were mostly associated to immune functions, while downregulated ones mainly involved metabolism-related genes. Genetic response appeared clustered in a few groups of genes with similar expression profiles along the temporal series. The information obtained will aid to understand the turbot immune response and will specifically be valuable to develop strategies of defense to P. dicentrarchi to achieve more resistant broodstocks for turbot industry.
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http://dx.doi.org/10.1007/s10126-012-9440-9DOI Listing
October 2012

Gene expression profiles of the spleen, liver, and head kidney in turbot (Scophthalmus maximus) along the infection process with Aeromonas salmonicida using an immune-enriched oligo-microarray.

Mar Biotechnol (NY) 2011 Dec 19;13(6):1099-114. Epub 2011 Apr 19.

Departamento de Genética, Facultad de Veterinaria, Campus de Lugo, Universidad de Santiago de Compostela, 27002 Lugo, Spain.

We evaluated the expression profiles of turbot in the spleen, liver, and head kidney across five temporal points of the Aeromonas salmonicida infection process using an 8 × 15 K Agilent oligo-microarray. The microarray included 2,176 different fivefold replicated gene probes designed from a turbot 3' sequenced EST database. We were able to identify 471 differentially expressed (DE) genes (17.3% of the whole microarray), 223 in the spleen, 246 in the liver, and 125 in the head kidney, in at least one of the five temporal points sampled for each organ. Most of these genes could be annotated (83.0%) and functionally categorized using Gene Ontology terms (69.1%) after the additional sequencing of DE genes from the 5' end. Many DE genes were related to innate and acquired immune functions in accordance to previous studies with this pathogen in other fish species. A high proportion of DE genes were organ specific (77.1%), but their associated GO functions were rather similar in the three organs. The most striking difference in functional distribution was observed between the up- and down-regulated gene groups. Up-regulated genes were mostly associated to key immune functions while down-regulated ones mainly involved metabolism- and transport-related genes. Genetic response appeared clustered in groups of genes with similar expression profiles along the temporal series. The spleen showed the most clustering while the liver and head kidney displayed a higher diversification. The information obtained will aid to understand the turbot immune response and will specifically be valuable to develop strategies of defense to A. salmonicida to achieve more resistant broodstocks for turbot industry.
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http://dx.doi.org/10.1007/s10126-011-9374-7DOI Listing
December 2011

Design and performance of a turbot (Scophthalmus maximus) oligo-microarray based on ESTs from immune tissues.

Mar Biotechnol (NY) 2010 Aug 21;12(4):452-65. Epub 2009 Oct 21.

Departamento de Genética, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus de Lugo, 27002 Lugo, Spain.

An expressed sequence tag database from immune tissues was used to design the first high-density turbot (Scophthalmus maximus) oligo-microarray with the aim of identifying candidate genes for tolerance to pathogens. Specific oligonucleotides (60 mers) were successfully designed for 2,716 out of 3,482 unique sequences of the database. An Agilent custom oligo-microarray 8 x 15 k (five replicates/gene; eight microarrays/slide) was constructed. The performance of the microarray and the sources of variation along microarray analysis were examined on spleen pools of controls and Aeromonas salmonicida-challenged fish at 3 days postinfection. Only 48 out of 2,716 probes did not show signal of hybridization on the 32 microarrays employed, thus demonstrating the consistency of the bioinformatic applications of our database. An asymmetric hierarchical design was employed to ascertain the noise associated with biological and technical (RNA extraction, labeling, hybridization, slide, and dye bias) factors using 1C and 2C labeling approaches. The high correlation coefficient between replicates at most factors tested demonstrated the high reproducibility of the signal. An analysis of random-effects variance revealed that technical variation was mostly negligible, and biological variation represented the main factor, even using pooled samples. One-color approach performed at least as well as 2C, suggesting their usefulness due to its higher design flexibility and lower cost. A relevant proportion of genes turn out to be differentially labeled depending on fluorophore, which alerts for the likely need of swapping replication in 2C experiments. A set of differentially expressed genes and enriched functions related to immune/defense response were detected at 3 days postchallenging.
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http://dx.doi.org/10.1007/s10126-009-9231-0DOI Listing
August 2010

Ancestry analysis in the 11-M Madrid bomb attack investigation.

PLoS One 2009 Aug 11;4(8):e6583. Epub 2009 Aug 11.

Forensic Genetics Unit, Institute of Legal Medicine, University of Santiago de Compostela, Santiago de Compostela, Galicia, Spain.

The 11-M Madrid commuter train bombings of 2004 constituted the second biggest terrorist attack to occur in Europe after Lockerbie, while the subsequent investigation became the most complex and wide-ranging forensic case in Spain. Standard short tandem repeat (STR) profiling of 600 exhibits left certain key incriminatory samples unmatched to any of the apprehended suspects. A judicial order to perform analyses of unmatched samples to differentiate European and North African ancestry became a critical part of the investigation and was instigated to help refine the search for further suspects. Although mitochondrial DNA (mtDNA) and Y-chromosome markers routinely demonstrate informative geographic differentiation, the populations compared in this analysis were known to show a proportion of shared mtDNA and Y haplotypes as a result of recent gene-flow across the western Mediterranean, while any two loci can be unrepresentative of the ancestry of an individual as a whole. We based our principal analysis on a validated 34plex autosomal ancestry-informative-marker single nucleotide polymorphism (AIM-SNP) assay to make an assignment of ancestry for DNA from seven unmatched case samples including a handprint from a bag containing undetonated explosives together with personal items recovered from various locations in Madrid associated with the suspects. To assess marker informativeness before genotyping, we predicted the probable classification success for the 34plex assay with standard error estimators for a naïve Bayesian classifier using Moroccan and Spanish training sets (each n = 48). Once misclassification error was found to be sufficiently low, genotyping yielded seven near-complete profiles (33 of 34 AIM-SNPs) that in four cases gave probabilities providing a clear assignment of ancestry. One of the suspects predicted to be North African by AIM-SNP analysis of DNA from a toothbrush was identified late in the investigation as Algerian in origin. The results achieved illustrate the benefit of adding specialized marker sets to provide enhanced scope and power to an already highly effective system of DNA analysis for forensic identification.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0006583PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719087PMC
August 2009

Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens.

BMC Vet Res 2008 Sep 25;4:37. Epub 2008 Sep 25.

Departamento de Genética, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus de Lugo, 27002 Lugo, Spain.

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.
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http://dx.doi.org/10.1186/1746-6148-4-37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2569028PMC
September 2008

A microsatellite genetic map of the turbot (Scophthalmus maximus).

Genetics 2007 Dec;177(4):2457-67

Department of Genetics, University of Santiago de Compostela, Lugo, Spain.

A consensus microsatellite-based linkage map of the turbot (Scophthalmus maximus) was constructed from two unrelated families. The mapping panel was derived from a gynogenetic family of 96 haploid embryos and a biparental diploid family of 85 full-sib progeny with known linkage phase. A total of 242 microsatellites were mapped in 26 linkage groups, six markers remaining unlinked. The consensus map length was 1343.2 cM, with an average distance between markers of 6.5 +/- 0.5 cM. Similar length of female and male maps was evidenced. However, the mean recombination at common intervals throughout the genome revealed significant differences between sexes, approximately 1.6 times higher in the female than in the male. The comparison of turbot microsatellite flanking sequences against the Tetraodon nigroviridis genome revealed 55 significant matches, with a mean length of 102 bp and high sequence similarity (81-100%). The comparative mapping revealed significant syntenic regions among fish species. This study represents the first linkage map in the turbot, one of the most important flatfish in European aquaculture. This map will be suitable for QTL identification of productive traits in this species and for further evolutionary studies in fish and vertebrate species.
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http://dx.doi.org/10.1534/genetics.107.075416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2219478PMC
December 2007