Large scale sequence-based screen for recessive variants allows for identification and monitoring of rare deleterious variants in pigs.

Most deleterious variants are recessive and segregate at relatively low frequency. Therefore, high sample sizes are required to identify these variants. In this study we report a large-scale sequence based genome-wide association study (GWAS) in pigs, with a total of 120,000 Large White and 80,000 Synthetic breed animals imputed to sequence using a reference population of approximately 1,100 whole genome sequenced pigs. We imputed over 20 million variants with high accuracies (R2>0.9) even for low frequency variants (1-5% minor allele frequency). This sequence-based analysis revealed a total of 14 additive and 9 non-additive significant quantitative trait loci (QTLs) for growth rate and backfat thickness. With the non-additive (recessive) model, we identified a deleterious missense SNP in the CDHR2 gene reducing growth rate and backfat in homozygous Large White animals. For the Synthetic breed, we revealed a QTL on chromosome 15 with a frameshift variant in the OBSL1 gene. This QTL has a major impact on both growth rate and backfat, resembling human 3M-syndrome 2 which is related to the same gene. With the additive model, we confirmed known QTLs on chromosomes 1 and 5 for both breeds, including variants in the MC4R and CCND2 genes. On chromosome 1, we disentangled a complex QTL region with multiple variants affecting both traits, harboring 4 independent QTLs in the span of 5 Mb. Together we present a large scale sequence-based association study that provides a key resource to scan for novel variants at high resolution for breeding and to further reduce the frequency of deleterious alleles at an early stage in the breeding program.

Detailed molecular and epigenetic characterization of the pig IPEC-J2 and chicken SL-29 cell lines.

The pig IPEC-J2 and chicken SL-29 cell lines are of interest because of their untransformed nature and wide use in functional studies. Molecular characterization of these cell lines is important to gain insight into possible molecular aberrations. The aim of this paper is to provide a molecular and epigenetic characterization of the IPEC-J2 and SL-29 cell lines, a cell-line reference for the FAANG community, and future biomedical research. Whole genome sequencing, gene expression, DNA methylation, chromatin accessibility, and ChIP-seq of four histone marks (H3K4me1, H3K4me3, H3K27ac, H3K27me3) and an insulator (CTCF) are used to achieve these aims. Heteroploidy (aneuploidy) of various chromosomes was observed from whole genome sequencing analysis in both cell lines. Furthermore, higher gene expression for genes located on chromosomes with aneuploidy in comparison to diploid chromosomes was observed. Regulatory complexity of gene expression, DNA methylation, and chromatin accessibility was investigated through an integrative approach.

Corrigendum: A novel loss-of-function variant in transmembrane protein 263 (TMEM263) of autosomal dwarfism in chicken.

[This corrects the article DOI: 10.3389/fgene.2018.00193.].

Ancient globetrotters-connectivity and putative native ranges of two cosmopolitan biofouling amphipods.

The geographic distributions of some coastal marine species have appeared as cosmopolitan ever since they were first scientifically documented. In particular, for many benthic species that are associated with anthropogenic substrata, there is much speculation as to whether or not their broad distributions can be explained by natural mechanisms of dispersal. Here, we focused on two congeneric coastal crustaceans with cosmopolitan distributions-the tube-dwelling amphipods Jassa marmorata and Jassa slatteryi. Both species are common elements of marine biofouling on nearly all kinds of artificial hard substrata in temperate to warm seas. We hypothesized that the two species' modern occurrences across the oceans are the result of human shipping activities that started centuries ago. Mitochondrial DNA sequences of the CO1 fragment of specimens from distinct marine regions around the world were analysed, evaluating genetic structure and migration models and making inferences on putative native ranges of the two Jassa species. Populations of both species exhibited considerable genetic diversity with differing levels of geographic structure. For both species, at least two dominant haplotypes were shared among several geographic populations. Rapid demographic expansion and high migration rates between geographically distant regions support a scenario of ongoing dispersal all over the world. Our findings indicate that the likely former native range of J. marmorata is the Northwest Atlantic, whereas the likely former native range of J. slatteryi is the Northern Pacific region. As corroborated by the genetic connectivity between populations, shipping still appears to be the more successful vector of the two species' dispersal when compared to natural mechanisms. Historical invasion events that likely started centuries ago, along with current ongoing dispersal, confirm these species' identities as true "neocosmopolitans".

Parallel Genetic Origin of Foot Feathering in Birds.

Understanding the genetic basis of similar phenotypes shared between lineages is a long-lasting research interest. Even though animal evolution offers many examples of parallelism, for many phenotypes little is known about the underlying genes and mutations. We here use a combination of whole-genome sequencing, expression analyses, and comparative genomics to study the parallel genetic origin of ptilopody (Pti) in chicken. Ptilopody (or foot feathering) is a polygenic trait that can be observed in domesticated and wild avian species and is characterized by the partial or complete development of feathers on the ankle and feet. In domesticated birds, ptilopody is easily selected to fixation, though extensive variation in the type and level of feather development is often observed. By means of a genome-wide association analysis, we identified two genomic regions associated with ptilopody. At one of the loci, we identified a 17-kb deletion affecting PITX1 expression, a gene known to encode a transcription regulator of hindlimb identity and development. Similarly to pigeon, at the second loci, we observed ectopic expression of TBX5, a gene involved in forelimb identity and a key determinant of foot feather development. We also observed that the trait evolved only once as foot-feathered birds share the same haplotype upstream TBX5. Our findings indicate that in chicken and pigeon ptilopody is determined by the same set of genes that affect similar molecular pathways. Our study confirms that ptilopody has evolved through parallel evolution in chicken and pigeon.

Detection of a Frameshift Deletion in the SPTBN4 Gene Leads to Prevention of Severe Myopathy and Postnatal Mortality in Pigs.

Piglet mortality is a complex phenotype that depends on the environment, selection on piglet health, but also on the interaction between the piglet and sow. However, also monogenic recessive defects contribute to piglet mortality. Selective breeding has decreased overall piglet mortality by improving both mothering abilities and piglet viability. However, variants underlying recessive monogenic defects are usually not well captured within the breeding values, potentially drifting to higher frequency as a result of intense selection or genetic drift. This study describes the identification by whole-genome sequencing of a recessive 16-bp deletion in the SPTBN4 gene causing postnatal mortality in a pig breeding line. The deletion induces a frameshift and a premature stop codon, producing an impaired and truncated spectrin beta non-erythrocytic 4 protein (SPTBN4). Applying medium density single nucleotide polymorphism (SNP) data available for all breeding animals, a pregnant carrier sow sired by a carrier boar was identified. Of the resulting piglets, two confirmed homozygous piglets suffered from severe myopathy, hind-limb paralysis, and tremors. Histopathological examination showed dispersed degeneration and decrease of cross-striations in the dorsal and hind-limb muscle fibers of the affected piglets. Hence, the affected piglets are unable to walk or drink, usually resulting in death within a few hours after birth. This study demonstrates how growing genomic resources in pig breeding can be applied to identify rare syndromes in breeding populations, that are usually poorly documented and often are not even known to have a genetic basis. The study allows to prevent carrier-by-carrier matings, thereby gradually decreasing the frequency of the detrimental allele and avoiding the birth of affected piglets, improving animal welfare. Finally, these "natural knockouts" increase our understanding of gene function within the mammalian clade, and provide a potential model for human disease.

The Genomic Complexity of a Large Inversion in Great Tits.

Chromosome inversions have clear effects on genome evolution and have been associated with speciation, adaptation, and the evolution of the sex chromosomes. In birds, these inversions may play an important role in hybridization of species and disassortative mating. We identified a large (≈64 Mb) inversion polymorphism in the great tit (Parus major) that encompasses almost 1,000 genes and more than 90% of Chromosome 1A. The inversion occurs at a low frequency in a set of over 2,300 genotyped great tits in the Netherlands with only 5% of the birds being heterozygous for the inversion. In an additional analysis of 29 resequenced birds from across Europe, we found two heterozygotes. The likely inversion breakpoints show considerable genomic complexity, including multiple copy number variable segments. We identified different haplotypes for the inversion, which differ in the degree of recombination in the center of the chromosome. Overall, this remarkable genetic variant is widespread among distinct great tit populations and future studies of the inversion haplotype, including how it affects the fitness of carriers, may help to understand the mechanisms that maintain it.

Balancing selection on a recessive lethal deletion with pleiotropic effects on two neighboring genes in the porcine genome.

Livestock populations can be used to study recessive defects caused by deleterious alleles. The frequency of deleterious alleles including recessive lethal alleles can stay at high or moderate frequency within a population, especially if recessive lethal alleles exhibit an advantage for favourable traits in heterozygotes. In this study, we report such a recessive lethal deletion of 212kb (del) within the BBS9 gene in a breeding population of pigs. The deletion produces a truncated BBS9 protein expected to cause a complete loss-of-function, and we find a reduction of approximately 20% on the total number of piglets born from carrier by carrier matings. Homozygous del/del animals die mid- to late-gestation, as observed from high increase in numbers of mummified piglets resulting from carrier-by-carrier crosses. The moderate 10.8% carrier frequency (5.4% allele frequency) in this pig population suggests an advantage on a favourable trait in heterozygotes. Indeed, heterozygous carriers exhibit increased growth rate, an important selection trait in pig breeding. Increased growth and appetite together with a lower birth weight for carriers of the BBS9 null allele in pigs is analogous to the phenotype described in human and mouse for (naturally occurring) BBS9 null-mutants. We show that fetal death, however, is induced by reduced expression of the downstream BMPER gene, an essential gene for normal foetal development. In conclusion, this study describes a lethal 212kb deletion with pleiotropic effects on two different genes, one resulting in fetal death in homozygous state (BMPER), and the other increasing growth (BBS9) in heterozygous state. We provide strong evidence for balancing selection resulting in an unexpected high frequency of a lethal allele in the population. This study shows that the large amounts of genomic and phenotypic data routinely generated in modern commercial breeding programs deliver a powerful tool to monitor and control lethal alleles much more efficiently.

Copy number variations in Friesian horses and genetic risk factors for insect bite hypersensitivity.

BACKGROUND: Many common and relevant diseases affecting equine welfare have yet to be tested regarding structural variants such as copy number variations (CNVs). CNVs make up a substantial proportion of total genetic variability in populations of many species, resulting in more sequence differences between individuals than SNPs. Associations between CNVs and disease phenotypes have been established in several species, but equine CNV studies have been limited. Aim of this study was to identify CNVs and to perform a genome-wide association (GWA) study in Friesian horses to identify genomic loci associated with insect bite hypersensitivity (IBH), a common seasonal allergic dermatitis observed in many horse breeds worldwide. RESULTS: Genotypes were obtained using the Axiom® Equine Genotyping Array containing 670,796 SNPs. After quality control of genotypes, 15,041 CNVs and 5350 CNV regions (CNVRs) were identified in 222 Friesian horses. Coverage of the total genome by CNVRs was 11.2% with 49.2% of CNVRs containing genes. 58.0% of CNVRs were novel (i.e. so far only identified in Friesian horses). A SNP- and CNV-based GWA analysis was performed, where about half of the horses were affected by IBH. The SNP-based analysis showed a highly significant association between the MHC region on ECA20 and IBH in Friesian horses. Associations between the MHC region on ECA20 and IBH were also detected based on the CNV-based analysis. However, CNVs associated with IBH in Friesian horses were not often in close proximity to SNPs identified to be associated with IBH. CONCLUSIONS: CNVs were identified in a large sample of the Friesian horse population, thereby contributing to our knowledge on CNVs in horses and facilitating our understanding of the equine genome and its phenotypic expression. A clear association was identified between the MHC region on ECA20 and IBH in Friesian horses based on both SNP- and CNV-based GWA studies. These results imply that MHC contributes to IBH sensitivity in Friesian horses. Although subsequent analyses are needed for verification, nucleotide differences, as well as more complex structural variations like CNVs, seem to contribute to IBH sensitivity. IBH should be considered as a common disease with a complex genomic architecture.

A Novel Loss-of-Function Variant in Transmembrane Protein 263 (TMEM263) of Autosomal Dwarfism in Chicken.

Autosomal dwarfism (adw) in chickens is a growth deficiency caused by a recessive mutation. Characteristic for adw is an approximately 30% growth reduction with short shank. The adw variant was first recognized in the Cornell K-strain of White Leghorns, but the genetic causal variant remained unknown. To identify the causal variant underlying the adw phenotype, fine mapping was conducted on chromosome 1, within 52-56 Mb. This region was known to harbor the causal variant from previous linkage studies. We compared whole-genome sequence data of this region from normal-sized and adw chickens in order to find the unique causal variant. We identified a novel nonsense mutation NP_001006244.1:p.(Trp59∗), in the transmembrane protein 263 gene (TMEM263), completely associated with adw. The nonsense mutation truncates the transmembrane protein within the membrane-spanning domain, expected to cause a dysfunctional protein. TMEM263 is reported to be associated with bone mineral deposition in humans, and the protein shows interaction with growth hormone 1 (GH1). Our study presents molecular genetic evidence for a novel loss-of-function variant, which likely alters body growth and development in autosomal dwarf chicken.

CNVs are associated with genomic architecture in a songbird.

BACKGROUND: Understanding variation in genome structure is essential to understand phenotypic differences within populations and the evolutionary history of species. A promising form of this structural variation is copy number variation (CNV). CNVs can be generated by different recombination mechanisms, such as non-allelic homologous recombination, that rely on specific characteristics of the genome architecture. These structural variants can therefore be more abundant at particular genes ultimately leading to variation in phenotypes under selection. Detailed characterization of CNVs therefore can reveal evolutionary footprints of selection and provide insight in their contribution to phenotypic variation in wild populations. RESULTS: Here we use genotypic data from a long-term population of great tits (Parus major), a widely studied passerine bird in ecology and evolution, to detect CNVs and identify genomic features prevailing within these regions. We used allele intensities and frequencies from high-density SNP array data from 2,175 birds. We detected 41,029 CNVs concatenated into 8,008 distinct CNV regions (CNVRs). We successfully validated 93.75% of the CNVs tested by qPCR, which were sampled at different frequencies and sizes. A mother-daughter family structure allowed for the evaluation of the inheritance of a number of these CNVs. Thereby, only CNVs with 40 probes or more display segregation in accordance with Mendelian inheritance, suggesting a high rate of false negative calls for smaller CNVs. As CNVRs are a coarse-grained map of CNV loci, we also inferred the frequency of coincident CNV start and end breakpoints. We observed frequency-dependent enrichment of these breakpoints at homologous regions, CpG sites and AT-rich intervals. A gene ontology enrichment analyses showed that CNVs are enriched in genes underpinning neural, cardiac and ion transport pathways. CONCLUSION: Great tit CNVs are present in almost half of the genes and prominent at repetitive-homologous and regulatory regions. Although overlapping genes under selection, the high number of false negatives make neutrality or association tests on CNVs detected here difficult. Therefore, CNVs should be further addressed in the light of their false negative rate and architecture to improve the comprehension of their association with phenotypes and evolutionary history.

Dwarfism with joint laxity in Friesian horses is associated with a splice site mutation in B4GALT7.

BACKGROUND: Inbreeding and population bottlenecks in the ancestry of Friesian horses has led to health issues such as dwarfism. The limbs of dwarfs are short and the ribs are protruding inwards at the costochondral junction, while the head and back appear normal. A striking feature of the condition is the flexor tendon laxity that leads to hyperextension of the fetlock joints. The growth plates of dwarfs display disorganized and thickened chondrocyte columns. The aim of this study was to identify the gene defect that causes the recessively inherited trait in Friesian horses to understand the disease process at the molecular level. RESULTS: We have localized the genetic cause of the dwarfism phenotype by a genome wide approach to a 3 Mb region on the p-arm of equine chromosome 14. The DNA of two dwarfs and one control Friesian horse was sequenced completely and we identified the missense mutation ECA14:g.4535550C > T that cosegregated with the phenotype in all Friesians analyzed. The mutation leads to the amino acid substitution p.(Arg17Lys) of xylosylprotein beta 1,4-galactosyltransferase 7 encoded by B4GALT7. The protein is one of the enzymes that synthesize the tetrasaccharide linker between protein and glycosaminoglycan moieties of proteoglycans of the extracellular matrix. The mutation not only affects a conserved arginine codon but also the last nucleotide of the first exon of the gene and we show that it impedes splicing of the primary transcript in cultured fibroblasts from a heterozygous horse. As a result, the level of B4GALT7 mRNA in fibroblasts from a dwarf is only 2 % compared to normal levels. Mutations in B4GALT7 in humans are associated with Ehlers-Danlos syndrome progeroid type 1 and Larsen of Reunion Island syndrome. Growth retardation and ligamentous laxity are common manifestations of these syndromes. CONCLUSIONS: We suggest that the identified mutation of equine B4GALT7 leads to the typical dwarfism phenotype in Friesian horses due to deficient splicing of transcripts of the gene. The mutated gene implicates the extracellular matrix in the regular organization of chrondrocyte columns of the growth plate. Conservation of individual amino acids may not be necessary at the protein level but instead may reflect underlying conservation of nucleotide sequence that are required for efficient splicing.

A nonsense mutation in B3GALNT2 is concordant with hydrocephalus in Friesian horses.

BACKGROUND: Hydrocephalus in Friesian horses is a developmental disorder that often results in stillbirth of affected foals and dystocia in dams. The occurrence is probably related to a founder effect and inbreeding in the population. The aim of our study was to find genomic associations, to investigate the mode of inheritance, to allow a DNA test for hydrocephalus in Friesian horses to be developed. In case of a monogenic inheritance we aimed to identify the causal mutation. RESULTS: A genome-wide association study of hydrocephalus in 13 cases and 69 controls using 29,720 SNPs indicated the involvement of a region on ECA1 (P <1.68 × 10(-6)). Next generation DNA sequence analysis of 4 cases and 6 controls of gene exons within the region revealed a mutation in ß-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2) as the likely cause of hydrocephalus in Friesian horses. The nonsense mutation XM_001491545 c.1423C>T corresponding to XP_001491595 p.Gln475* was identical to a B3GALNT2 mutation identified in a human case of muscular dystrophy-dystroglycanopathy with hydrocephalus. All 16 available cases and none of the controls were homozygous for the mutation, and all 17 obligate carriers (= dams of cases) were heterozygous. A random sample of the Friesian horse population (n = 865) was tested for the mutation in a commercial laboratory. One-hundred and forty-seven horses were carrier and 718 horses were homozygous for the normal allele; the estimated allele frequency in the Friesian horse population is 0.085. CONCLUSIONS: Hydrocephalus in Friesian horses has an autosomal recessive mode of inheritance. A nonsense mutation XM_001491545 c.1423C>T corresponding to XP_001491595 p.Gln475* in B3GALNT2 (1:75,859,296-75,909,376) is concordant with hydrocephalus in Friesian horses. Application of a DNA test in the breeding programme will reduce the losses caused by hydrocephalus in the Friesian horse population.

Analyses of pig genomes provide insight into porcine demography and evolution.

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model.

The design and cross-population application of a genome-wide SNP chip for the great tit Parus major.

The vast amount of phenotypic information collected in some wild animal populations makes them extremely valuable for unravelling the genetics of ecologically important traits and understanding how populations adapt to changes in their environment. Next generation sequencing has revolutionized the development of large marker panels in species previously lacking genomic resources. In this study, a unique genomics toolkit was developed for the great tit (Parus major), a model species in ecology and behavioural biology. This toolkit consists of nearly 100,000 SNPs, over 250 million nucleotides of assembled genomic DNA and more than 80 million nucleotides of assembled expressed sequences. A SNP chip with 9193 SNP markers expected to be spaced evenly along the great tit genome was used to genotype 4702 birds from two of the most intensively studied natural vertebrate populations [Wytham Woods/Bagley Woods (United Kingdom) and de Hoge Veluwe/Westerheide (The Netherlands)]. We show that (i) SNPs identified in either of the two populations have a high genotyping success in the other population, (ii) the minor allele frequencies of the SNPs are highly correlated between the two populations and (iii) despite this high correlation, a large number of SNPs display significant differentiation (F(ST) ) between the populations, with an overrepresentation of genes involved in cardiovascular development close to these SNPs. The developed resources provide the basis for unravelling the genetics of important traits in many long-term studies of great tits. More generally, the protocols and pitfalls encountered will be of use for those developing similar resources.

The imprinted gene DIO3 is a candidate gene for litter size in pigs.

Genomic imprinting is an important epigenetic phenomenon, which on the phenotypic level can be detected by the difference between the two heterozygote classes of a gene. Imprinted genes are important in both the development of the placenta and the embryo, and we hypothesized that imprinted genes might be involved in female fertility traits. We therefore performed an association study for imprinted genes related to female fertility traits in two commercial pig populations. For this purpose, 309 SNPs in fifteen evolutionary conserved imprinted regions were genotyped on 689 and 1050 pigs from the two pig populations. A single SNP association study was used to detect additive, dominant and imprinting effects related to four reproduction traits; total number of piglets born, the number of piglets born alive, the total weight of the piglets born and the total weight of the piglets born alive. Several SNPs showed significant (q-value < 0.10) additive and dominant effects and one SNP showed a significant imprinting effect. The SNP with a significant imprinting effect is closely linked to DIO3, a gene involved in thyroid metabolism. The imprinting effect of this SNP explained approximately 1.6% of the phenotypic variance, which corresponded to approximately 15.5% of the additive genetic variance. In the other population, the imprinting effect of this QTL was not significant (q-value > 0.10), but had a similar effect as in the first population. The results of this study indicate a possible association between the imprinted gene DIO3 and female fertility traits in pigs.

Structural variation in the chicken genome identified by paired-end next-generation DNA sequencing of reduced representation libraries.

BACKGROUND: Variation within individual genomes ranges from single nucleotide polymorphisms (SNPs) to kilobase, and even megabase, sized structural variants (SVs), such as deletions, insertions, inversions, and more complex rearrangements. Although much is known about the extent of SVs in humans and mice, species in which they exert significant effects on phenotypes, very little is known about the extent of SVs in the 2.5-times smaller and less repetitive genome of the chicken. RESULTS: We identified hundreds of shared and divergent SVs in four commercial chicken lines relative to the reference chicken genome. The majority of SVs were found in intronic and intergenic regions, and we also found SVs in the coding regions. To identify the SVs, we combined high-throughput short read paired-end sequencing of genomic reduced representation libraries (RRLs) of pooled samples from 25 individuals and computational mapping of DNA sequences from a reference genome. CONCLUSION: We provide a first glimpse of the high abundance of small structural genomic variations in the chicken. Extrapolating our results, we estimate that there are thousands of rearrangements in the chicken genome, the majority of which are located in non-coding regions. We observed that structural variation contributes to genetic differentiation among current domesticated chicken breeds and the Red Jungle Fowl. We expect that, because of their high abundance, SVs might explain phenotypic differences and play a role in the evolution of the chicken genome. Finally, our study exemplifies an efficient and cost-effective approach for identifying structural variation in sequenced genomes.

Genome-wide SNP detection in the great tit Parus major using high throughput sequencing.

Identifying genes that underlie ecological traits will open exiting possibilities to study gene-environment interactions in shaping phenotypes and in measuring natural selection on genes. Evolutionary ecology has been pursuing these objectives for decades, but they come into reach now that next generation sequencing technologies have dramatically lowered the costs to obtain the genomic sequence information that is currently lacking for most ecologically important species. Here we describe how we generated over 2 billion basepairs of novel sequence information for an ecological model species, the great tit Parus major. We used over 16 million short sequence reads for the de novo assembly of a reference sequence consisting of 550 000 contigs, covering 2.5% of the genome of the great tit. This reference sequence was used as the scaffold for mapping of the sequence reads, which allowed for the detection of over 20 000 novel single nucleotide polymorphisms. Contigs harbouring 4272 of the single nucleotide polymorphisms could be mapped to a unique location on the recently sequenced zebra finch genome. Of all the great tit contigs, significantly more were mapped to the microchromosomes than to the intermediate and the macrochromosomes of the zebra finch, indicating a higher overall level of sequence conservation on the microchromosomes than on the other types of chromosomes. The large number of great tit contigs that can be aligned to the zebra finch genome shows that this genome provides a valuable framework for large scale genetics, e.g. QTL mapping or whole genome association studies, in passerines.

Large scale single nucleotide polymorphism discovery in unsequenced genomes using second generation high throughput sequencing technology: applied to turkey.

BACKGROUND: The development of second generation sequencing methods has enabled large scale DNA variation studies at moderate cost. For the high throughput discovery of single nucleotide polymorphisms (SNPs) in species lacking a sequenced reference genome, we set-up an analysis pipeline based on a short read de novo sequence assembler and a program designed to identify variation within short reads. To illustrate the potential of this technique, we present the results obtained with a randomly sheared, enzymatically generated, 2-3 kbp genome fraction of six pooled Meleagris gallopavo (turkey) individuals. RESULTS: A total of 100 million 36 bp reads were generated, representing approximately 5-6% (approximately 62 Mbp) of the turkey genome, with an estimated sequence depth of 58. Reads consisting of bases called with less than 1% error probability were selected and assembled into contigs. Subsequently, high throughput discovery of nucleotide variation was performed using sequences with more than 90% reliability by using the assembled contigs that were 50 bp or longer as the reference sequence. We identified more than 7,500 SNPs with a high probability of representing true nucleotide variation in turkeys. Increasing the reference genome by adding publicly available turkey BAC-end sequences increased the number of SNPs to over 11,000. A comparison with the sequenced chicken genome indicated that the assembled turkey contigs were distributed uniformly across the turkey genome. Genotyping of a representative sample of 340 SNPs resulted in a SNP conversion rate of 95%. The correlation of the minor allele count (MAC) and observed minor allele frequency (MAF) for the validated SNPs was 0.69. CONCLUSION: We provide an efficient and cost-effective approach for the identification of thousands of high quality SNPs in species currently lacking a sequenced genome and applied this to turkey. The methodology addresses a random fraction of the genome, resulting in an even distribution of SNPs across the targeted genome.

Application of massive parallel sequencing to whole genome SNP discovery in the porcine genome.

BACKGROUND: Although the Illumina 1 G Genome Analyzer generates billions of base pairs of sequence data, challenges arise in sequence selection due to the varying sequence quality. Therefore, in the framework of the International Porcine SNP Chip Consortium, this pilot study aimed to evaluate the impact of the quality level of the sequenced bases on mapping quality and identification of true SNPs on a large scale. RESULTS: DNA pooled from five animals from a commercial boar line was digested with DraI; 150-250-bp fragments were isolated and end-sequenced using the Illumina 1 G Genome Analyzer, yielding 70,348,064 sequences 36-bp long. Rules were developed to select sequences, which were then aligned to unique positions in a reference genome. Sequences were selected based on quality, and three thresholds of sequence quality (SQ) were compared. The highest threshold of SQ allowed identification of a larger number of SNPs (17,489), distributed widely across the pig genome. In total, 3,142 SNPs were validated with a success rate of 96%. The correlation between estimated minor allele frequency (MAF) and genotyped MAF was moderate, and SNPs were highly polymorphic in other pig breeds. Lowering the SQ threshold and maintaining the same criteria for SNP identification resulted in the discovery of fewer SNPs (16,768), of which 259 were not identified using higher SQ levels. Validation of SNPs found exclusively in the lower SQ threshold had a success rate of 94% and a low correlation between estimated MAF and genotyped MAF. Base change analysis suggested that the rate of transitions in the pig genome is likely to be similar to that observed in humans. Chromosome X showed reduced nucleotide diversity relative to autosomes, as observed for other species. CONCLUSION: Large numbers of SNPs can be identified reliably by creating strict rules for sequence selection, which simultaneously decreases sequence ambiguity. Selection of sequences using a higher SQ threshold leads to more reliable identification of SNPs. Lower SQ thresholds can be used to guarantee sufficient sequence coverage, resulting in high success rate but less reliable MAF estimation. Nucleotide diversity varies between porcine chromosomes, with the X chromosome showing less variation as observed in other species.