A gene-based high-resolution comparative radiation hybrid map as a framework for genome sequence assembly of a bovine chromosome 6 region associated with QTL for growth, body composition, and milk performance traits

BACKGROUND: A number of different quantitative trait loci (QTL) for various phenotypic traits, including milk production, functional, and conformation traits in dairy cattle as well as growth and body composition traits in meat cattle, have been mapped consistently in the middle region of bovine chromosome 6 (BTA6). Dense genetic and physical maps and, ultimately, a fully annotated genome sequence as well as their mutual connections are required to efficiently identify genes and gene variants responsible for genetic variation of phenotypic traits. A comprehensive high-resolution gene-rich map linking densely spaced bovine markers and genes to the annotated human genome sequence is required as a framework to facilitate this approach for the region on BTA6 carrying the QTL. RESULTS: Therefore, we constructed a high-resolution radiation hybrid (RH) map for the QTL containing chromosomal region of BTA6. This new RH map with a total of 234 loci including 115 genes and ESTs displays a substantial increase in loci density compared to existing physical BTA6 maps. Screening the available bovine genome sequence resources, a total of 73 loci could be assigned to sequence contigs, which were already identified as specific for BTA6. For 43 loci, corresponding sequence contigs, which were not yet placed on the bovine genome assembly, were identified. In addition, the improved potential of this high-resolution RH map for BTA6 with respect to comparative mapping was demonstrated. Mapping a large number of genes on BTA6 and cross-referencing them with map locations in corresponding syntenic multi-species chromosome segments (human, mouse, rat, dog, chicken) achieved a refined accurate alignment of conserved segments and evolutionary breakpoints across the species included. CONCLUSION: The gene-anchored high-resolution RH map (1 locus/300 kb) for the targeted region of BTA6 presented here will provide a valuable platform to guide high-quality assembling and annotation of the currently existing bovine genome sequence draft to establish the final architecture of BTA6. Hence, a sequence-based map will provide a key resource to facilitate prospective continued efforts for the selection and validation of relevant positional and functional candidates underlying QTL for milk production and growth-related traits mapped on BTA6 and on similar chromosomal regions from evolutionary closely related species like sheep and goat. Furthermore, the high-resolution sequence-referenced BTA6 map will enable precise identification of multi-species conserved chromosome segments and evolutionary breakpoints in mammalian phylogenetic studies

\u3ci\u3eAMBYSTOMA\u3c/i\u3e: PERSPECTIVES ON ADAPTATION AND THE EVOLUTION OF VERTEBRATE GENOMES

Tiger salamanders, and especially the Mexican axolotl (Ambystoma mexicanum), are important model organisms in biological research. This dissertation describes new genomic resources and scientific results that greatly extend the utility of tiger salamanders. With respect to new resources, this dissertation describes the development of expressed sequence tags and assembled contigs, a comparative genome map, a web-portal that makes genomic information freely available to the scientific community, and a computer program that compares structure features of organism genomes. With respect to new scientific results, this dissertation describes a quantitative trait locus that is associated with ecologically and evolutionarily relevant variation in developmental timing, the evolutionary history of the tiger salamander genome in relation to other vertebrate genomes, the likely origin of amniote sex chromosomes, and the identification of the Mexican axolotl sex-determining locus. This dissertation is concluded with a brief outline of future research directions that can extend from the works that are presented here

Detecting parent of origin and dominant QTL in a two-generation commercial poultry pedigree using variance component methodology

<p>Abstract</p> <p>Introduction</p> <p>Variance component QTL methodology was used to analyse three candidate regions on chicken chromosomes 1, 4 and 5 for dominant and parent-of-origin QTL effects. Data were available for bodyweight and conformation score measured at 40 days from a two-generation commercial broiler dam line. One hundred dams were nested in 46 sires with phenotypes and genotypes on 2708 offspring. Linear models were constructed to simultaneously estimate fixed, polygenic and QTL effects. Different genetic models were compared using likelihood ratio test statistics derived from the comparison of full with reduced or null models. Empirical thresholds were derived by permutation analysis.</p> <p>Results</p> <p>Dominant QTL were found for bodyweight on chicken chromosome 4 and for bodyweight and conformation score on chicken chromosome 5. Suggestive evidence for a maternally expressed QTL for bodyweight and conformation score was found on chromosome 1 in a region corresponding to orthologous imprinted regions in the human and mouse.</p> <p>Conclusion</p> <p>Initial results suggest that variance component analysis can be applied within commercial populations for the direct detection of segregating dominant and parent of origin effects.</p

Evolution and gene regulation of the genomic imprinting mechanism

Genomic imprinting describes an epigenetic mechanism by which genes are active or silent depending on their parental origin. Imprinting exists in plants and mammals, but how this monoallelic expression mechanism has evolved is not understood at the molecular level. Here I describe the mapping, sequencing and analysis of vertebrate orthologous imprinted regions spanning 11.5 Mb of genomic sequence from species with and without genomic imprinting. In eutherian (placental) mammals, imprinting can be regulated by differential DNA methylation, non-coding RNAs, enhancers and insulator elements. The systematic sequence comparison of the IGF2-H19 imprinting cluster, in eutherians and marsupials (tammar wallaby and opossum), has revealed the presence of the enigmatic non-coding RNA H19 in marsupials. Furthermore, we have characterised the marsupial H19 expression status and identified key regulatory elements required for the germline imprinting of the neighbouring IGF2 gene. All the major hallmarks of the imprinting mechanism of the IGF2-H19 locus were found to be conserved in therian mammals. In mammals, this imprinting system is therefore the most conserved germline derived epigenetic mechanism discovered so far. The high-quality genomic sequences have provided early glimpses of the genomic landscapes for species such as the monotreme platypus and marsupial tammar wallaby for which little was previously known. Comparative sequence analysis was used to identify candidate regulatory elements in the neighbouring imprinting centre 1 and 2 regions of human chromosome 11p15.5. Nine novel enhancer elements were identified following in vitro gene-reporter assays and correlation of conserved sequences with recent ENCODE data revealed probable functions for a further 24 elements. This project has led to the formation of the Sequence Analysis of Vertebrate Orthologous Imprinted Regions (SAVOIR) consortium and resources developed here are being used by the imprinting community to further our knowledge of the evolution of the genomic imprinting mechanism

The complete sequence of human chromosome 5

Chromosome 5 is one of the largest human chromosomes yet has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding and syntenic conservation with non-mammalian vertebrates, suggesting they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-encoding genes including the protocadherin and interleukin gene families and the first complete versions of each of the large chromosome 5 specific internal duplications. These duplications are very recent evolutionary events and play a likely mechanistic role, since deletions of these regions are the cause of debilitating disorders including spinal muscular atrophy (SMA)

Genetic variation in the chicken genome: insights in selection

The chicken currently provides more than a quarter of the meat and nearly all eggs produced worldwide. For future improvements in production traits and animal welfare as well as to address future consumer demands it is necessary to understand the etiology and biology underlying production traits and diseases. The primary aim of the research described in this thesis was to investigate the utility of several molecular approaches to identify causative variants underlying a variety of traits in the chicken. The general introduction in chapter 1 provides an overview of the domestication history of the chicken - with a particular focus on commercial chicken breeds - and describes the importance to identify causative variants underlying production traits and diseases. Furthermore, several different molecular techniques and methods are introduced that are being used to detect causative variants underlying monogenic and polygenic traits. Linkage maps are essential for linkage analysis, important to study recombination rates and recombination hotspots within the genome and can assist in the sequence assembly of genomes. In chapter 2 we describe the construction of a new high-resolution linkage map of the chicken genome based on two chicken populations with a total of 1619 individuals. The two populations used are a purebred broiler line and a broiler x broiler cross. This high resolution allowed accurate identification of recombination hotspots in the chicken genome, including sex specific recombination. Furthermore, to improve the current reference genome (WASHUC2), 613 unmapped markers were included in the genome-wide assay that included a total of 17,790 SNPs. The resulting linkage map comprises 13,340 SNPs, of which 360 had not been assigned to a known chromosome on chicken genome build WASHUC2. The resulting linkage map is composed of 31 linkage groups, with a total length of 3,054 cM for the sex-average map of the combined population. Regional differences in recombination hotspots between the two mapping populations were observed for several chromosomes near the telomere of the p arm. The sex-specific analysis revealed that these regional differences were mainly caused by female-specific recombination hotspots in the broiler × broiler cross. In chapter 3 we describe the molecular characterization of the locus causing the late feathering phenotype; a monogenic trait in chicken that results in a delayed emergence of flight feathers at hatch. The late feathering phenotype is beneficial to breeders as it can be used for sex typing at hatch. The locus has, therefore, been extensively used in diverse commercial chicken breeds. However, a retrovirus closely linked to the late feathering allele causes a negative pleiotropic effect on egg production and causes viral infections. Within this chapter we describe the identification of a 180 kb tandem duplication in the late feathering allele using a quantitative PCR approach. The tandem duplication results in the partial duplication of two genes; the prolactin receptor and the gene encoding sperm flagellar protein 2. Sequence analysis revealed that the duplication is identical in broiler, white egg-layer, and brown egg-layer lines. This information was also used to design a molecular test to detect this duplication, particularly in heterozygous individuals. The recent advances in massive parallel sequencing technologies have enabled rapid and cost-effective detection of all genetic variants within genomes. The detection of all genetic variants within a genome has further increased our ability to identify causative variants underlying quantitative trait loci (QTL). In chapter 4, we combined a genome-wide association study with whole-genome resequencing to identify causative variants underlying the pulmonary hypertension syndrome (PHS), a polygenic trait in chicken. PHS is a metabolic disease that has been linked to intense selection on growth rate and feed conversion ratio of modern broilers (meat-type chicken). PHS has become one of the most frequent causes of mortality within the broiler industry and leads to substantial economic losses and reduced animal welfare. In total, 18 QTL regions were identified in the genome-wide association study. In order to detect causative variants underlying these QTL regions, we sequenced the genomes of twelve individuals. To maximize the detection of causative variants we selected the individuals based on extreme phenotypes for PHS. Within 8 QTL regions we identified a total of 10 genes that contain at least one variant that is predicted to affect protein function. Moreover, 7.62 million SNPs were detected within the twelve animals compared to the reference genome. These markers can be used in the development of future genome-wide assays. Genomic regions that have undergone selection should contain loci that influence important phenotypic traits and will, therefore, include causative variant(s) that could aid in further future improvement of production traits and disease resistance. In chapter 5, we applied hitch-hiking mapping to make a broad assessment of the effects of selection histories in domesticated chicken. Towards this end, we sampled commercial chickens representing all major breeding goals from multiple breeding companies. In addition, we sampled non-commercial chicken diversity by sampling almost all recognized traditional Dutch breeds and a representative sample of breeds from China. The broad sample of 67 commercial and non-commercial breeds were assessed for signatures of selection in the genome using information of 57,636 SNPs that were genotyped on pooled DNA samples. Our approach demonstrates the strength of including many different populations with similar, and breed groups with different selection histories to reduce stochastic effects based on single populations. The detection of regions of putative selection resulted in the identification of several candidate genes that could aid in further improvement of production traits and disease resistance. Finally, the general discussion in chapter 6 describes the main findings of this thesis. In this chapter recommendations are given for the best strategies to detect causative variants underlying monogenic or polygenic traits. All strategies can benefit substantially from the recent developments in massive parallel sequencing, although the high costs of this method currently prevent large scale studies. In order to perform powerful and cost-effective studies, several strategies are discussed that combine massive parallel sequencing with other existing methods and techniques. Furthermore, the limitations of the different strategies are addressed, as well as the improvements needed in the near future to identify causative variants underlying a variety of traits in, but not limited to, the chicken.   </p

An initial comparative map of copy number variations in the goat (Capra hircus) genome

<p>Abstract</p> <p>Background</p> <p>The goat (<it>Capra hircus</it>) represents one of the most important farm animal species. It is reared in all continents with an estimated world population of about 800 million of animals. Despite its importance, studies on the goat genome are still in their infancy compared to those in other farm animal species. Comparative mapping between cattle and goat showed only a few rearrangements in agreement with the similarity of chromosome banding. We carried out a cross species cattle-goat array comparative genome hybridization (aCGH) experiment in order to identify copy number variations (CNVs) in the goat genome analysing animals of different breeds (Saanen, Camosciata delle Alpi, Girgentana, and Murciano-Granadina) using a tiling oligonucleotide array with ~385,000 probes designed on the bovine genome.</p> <p>Results</p> <p>We identified a total of 161 CNVs (an average of 17.9 CNVs per goat), with the largest number in the Saanen breed and the lowest in the Camosciata delle Alpi goat. By aggregating overlapping CNVs identified in different animals we determined CNV regions (CNVRs): on the whole, we identified 127 CNVRs covering about 11.47 Mb of the virtual goat genome referred to the bovine genome (0.435% of the latter genome). These 127 CNVRs included 86 loss and 41 gain and ranged from about 24 kb to about 1.07 Mb with a mean and median equal to 90,292 bp and 49,530 bp, respectively. To evaluate whether the identified goat CNVRs overlap with those reported in the cattle genome, we compared our results with those obtained in four independent cattle experiments. Overlapping between goat and cattle CNVRs was highly significant (P < 0.0001) suggesting that several chromosome regions might contain recurrent interspecies CNVRs. Genes with environmental functions were over-represented in goat CNVRs as reported in other mammals.</p> <p>Conclusions</p> <p>We describe a first map of goat CNVRs. This provides information on a comparative basis with the cattle genome by identifying putative recurrent interspecies CNVs between these two ruminant species. Several goat CNVs affect genes with important biological functions. Further studies are needed to evaluate the functional relevance of these CNVs and their effects on behavior, production, and disease resistance traits in goats.</p