Integrating the markers Pan I and haemoglobin with the genetic linkage map of Atlantic cod (Gadus morhua)

<p>Abstract</p> <p>Background</p> <p>Haemoglobin (Hb) and pantophysin (Pan I) markers have been used intensively in population studies of Atlantic cod (<it>Gadus morhua</it>) and in the analysis of traits such as temperature tolerance, growth characteristics and sexual maturation. We used an Illumina GoldenGate panel and the KASPar SNP genotyping system to analyse SNPs in three Atlantic cod families, one of which was polymorphic at the Hb β1 locus, and to generate a genetic linkage map integrating Pan I and multiple Hb loci.</p> <p>Findings</p> <p>Data generated allowed the mapping of nine Hb loci, the Pan I locus, and other 122 SNPs onto an existing linkage genetic map for Atlantic cod. Four Hb genes (i.e. α1, α4, β1 and β5) have been mapped on linkage group (LG) 2 while the other five (i.e. α2, α3, β2, β3 and β4) were placed on LG18. Pan I was mapped on LG 1 using a newly developed KASPar assay for a SNP variable only in Pan I^Aallelic variants. The new linkage genetic map presented here comprises 1046 SNPs distributed between 23 linkage groups, with a length of 1145.6 cM. A map produced by forcing additional loci, resulting in a reduced goodness-of-fit for mapped markers, allowed the mapping of a total of 1300 SNPs. Finally, we compared our genetic linkage map data with the genetic linkage map data produced by a different group and identified 29 shared SNPs distributed on 10 different linkage groups.</p> <p>Conclusions</p> <p>The genetic linkage map presented here incorporates the marker Pan I, together with multiple Hb loci, and integrates genetic linkage data produced by two different research groups. This represents a useful resource to further explore if Pan I and Hbs or other genes underlie quantitative trait loci (QTL) for temperature sensitivity/tolerance or other phenotypes.</p

Development of a SNP resource and a genetic linkage map for Atlantic cod (Gadus morhua)

<p>Abstract</p> <p>Background</p> <p>Atlantic cod (<it>Gadus morhua</it>) is a species with increasing economic significance for the aquaculture industry. The genetic improvement of cod will play a critical role in achieving successful large-scale aquaculture. While many microsatellite markers have been developed in cod, the number of single nucleotide polymorphisms (SNPs) is currently limited. Here we report the identification of SNPs from sequence data generated by a large-scale expressed sequence tag (EST) program, focusing on fish originating from Canadian waters.</p> <p>Results</p> <p>A total of 97976 ESTs were assembled to generate 13448 contigs. We detected 4753 SNPs that met our selection criteria (depth of coverage ≥ 4 reads; minor allele frequency > 25%). 3072 SNPs were selected for testing. The percentage of successful assays was 75%, with 2291 SNPs amplifying correctly. Of these, 607 (26%) SNPs were monomorphic for all populations tested. In total, 64 (4%) of SNPs are likely to represent duplicated genes or highly similar members of gene families, rather than alternative alleles of the same gene, since they showed a high frequency of heterozygosity. The remaining polymorphic SNPs (1620) were categorised as validated SNPs. The mean minor allele frequency of the validated loci was 0.258 (± 0.141). Of the 1514 contigs from which validated SNPs were selected, 31% have a significant blast hit. For the SNPs predicted to occur in coding regions (141), we determined that 36% (51) are non-synonymous. Many loci (1033 SNPs; 64%) are polymorphic in all populations tested. However a small number of SNPs (184) that are polymorphic in the Western Atlantic were monomorphic in fish tested from three European populations. A preliminary linkage map has been constructed with 23 major linkage groups and 924 mapped SNPs.</p> <p>Conclusions</p> <p>These SNPs represent powerful tools to accelerate the genetic improvement of cod aquaculture. They have been used to build a genetic linkage map that can be applied to quantitative trait locus (QTL) discovery. Since these SNPs were generated from ESTs, they are linked to specific genes. Genes that map within QTL intervals can be prioritized for testing to determine whether they contribute to observed phenotypes.</p

Complete Sequence and Analysis of the Mitochondrial Genome of Hemiselmis andersenii CCMP644 (Cryptophyceae)

<p>Abstract</p> <p>Background</p> <p>Cryptophytes are an enigmatic group of unicellular eukaryotes with plastids derived by secondary (i.e., eukaryote-eukaryote) endosymbiosis. Cryptophytes are unusual in that they possess four genomes–a host cell-derived nuclear and mitochondrial genome and an endosymbiont-derived plastid and 'nucleomorph' genome. The evolutionary origins of the host and endosymbiont components of cryptophyte algae are at present poorly understood. Thus far, a single complete mitochondrial genome sequence has been determined for the cryptophyte <it>Rhodomonas salina</it>. Here, the second complete mitochondrial genome of the cryptophyte alga <it>Hemiselmis andersenii </it>CCMP644 is presented.</p> <p>Results</p> <p>The <it>H. andersenii </it>mtDNA is 60,553 bp in size and encodes 30 structural RNAs and 36 protein-coding genes, all located on the same strand. A prominent feature of the genome is the presence of a ~20 Kbp long intergenic region comprised of numerous tandem and dispersed repeat units of between 22–336 bp. Adjacent to these repeats are 27 copies of palindromic sequences predicted to form stable DNA stem-loop structures. One such stem-loop is located near a GC-rich and GC-poor region and may have a regulatory function in replication or transcription. The <it>H. andersenii </it>mtDNA shares a number of features in common with the genome of the cryptophyte <it>Rhodomonas salina</it>, including general architecture, gene content, and the presence of a large repeat region. However, the <it>H. andersenii </it>mtDNA is devoid of inverted repeats and introns, which are present in <it>R. salina</it>. Comparative analyses of the suite of tRNAs encoded in the two genomes reveal that the <it>H. andersenii </it>mtDNA has lost or converted its original <it>trnK(uuu) </it>gene and possesses a <it>trnS</it>-derived '<it>trnK(uuu)</it>', which appears unable to produce a functional tRNA. Mitochondrial protein coding gene phylogenies strongly support a variety of previously established eukaryotic groups, but fail to resolve the relationships among higher-order eukaryotic lineages.</p> <p>Conclusion</p> <p>Comparison of the <it>H. andersenii </it>and <it>R. salina </it>mitochondrial genomes reveals a number of cryptophyte-specific genomic features, most notably the presence of a large repeat-rich intergenic region. However, unlike <it>R. salina</it>, the <it>H. andersenii </it>mtDNA does not possess introns and lacks a Lys-tRNA, which is presumably imported from the cytosol.</p

Heat-shock responsive genes identified and validated in Atlantic cod (Gadus morhua) liver, head kidney and skeletal muscle using genomic techniques

Background: Daily and seasonal changes in temperature are challenges that fish within aquaculture settings cannot completely avoid, and are known to elicit complex organismal and cellular stress responses. We conducted a large-scale gene discovery and transcript expression study in order to better understand the genes that are potentially involved in the physiological and cellular aspects of stress caused by heat-shock. We used suppression subtractive hybridization (SSH) cDNA library construction and characterization to identify transcripts that were dysregulated by heat-shock in liver, skeletal muscle and head kidney of Atlantic cod. These tissues were selected due to their roles in metabolic regulation, locomotion and growth, and immune function, respectively. Fish were exposed for 3 hours to an 8°C elevation in temperature, and then allowed to recover for 24 hours at the original temperature (i.e. 10°C). Tissue samples obtained before heat-shock (BHS), at the cessation of heat-shock (CS), and 3, 12, and 24 hours after the cessation of heat-shock (ACS), were used for reciprocal SSH library construction and quantitative reverse transcription - polymerase chain reaction (QPCR) analysis of gene expression using samples from a group that was transferred but not heat-shocked (CT) as controls.Results: We sequenced and characterized 4394 ESTs (1524 from liver, 1451 from head kidney and 1419 from skeletal muscle) from three "forward subtracted" libraries (enriched for genes up-regulated by heat-shock) and 1586 from the liver "reverse subtracted" library (enriched for genes down-regulated by heat-shock), for a total of 5980 ESTs. Several cDNAs encoding putative chaperones belonging to the heat-shock protein (HSP) family were found in these libraries, and "protein folding" was among the gene ontology (GO) terms with the highest proportion in the libraries. QPCR analysis of HSP90α and HSP70-1 (synonym: HSPA1A) mRNA expression showed significant up-regulation in all three tissues studied. These transcripts were more than 100-fold up-regulated in liver following heat-shock. We also identified HSP47, GRP78 and GRP94-like transcripts, which were significantly up-regulated in all 3 tissues studied. Toll-like receptor 22 (TLR22) transcript, found in the liver reverse SSH library, was shown by QPCR to be significantly down-regulated in the head kidney after heat-shock.Conclusion: Chaperones are an important part of the cellular response to stress, and genes identified in this work may play important roles in resistance to thermal-stress. Moreover, the transcript for one key immune response gene (TLR22) was down-regulated by heat-shock, and this down-regulation may be a component of heat-induced immunosuppression

Atlantic cod (Gadus morhua) hemoglobin genes: multiplicity and polymorphism

Background: Hemoglobin (Hb) polymorphism, assessed by protein gel electrophoresis, has been used almost exclusively to characterize the genetic structure of Atlantic cod (Gadus morhua) populations and to establish correlations with phenotypic traits such as Hb oxygen binding capacity, temperature tolerance and growth characteristics. The genetic system used to explain the results of gel electrophoresis entails the presence of one polymorphic locus with two major alleles (HbI-1; HbI-2). However, vertebrates have more than one gene encoding Hbs and recent studies have reported that more than one Hb gene is present in Atlantic cod. These observations prompted us to re-evaluate the number of Hb genes expressed in Atlantic cod, and to perform an in depth search for polymorphisms that might produce relevant phenotypes for breeding programs. Results: Analysis of Expressed Sequence Tags (ESTs) led to the identification of nine distinct Hb transcripts; four corresponding to the α Hb gene family and five to the β Hb gene family. To gain insights about the Hb genes encoding these transcripts, genomic sequence data was generated from heterozygous (HbI-1/2) parents and fifteen progeny; five of each HbI type, i.e., HbI-1/1, HbI-1/2 and HbI-2/2. β Hb genes displayed more polymorphism than α Hb genes. Two major allele types (β1A and β1B) that differ by two linked non-synonymous substitutions (Met55Val and Lys62Ala) were found in the β1 Hb gene, and the distribution of these β1A and β1B alleles among individuals was congruent with that of the HbI-1 and HbI-2 alleles determined by protein gel electrophoresis. RT-PCR and Q-PCR analysis of the nine Hb genes indicates that all genes are expressed in adult fish, but their level of expression varies greatly; higher expression of almost all Hb genes was found in individuals displaying the HbI-2/2 electrophoretic type. Conclusion: This study indicates that more Hb genes are present and expressed in adult Atlantic cod than previously documented. Our finding that nine Hb genes are expressed simultaneously in adult fish suggests that Atlantic cod, similarly to fish such as rainbow trout, carp, and goldfish, might be able to respond to environmental challenges such as chronic hypoxia or long-term changes in temperature by altering the level of expression of these genes. In this context, the role of the non-conservative substitution Lys62Ala found in the β1 Hb gene, which appears to explain the occurrence of the HbI-1 and HbI-2 alleles described by gel electrophoresis, and which was found to be present in other fish such as eel, emerald rockcod, rainbow trout and moray, requires further investigation

Development and Experimental Validation of a 20K Atlantic Cod (Gadus morhua) Oligonucleotide Microarray Based on a Collection of over 150,000 ESTs

The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research

Mutations in the UBIAD1 Gene, Encoding a Potential Prenyltransferase, Are Causal for Schnyder Crystalline Corneal Dystrophy

Schnyder crystalline corneal dystrophy (SCCD, MIM 121800) is a rare autosomal dominant disease characterized by progressive opacification of the cornea resulting from the local accumulation of lipids, and associated in some cases with systemic dyslipidemia. Although previous studies of the genetics of SCCD have localized the defective gene to a 1.58 Mbp interval on chromosome 1p, exhaustive sequencing of positional candidate genes has thus far failed to reveal causal mutations. We have ascertained a large multigenerational family in Nova Scotia affected with SCCD in which we have confirmed linkage to the same general area of chromosome 1. Intensive fine mapping in our family revealed a 1.3 Mbp candidate interval overlapping that previously reported. Sequencing of genes in our interval led to the identification of five putative causal mutations in gene UBIAD1, in our family as well as in four other small families of various geographic origins. UBIAD1 encodes a potential prenyltransferase, and is reported to interact physically with apolipoprotein E. UBIAD1 may play a direct role in intracellular cholesterol biochemistry, or may prenylate other proteins regulating cholesterol transport and storage

Mitochondrial ATPase : biochemical and molecular genetic analysis

S.cerevisiae mutants were isolated showing nuclear-coded resistance to the antibiotic venturicidin, a known F₀ATPase binding antibiotic. Two types of mutant were identified, one of which had cross-resistance to a variety of antibiotics and appeared linked to the leu₁ locus on chromosome VII, and one which was cross-resistant to chloramphenicol only and not linked to leu₁. The level of resistance to venturicidin was not increased in isolated mitochondria, therefore resistance shown in both groups is believed to be due to a decrease in plasma membrane permeability to these antibiotics. The fluorescence properties of several organotin compounds, derivatives of the substituted flavones 3-hydroxyflavone (hof) and penta-hydroxyflavone (morin), were investigated on incubation with rat liver mitochondria. The compound Bu₂SnBr(of) was found to show fluorescence enhancement when added to mitochondrial preparations, which could be lowered by addition of the non-fluorescent compound Bu3SnAc. Addition of Bu₂SnBr(of) did not affect mitochondrial membrane potential, and conversely the energetic state of the mitochondrial inner membrane had no effect on Bu₂SnBr(of) fluorescence. This compound was shown to be an inhibitor of mitochondrial ATPase, and is thought to have its binding site on the F₀ moiety of that enzyme complex. The nuclear gene causing respiratory deficiency in the complementation group G57 was cloned and sequenced. This gene (PET₅₇) encoded a protein of 36 Kdal which did not show significant homology with any known protein. The mutant strain was deficient in mitochondrial ATPase activity, but the major F₁ATPase subunits were detected in mutant mitochondria, although in reduced amounts. Mutant F₁ showed abnormal membrane binding and could not be isolated by standard methods. The protein encoded by the gene PET₅₇ is transported into mitochondria and is thought to contribute to processing or assembly of one or more of the cytoplasmic subunits of the F₁F₀ATPase complex

Entering the post-genomic era of malaria research

The sequencing of the genome of Plasmodium falciparum promises to revolutionize the way in which malaria research will be carried out. Beyond simple gene discovery, the genome sequence will facilitate the comprehensive determination of the parasite?s gene expression during its developmental phases, pathology, and in response to environmental variables, such as drug treatment and host genetic background. This article reviews the current status of the P. falciparum genome sequencing project and the unique insights it has generated. We also summarize the application of bioinformatics and analytical tools that have been developed for functional genomics. The aim of these activities is the rational, information-based identification of new therapeutic strategies and targets, based on a thorough insight into the biology of Plasmodium spp