Gene expression identifies metabolic and functional differences between intramuscular and subcutaneous adipocytes in cattle.

BACKGROUND: This study used a genome-wide screen of gene expression to better understand the metabolic and functional differences between commercially valuable intramuscular fat (IMF) and commercially wasteful subcutaneous (SC) fat depots in Bos taurus beef cattle. RESULTS: We confirmed many findings previously made at the biochemical level and made new discoveries. The fundamental lipogenic machinery, such as ACACA and FASN encoding the rate limiting Acetyl CoA carboxylase and Fatty Acid synthase were expressed at 1.6-1.8 fold lower levels in IMF, consistent with previous findings. The FA elongation pathway including the rate limiting ELOVL6 was also coordinately downregulated in IMF compared to SC as expected. A 2-fold lower expression in IMF of ACSS2 encoding Acetyl Coenzyme A synthetase is consistent with utilisation of less acetate for lipogenesis in IMF compared to SC as previously determined using radioisotope incorporation. Reduced saturation of fat in the SC depot is reflected by 2.4 fold higher expression of the SCD gene encoding the Δ9 desaturase enzyme. Surprisingly, CH25H encoding the cholesterol 25 hydroxylase enzyme was ~ 36 fold upregulated in IMF compared to SC. Moreover, its expression in whole muscle tissue appears representative of the proportional representation of bovine marbling adipocytes. This suite of observations prompted quantification of a set of oxysterols (oxidised forms of cholesterol) in the plasma of 8 cattle exhibiting varying IMF. Using Liquid Chromatography-Mass Spectrometry (LC-MS) we found the levels of several oxysterols were significantly associated with multiple marbling measurements across the musculature, but (with just one exception) no other carcass phenotypes. CONCLUSIONS: These data build on our molecular understanding of ruminant fat depot biology and suggest oxysterols represent a promising circulating biomarker for cattle marbling.

Polygenic and sex specific architecture for two maturation traits in farmed Atlantic salmon.

BACKGROUND: A key developmental transformation in the life of all vertebrates is the transition to sexual maturity, whereby individuals are capable of reproducing for the first time. In the farming of Atlantic salmon, early maturation prior to harvest size has serious negative production impacts. RESULTS: We report genome wide association studies (GWAS) using fish measured for sexual maturation in freshwater or the marine environment. Genotypic data from a custom 50 K single nucleotide polymorphism (SNP) array was used to identify 13 significantly associated SNP for freshwater maturation with the most strongly associated on chromosomes 10 and 11. A higher number of associations (48) were detected for marine maturation, and the two peak loci were found to be the same for both traits. The number and broad distribution of GWAS hits confirmed a highly polygenetic nature, and GWAS performed separately within males and females revealed sex specific genetic behaviour for loci co-located with positional candidate genes phosphatidylinositol-binding clathrin assembly protein-like (picalm) and membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2 (magi2). CONCLUSIONS: The results extend earlier work and have implications for future applied breeding strategies to delay maturation in this important aquaculture species.

Non-synonymous mutations mapped to chromosome X associated with andrological and growth traits in beef cattle.

BACKGROUND: Previous genome-wide association analyses identified QTL regions in the X chromosome for percentage of normal sperm and scrotal circumference in Brahman and Tropical Composite cattle. These traits are important to be studied because they are indicators of male fertility and are correlated with female sexual precocity and reproductive longevity. The aim was to investigate candidate genes in these regions and to identify putative causative mutations that influence these traits. In addition, we tested the identified mutations for female fertility and growth traits. RESULTS: Using a combination of bioinformatics and molecular assay technology, twelve non-synonymous SNPs in eleven genes were genotyped in a cattle population. Three and nine SNPs explained more than 1% of the additive genetic variance for percentage of normal sperm and scrotal circumference, respectively. The SNPs that had a major influence in percentage of normal sperm were mapped to LOC100138021 and TAF7L genes; and in TEX11 and AR genes for scrotal circumference. One SNP in TEX11 was explained ~13% of the additive genetic variance for scrotal circumference at 12 months. The tested SNP were also associated with weight measurements, but not with female fertility traits. CONCLUSIONS: The strong association of SNPs located in X chromosome genes with male fertility traits validates the QTL. The implicated genes became good candidates to be used for genetic evaluation, without detrimentally influencing female fertility traits.

Next-generation sequencing: a challenge to meet the increasing demand for training workshops in Australia.

The widespread adoption of high-throughput next-generation sequencing (NGS) technology among the Australian life science research community is highlighting an urgent need to up-skill biologists in tools required for handling and analysing their NGS data. There is currently a shortage of cutting-edge bioinformatics training courses in Australia as a consequence of a scarcity of skilled trainers with time and funding to develop and deliver training courses. To address this, a consortium of Australian research organizations, including Bioplatforms Australia, the Commonwealth Scientific and Industrial Research Organisation and the Australian Bioinformatics Network, have been collaborating with EMBL-EBI training team. A group of Australian bioinformaticians attended the train-the-trainer workshop to improve training skills in developing and delivering bioinformatics workshop curriculum. A 2-day NGS workshop was jointly developed to provide hands-on knowledge and understanding of typical NGS data analysis workflows. The road show-style workshop was successfully delivered at five geographically distant venues in Australia using the newly established Australian NeCTAR Research Cloud. We highlight the challenges we had to overcome at different stages from design to delivery, including the establishment of an Australian bioinformatics training network and the computing infrastructure and resource development. A virtual machine image, workshop materials and scripts for configuring a machine with workshop contents have all been made available under a Creative Commons Attribution 3.0 Unported License. This means participants continue to have convenient access to an environment they had become familiar and bioinformatics trainers are able to access and reuse these resources.

Whole-genome resequencing uncovers molecular signatures of natural and sexual selection in wild bighorn sheep.

The identification of genes influencing fitness is central to our understanding of the genetic basis of adaptation and how it shapes phenotypic variation in wild populations. Here, we used whole-genome resequencing of wild Rocky Mountain bighorn sheep (Ovis canadensis) to >50-fold coverage to identify 2.8 million single nucleotide polymorphisms (SNPs) and genomic regions bearing signatures of directional selection (i.e. selective sweeps). A comparison of SNP diversity between the X chromosome and the autosomes indicated that bighorn males had a dramatically reduced long-term effective population size compared to females. This probably reflects a long history of intense sexual selection mediated by male-male competition for mates. Selective sweep scans based on heterozygosity and nucleotide diversity revealed evidence for a selective sweep shared across multiple populations at RXFP2, a gene that strongly affects horn size in domestic ungulates. The massive horns carried by bighorn rams appear to have evolved in part via strong positive selection at RXFP2. We identified evidence for selection within individual populations at genes affecting early body growth and cellular response to hypoxia; however, these must be interpreted more cautiously as genetic drift is strong within local populations and may have caused false positives. These results represent a rare example of strong genomic signatures of selection identified at genes with known function in wild populations of a nonmodel species. Our results also showcase the value of reference genome assemblies from agricultural or model species for studies of the genomic basis of adaptation in closely related wild taxa.

Genomic correlation: harnessing the benefit of combining two unrelated populations for genomic selection.

BACKGROUND: The success of genomic selection in animal breeding hinges on the availability of a large reference population on which genomic-based predictions of additive genetic or breeding values are built. Here, we explore the benefit of combining two unrelated populations into a single reference population. METHODS: The datasets consisted of 1829 Brahman and 1973 Tropical Composite cattle with measurements on five phenotypes relevant to tropical adaptation and genotypes for 71,726 genome-wide single nucleotide polymorphisms (SNPs). The underlying genomic correlation for the same phenotype across the two breeds was explored on the basis of consistent linkage disequilibrium (LD) phase and marker effects in both breeds. RESULTS: The proportion of genetic variance explained by the entire set of SNPs ranged from 37.5 to 57.6 %. Estimated genomic correlations were drastically affected by the process used to select SNPs and went from near 0 to more than 0.80 for most traits when using the set of SNPs with significant effects and the same LD phase in the two breeds. We found that, by carefully selecting the subset of SNPs, the missing heritability can be largely recovered and accuracies in genomic predictions can be improved six-fold. However, the increases in accuracy might come at the expense of large biases. CONCLUSIONS: Our results offer hope for the effective implementation of genomic selection schemes in situations where the number of breeds is large, the sample size within any single breed is small and the breeding objective includes many phenotypes.

Information compression exploits patterns of genome composition to discriminate populations and highlight regions of evolutionary interest.

BACKGROUND: Genomic information allows population relatedness to be inferred and selected genes to be identified. Single nucleotide polymorphism microarray (SNP-chip) data, a proxy for genome composition, contains patterns in allele order and proportion. These patterns can be quantified by compression efficiency (CE). In principle, the composition of an entire genome can be represented by a CE number quantifying allele representation and order. RESULTS: We applied a compression algorithm (DEFLATE) to genome-wide high-density SNP data from 4,155 human, 1,800 cattle, 1,222 sheep, 81 dogs and 49 mice samples. All human ethnic groups can be clustered by CE and the clusters recover phylogeography based on traditional fixation index (FST) analyses. CE analysis of other mammals results in segregation by breed or species, and is sensitive to admixture and past effective population size. This clustering is a consequence of individual patterns such as runs of homozygosity. Intriguingly, a related approach can also be used to identify genomic loci that show population-specific CE segregation. A high resolution CE 'sliding window' scan across the human genome, organised at the population level, revealed genes known to be under evolutionary pressure. These include SLC24A5 (European and Gujarati Indian skin pigmentation), HERC2 (European eye color), LCT (European and Maasai milk digestion) and EDAR (Asian hair thickness). We also identified a set of previously unidentified loci with high population-specific CE scores including the chromatin remodeler SCMH1 in Africans and EDA2R in Asians. Closer inspection reveals that these prioritised genomic regions do not correspond to simple runs of homozygosity but rather compositionally complex regions that are shared by many individuals of a given population. Unlike FST, CE analyses do not require ab initio population comparisons and are amenable to the hemizygous X chromosome. CONCLUSIONS: We conclude with a discussion of the implications of CE for a complex systems science view of genome evolution. CE allows one to clearly visualise the evolution of individual genomes and populations through a formal, mathematically-rigorous information space. Overall, CE makes a set of biological predictions, some of which are unique and await functional validation.

Evidence for positive selection of taurine genes within a QTL region on chromosome X associated with testicular size in Australian Brahman cattle.

BACKGROUND: Previous genome-wide association studies have identified significant regions of the X chromosome associated with reproductive traits in two Bos indicus-influenced breeds: Brahman cattle and Tropical Composites. Two QTL regions on this chromosome were identified in both breeds as strongly associated with scrotal circumference measurements, a reproductive trait previously shown to be useful for selection of young bulls. Scrotal circumference is genetically correlated with early age at puberty in both male and female offspring. These QTL were located at positions 69-77 and 81-92 Mb respectively, large areas each to which a significant number of potential candidate genes were mapped. RESULTS: To further characterise these regions, a bioinformatic approach was undertaken to identify novel non-synonymous SNP within the QTL regions of interest in Brahman cattle. After SNP discovery, we used conventional molecular assay technologies to perform studies of two candidate genes in both breeds. Non-synonymous SNP mapped to Testis-expressed gene 11 (Tex11) were associated (P < 0.001) with scrotal circumference in both breeds, and associations with percentage of normal sperm cells were also observed (P < 0.05). Evidence for recent selection was found as Tex11 SNP form a haplotype segment of Bos taurus origin that is retained within Brahman and Tropical Composite cattle with greatest reproductive potential. CONCLUSIONS: Association of non-synonymous SNP presented here are a first step to functional genetic studies. Bovine species may serve as a model for studying the role of Tex11 in male fertility, warranting further in-depth molecular characterisation.

Multi-breed genomic predictions and functional variants for fertility of tropical bulls.

Worldwide, most beef breeding herds are naturally mated. As such, the ability to identify and select fertile bulls is critically important for both productivity and genetic improvement. Here, we collected ten fertility-related phenotypes for 6,063 bulls from six tropically adapted breeds. Phenotypes were comprised of four bull conformation traits and six traits directly related to the quality of the bull's semen. We also generated high-density DNA genotypes for all the animals. In total, 680,758 single nucleotide polymorphism (SNP) genotypes were analyzed. The genomic correlation of the same trait observed in different breeds was positive for scrotal circumference and sheath score on most breed comparisons, but close to zero for the percentage of normal sperm, suggesting a divergent genetic background for this trait. We confirmed the importance of a breed being present in the reference population to the generation of accurate genomic estimated breeding values (GEBV) in an across-breed validation scenario. Average GEBV accuracies varied from 0.19 to 0.44 when the breed was not included in the reference population. The range improved to 0.28 to 0.59 when the breed was in the reference population. Variants associated with the gene HDAC4, six genes from the spermatogenesis-associated (SPATA) family of proteins, and 29 transcription factors were identified as candidate genes. Collectively these results enable very early in-life selection for bull fertility traits, supporting genetic improvement strategies currently taking place within tropical beef production systems. This study also improves our understanding of the molecular basis of male fertility in mammals.

Genome assembly of the Australian black tiger shrimp (Penaeus monodon) reveals a novel fragmented IHHNV EVE sequence.

Shrimp are a valuable aquaculture species globally; however, disease remains a major hindrance to shrimp aquaculture sustainability and growth. Mechanisms mediated by endogenous viral elements have been proposed as a means by which shrimp that encounter a new virus start to accommodate rather than succumb to infection over time. However, evidence on the nature of such endogenous viral elements and how they mediate viral accommodation is limited. More extensive genomic data on Penaeid shrimp from different geographical locations should assist in exposing the diversity of endogenous viral elements. In this context, reported here is a PacBio Sequel-based draft genome assembly of an Australian black tiger shrimp (Penaeus monodon) inbred for 1 generation. The 1.89 Gbp draft genome is comprised of 31,922 scaffolds (N50: 496,398 bp) covering 85.9% of the projected genome size. The genome repeat content (61.8% with 30% representing simple sequence repeats) is almost the highest identified for any species. The functional annotation identified 35,517 gene models, of which 25,809 were protein-coding and 17,158 were annotated using interproscan. Scaffold scanning for specific endogenous viral elements identified an element comprised of a 9,045-bp stretch of repeated, inverted, and jumbled genome fragments of infectious hypodermal and hematopoietic necrosis virus bounded by a repeated 591/590 bp host sequence. As only near complete linear ∼4 kb infectious hypodermal and hematopoietic necrosis virus genomes have been found integrated in the genome of P. monodon previously, its discovery has implications regarding the validity of PCR tests designed to specifically detect such linear endogenous viral element types. The existence of joined inverted infectious hypodermal and hematopoietic necrosis virus genome fragments also provides a means by which hairpin double-stranded RNA could be expressed and processed by the shrimp RNA interference machinery.

Using paired-end sequences to optimise parameters for alignment of sequence reads against related genomes.

BACKGROUND: The advent of cheap high through-put sequencing methods has facilitated low coverage skims of a large number of organisms. To maximise the utility of the sequences, assembly into contigs and then ordering of those contigs is required. Whilst sequences can be assembled into contigs de novo, using assembled genomes of closely related organisms as a framework can considerably aid the process. However, the preferred search programs and parameters that will optimise the sensitivity and specificity of the alignments between the sequence reads and the framework genome(s) are not necessarily obvious. Here we demonstrate a process that uses paired-end sequence reads to choose an optimal program and alignment parameters. RESULTS: Unlike two single fragment reads, in paired-end sequence reads, such as BAC-end sequences, the two sequences in the pair have a known positional relationship in the original genome. This provides an additional level of confidence over match scores and e-values in the accuracy of the positional assignment of the reads in the comparative genome. Three commonly used sequence alignment programs: MegaBLAST, Blastz and PatternHunter were used to align a set of ovine BAC-end sequences against the equine genome assembly. A range of different search parameters, with a particular focus on contiguous and discontiguous seeds, were used for each program. The number of reads with a hit and the number of read pairs with hits for the two end sequences in the tail-to-tail paired-end configuration were plotted relative to the theoretical maximum expected curve. Of the programs tested, MegaBLAST with short contiguous seed lengths (word size 8-11) performed best in this particular task. In addition the data also provides estimates of the false positive and false negative rates, which can be used to determine the appropriate values of additional parameters, such as score cut-off, to balance sensitivity and specificity. To determine whether the approach also worked for the alignment of shorter reads, the first 240 bases of each BAC end sequence were also aligned to the equine genome. Again, contiguous MegaBLAST performed the best in optimising the sensitivity and specificity with which sheep BAC end reads map to the equine and bovine genomes. CONCLUSIONS: Paired-end reads, such as BAC-end sequences, provide an efficient mechanism to optimise sequence alignment parameters, for example for comparative genome assemblies, by providing an objective standard to evaluate performance.

Bovine proteins containing poly-glutamine repeats are often polymorphic and enriched for components of transcriptional regulatory complexes.

BACKGROUND: About forty human diseases are caused by repeat instability mutations. A distinct subset of these diseases is the result of extreme expansions of polymorphic trinucleotide repeats; typically CAG repeats encoding poly-glutamine (poly-Q) tracts in proteins. Polymorphic repeat length variation is also apparent in human poly-Q encoding genes from normal individuals. As these coding sequence repeats are subject to selection in mammals, it has been suggested that normal variations in some of these typically highly conserved genes are implicated in morphological differences between species and phenotypic variations within species. At present, poly-Q encoding genes in non-human mammalian species are poorly documented, as are their functions and propensities for polymorphic variation. RESULTS: The current investigation identified 178 bovine poly-Q encoding genes (Q ≥ 5) and within this group, 26 genes with orthologs in both human and mouse that did not contain poly-Q repeats. The bovine poly-Q encoding genes typically had ubiquitous expression patterns although there was bias towards expression in epithelia, brain and testes. They were also characterised by unusually large sizes. Analysis of gene ontology terms revealed that the encoded proteins were strongly enriched for functions associated with transcriptional regulation and many contributed to physical interaction networks in the nucleus where they presumably act cooperatively in transcriptional regulatory complexes. In addition, the coding sequence CAG repeats in some bovine genes impacted mRNA splicing thereby generating unusual transcriptional diversity, which in at least one instance was tissue-specific. The poly-Q encoding genes were prioritised using multiple criteria for their likelihood of being polymorphic and then the highest ranking group was experimentally tested for polymorphic variation within a cattle diversity panel. Extensive and meiotically stable variation was identified. CONCLUSIONS: Transcriptional diversity can potentially be generated in poly-Q encoding genes by the impact of CAG repeat tracts on mRNA alternative splicing. This effect, combined with the physical interactions of the encoded proteins in large transcriptional regulatory complexes suggests that polymorphic variations of proteins in these complexes have strong potential to affect phenotype.

Skeletal muscle specific genes networks in cattle.

While physiological differences across skeletal muscles have been described, the differential gene expression underlying them and the discovery of how they interact to perform specific biological processes are largely to be elucidated. The purpose of the present study was, firstly, to profile by cDNA microarrays the differential gene expression between two skeletal muscle types, Psoas major (PM) and Flexor digitorum (FD), in beef cattle and then to interpret the results in the context of a bovine gene coexpression network, detecting possible changes in connectivity across the skeletal muscle system. Eighty four genes were differentially expressed (DE) between muscles. Approximately 54% encoded metabolic enzymes and structural-contractile proteins. DE genes were involved in similar processes and functions, but the proportion of genes in each category varied within each muscle. A correlation matrix was obtained for 61 out of the 84 DE genes from a gene coexpression network. Different groups of coexpression were observed, the largest one having 28 metabolic and contractile genes, up-regulated in PM, and mainly encoding fast-glycolytic fibre structural components and glycolytic enzymes. In FD, genes related to cell support seemed to constitute its identity feature and did not positively correlate to the rest of DE genes in FD. Moreover, changes in connectivity for some DE genes were observed in the different gene ontologies. Our results confirm the existence of a muscle dependent transcription and coexpression pattern and suggest the necessity of integrating different muscle types to perform comprehensive networks for the transcriptional landscape of bovine skeletal muscle.

A genomics-informed, SNP association study reveals FBLN1 and FABP4 as contributing to resistance to fleece rot in Australian Merino sheep.

BACKGROUND: Fleece rot (FR) and body-strike of Merino sheep by the sheep blowfly Lucilia cuprina are major problems for the Australian wool industry, causing significant losses as a result of increased management costs coupled with reduced wool productivity and quality. In addition to direct effects on fleece quality, fleece rot is a major predisposing factor to blowfly strike on the body of sheep. In order to investigate the genetic drivers of resistance to fleece rot, we constructed a combined ovine-bovine cDNA microarray of almost 12,000 probes including 6,125 skin expressed sequence tags and 5,760 anonymous clones obtained from skin subtracted libraries derived from fleece rot resistant and susceptible animals. This microarray platform was used to profile the gene expression changes between skin samples of six resistant and six susceptible animals taken immediately before, during and after FR induction. Mixed-model equations were employed to normalize the data and 155 genes were found to be differentially expressed (DE). Ten DE genes were selected for validation using real-time PCR on independent skin samples. The genomic regions of a further 5 DE genes were surveyed to identify single nucleotide polymorphisms (SNP) that were genotyped across three populations for their associations with fleece rot resistance. RESULTS: The majority of the DE genes originated from the fleece rot subtracted libraries and over-representing gene ontology terms included defense response to bacterium and epidermis development, indicating a role of these processes in modulating the sheep's response to fleece rot. We focused on genes that contribute to the physical barrier function of skin, including keratins, collagens, fibulin and lipid proteins, to identify SNPs that were associated to fleece rot scores. CONCLUSIONS: We identified FBLN1 (fibulin) and FABP4 (fatty acid binding protein 4) as key factors in sheep's resistance to fleece rot. Validation of these markers in other populations could lead to vital tests for marker assisted selection that will ultimately increase the natural fleece rot resistance of Merino sheep.

Changed Patterns of Genomic Variation Following Recent Domestication: Selection Sweeps in Farmed Atlantic Salmon.

The introduction of wild Atlantic salmon into captivity, and their subsequent artificial selection for production traits, has caused phenotypic differences between domesticated fish and their wild counterparts. Identification of regions of the genome underling these changes offers the promise of characterizing the early biological consequences of domestication. In the current study, we sequenced a population of farmed European Atlantic salmon and compared the observed patterns of SNP variation to those found in conspecific wild populations. This identified 139 genomic regions that contained significantly elevated SNP homozygosity in farmed fish when compared to their wild counterparts. The most extreme was adjacent to versican, a gene involved in control of neural crest cell migration. To control for false positive signals, a second and independent dataset of farmed and wild European Atlantic salmon was assessed using the same methodology. A total of 81 outlier regions detected in the first dataset showed significantly reduced homozygosity within the second one, strongly suggesting the genomic regions identified are enriched for true selection sweeps. Examination of the associated genes identified a number previously characterized as targets of selection in other domestic species and that have roles in development, behavior and olfactory system. These include arcvf, sema6, errb4, id2-like, and 6n1-like genes. Finally, we searched for evidence of parallel sweeps using a farmed population of North American origin. This failed to detect a convincing overlap to the putative sweeps present in European populations, suggesting the factors that drive patterns of variation under domestication and early artificial selection were largely independent. This is the first analysis on domestication of aquaculture species exploiting whole-genome sequence data and resulted in the identification of sweeps common to multiple independent populations of farmed European Atlantic salmon.

A low-density SNP genotyping panel for the accurate prediction of cattle breeds.

Genomic tools to better define breed composition in agriculturally important species have sparked scientific and commercial industry interest. Knowledge of breed composition can inform multiple scientifically important decisions of industry application including DNA marker-assisted selection, identification of signatures of selection, and inference of product provenance to improve supply chain integrity. Genomic tools are expensive but can be economized by deploying a relatively small number of highly informative single-nucleotide polymorphisms (SNP) scattered evenly across the genome. Using resources from the 1000 Bull Genomes Project we established calibration (more stringent quality criteria; N = 1,243 cattle) and validation (less stringent; N = 864) data sets representing 17 breeds derived from both taurine and indicine bovine subspecies. Fifteen successively smaller panels (from 500,000 to 50 SNP) were built from those SNP in the calibration data that increasingly satisfied 2 criteria, high differential allele frequencies across the breeds as measured by average Euclidean distance (AED) and high uniformity (even spacing) across the physical genome. Those SNP awarded the highest AED were in or near genes previously identified as important signatures of selection in cattle such as LCORL, NCAPG, KITLG, and PLAG1. For each panel, the genomic breed composition (GBC) of each animal in the validation dataset was estimated using a linear regression model. A systematic exploration of the predictive accuracy of the various sized panels was then undertaken on the validation population using 3 benchmarking approaches: (1) % error (expressed relative to the estimated GBC made from over 1 million SNP), (2) % breed misassignment (expressed relative to each individual's breed recorded), and (3) Shannon's entropy of estimated GBC across the 17 target breeds. Our analyses suggest that a panel of just 250 SNP represents an adequate balance between accuracy and cost-only modest gains in accuracy are made as one increases panel density beyond this point.

A multiway analysis for identifying high integrity bovine BACs.

BACKGROUND: In large genomics projects involving many different types of analyses of bacterial artificial chromosomes (BACs), such as fingerprinting, end sequencing (BES) and full BAC sequencing there are many opportunities for the identities of BACs to become confused. However, by comparing the results from the different analyses, inconsistencies can be identified and a set of high integrity BACs preferred for future research can be defined. RESULTS: The location of each bovine BAC in the BAC fingerprint-based genome map and in the genome assembly were compared based on the reported BESs, and for a smaller number of BACs the full sequence. BACs with consistent positions in all three datasets, or if the full sequence was not available, for both the fingerprint map and BES-based alignments, were deemed to be correctly positioned. BACs with consistent BES-based and fingerprint-based locations, but with conflicting locations based on the fully sequenced BAC, appeared to have been misidentified during sequencing, and included a number of apparently swapped BACs. Inconsistencies between BES-based and fingerprint map positions identified thirty one plates from the CHORI-240 library that appear to have suffered substantial systematic problems during the end-sequencing of the BACs. No systematic problems were identified in the fingerprinting of the BACs. Analysis of BACs overlapping in the assembly identified a small overrepresentation of clones with substantial overlap in the library and a substantial enrichment of highly overlapping BACs on the same plate in the CHORI-240 library. More than half of these BACs appear to have been present as duplicates on the original BAC-library plates and thus should be avoided in subsequent projects. CONCLUSION: Our analysis shows that approximately 95% of the bovine CHORI-240 library clones with both a BAC fingerprint and two BESs mapping to the genome in the expected orientations (approximately 27% of all BACs) have consistent locations in the BAC fingerprint map and the genome assembly. We have developed a broadly applicable methodology for checking the integrity of BAC-based datasets even where only incomplete and partially assembled genomic sequence is available.

Analysis of the complement and molecular evolution of tRNA genes in cow.

BACKGROUND: Detailed information regarding the number and organization of transfer RNA (tRNA) genes at the genome level is becoming readily available with the increase of DNA sequencing of whole genomes. However the identification of functional tRNA genes is challenging for species that have large numbers of repetitive elements containing tRNA derived sequences, such as Bos taurus. Reliable identification and annotation of entire sets of tRNA genes allows the evolution of tRNA genes to be understood on a genomic scale. RESULTS: In this study, we explored the B. taurus genome using bioinformatics and comparative genomics approaches to catalogue and analyze cow tRNA genes. The initial analysis of the cow genome using tRNAscan-SE identified 31,868 putative tRNA genes and 189,183 pseudogenes, where 28,830 of the 31,868 predicted tRNA genes were classified as repetitive elements by the RepeatMasker program. We then used comparative genomics to further discriminate between functional tRNA genes and tRNA-derived sequences for the remaining set of 3,038 putative tRNA genes. For our analysis, we used the human, chimpanzee, mouse, rat, horse, dog, chicken and fugu genomes to predict that the number of active tRNA genes in cow lies in the vicinity of 439. Of this set, 150 tRNA genes were 100% identical in their sequences across all nine vertebrate genomes studied. Using clustering analyses, we identified a new tRNA-GlyCCC subfamily present in all analyzed mammalian genomes. We suggest that this subfamily originated from an ancestral tRNA-GlyGCC gene via a point mutation prior to the radiation of the mammalian lineages. Lastly, in a separate analysis we created phylogenetic profiles for each putative cow tRNA gene using a representative set of genomes to gain an overview of common evolutionary histories of tRNA genes. CONCLUSION: The use of a combination of bioinformatics and comparative genomics approaches has allowed the confident identification of a set of cow tRNA genes that will facilitate further studies in understanding the molecular evolution of cow tRNA genes.

Origin, evolution, and biological role of miRNA cluster in DLK-DIO3 genomic region in placental mammals.

MicroRNAs (miRNAs) are a rapidly growing family of small regulatory RNAs modulating gene expression in plants and animals. In animals, most of the miRNAs discovered in early studies were found to be evolutionarily conserved across the whole kingdom. More recent studies, however, have identified many miRNAs that are specific to a particular group of organisms or even a single species. These present a question about evolution of the individual miRNAs and their role in establishing and maintaining lineage-specific functions and characteristics. In this study, we describe a detailed analysis of the miRNA cluster (hereafter mir-379/mir-656 cluster) located within the imprinted DLK-DIO3 region on human chromosome 14. We show that orthologous miRNA clusters are present in all sequenced genomes of the placental (eutherian) mammals but not in the marsupial (metatherian), monotreme (prototherian), or any other vertebrate genomes. We provide evidence that the locus encompassing this cluster emerged in an early eutherian ancestor prior to the radiation of modern placental mammals by tandem duplication of the ancient precursor sequence. The original amplified cluster may have contained in excess of 250 miRNA precursor sequences, most of which now appear to be inactive. Examination of the eutherian genomes showed that the cluster has been maintained in evolution for approximately 100 Myr. Analysis of genes that contain predicted evolutionarily conserved targets for miRNAs from this cluster revealed significant overrepresentation of the Gene Ontology terms associated with biological processes such as neurogenesis, embryonic development, transcriptional regulation, and RNA metabolism. Consistent with these findings, a survey of the miRNA expression data within the cluster demonstrates a strong bias toward brain and placenta samples from adult organisms and some embryonic tissues. Our results suggest that emergence of the mir-379/mir-656 miRNA cluster was one of the factors that facilitated evolution of the placental mammals. Overrepresentation of genes involved in regulation of neurogenesis among predicted miRNAs targets indicates an important role of the mir-379/mir-656 cluster in this biological process in the placental mammals.

Greenlip Abalone (Haliotis laevigata) Genome and Protein Analysis Provides Insights into Maturation and Spawning.

Wild abalone (Family Haliotidae) populations have been severely affected by commercial fishing, poaching, anthropogenic pollution, environment and climate changes. These issues have stimulated an increase in aquaculture production; however production growth has been slow due to a lack of genetic knowledge and resources. We have sequenced a draft genome for the commercially important temperate Australian 'greenlip' abalone (Haliotis laevigata, Donovan 1808) and generated 11 tissue transcriptomes from a female adult abalone. Phylogenetic analysis of the greenlip abalone with reference to the Pacific abalone (Haliotis discus hannai) indicates that these abalone species diverged approximately 71 million years ago. This study presents an in-depth analysis into the features of reproductive dysfunction, where we provide the putative biochemical messenger components (neuropeptides) that may regulate reproduction including gonad maturation and spawning. Indeed, we isolate the egg-laying hormone neuropeptide and under trial conditions induce spawning at 80% efficiency. Altogether, we provide a solid platform for further studies aimed at stimulating advances in abalone aquaculture production. The H. laevigata genome and resources are made available to the public on the abalone 'omics website, http://abalonedb.org.