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Background: De novo mutations arising in the germline are a source of genetic variation and their discovery broadens our understanding of genetic disorders and evolutionary patterns. Although the number of de novo single nucleotide variants (dnSNVs) has been studied in a number of species, relatively little is known about the occurrence of de novo structural variants (dnSVs). In this study, we investigated 37 deeply sequenced pig trios from two commercial lines to identify dnSVs present in the offspring. The identified dnSVs were characterised by identifying their parent of origin, their functional annotations and characterizing sequence homology at the breakpoints. Results: We identified four swine germline dnSVs, all located in intronic regions of protein-coding genes. Our conservative, first estimate of the swine germline dnSV rate is 0.108 (95% CI 0.038–0.255) per generation (one dnSV per nine offspring), detected using short-read sequencing. Two detected dnSVs are clusters of mutations. Mutation cluster 1 contains a de novo duplication, a dnSNV and a de novo deletion. Mutation cluster 2 contains a de novo deletion and three de novo duplications, of which one is inverted. Mutation cluster 2 is 25 kb in size, whereas mutation cluster 1 (197 bp) and the other two individual dnSVs (64 and 573 bp) are smaller. Only mutation cluster 2 could be phased and is located on the paternal haplotype. Mutation cluster 2 originates from both micro-homology as well as non-homology mutation mechanisms, where mutation cluster 1 and the other two dnSVs are caused by mutation mechanisms lacking sequence homology. The 64 bp deletion and mutation cluster 1 were validated through PCR. Lastly, the 64 bp deletion and the 573 bp duplication were validated in sequenced offspring of probands with three generations of sequence data. Conclusions: Our estimate of 0.108 dnSVs per generation in the swine germline is conservative, due to our small sample size and restricted possibilities of dnSV

De novo mutations arising in the germline add to genetic variation. The number of de novo mutations occurring every generation, especially structural variants, has not been well studied in most species, including livestock. We used whole-genome sequencing from 46 pig trios from two commercial lines to identify de novo structural variants (dnSVs) present in the offspring. We characterised these dnSV by identifying their parent-of-origin, predicting their causal mechanisms, and identifying their functional annotations. We identified four dnSVs, including two clusters of mutations. One of these clusters contained a deletion, and three duplications, one of which was inverted. This cluster was the only dnSV that could be phased and was located in the paternal haplotype of the proband. All four identified dnSVs were located within the introns of genes. Our study is the first of its kind to identify and characterise dnSVs using whole genome shotgun sequence data in pigs.

Nearly 2000 SNPs associated with pig litter size traits have been reported based on genome-wide association studies (GWASs). The aims of this study were to gather and integrate previously reported associations between SNPs and five litter traits: total number born (TNB), number born alive (NBA), number of stillborn (SB), litter birth weight (LWT), and corpus luteum number (CLN), in order to evaluate their common genetic background and to perform a meta-analysis (MA) of GWASs for total number born (TNB) recorded for animals from five pig populations. In this study, the genes with the largest number of associations with evaluated litter traits were GABRG3, RBP7, PRKD1, and STXBP6. Only 21 genes out of 233 associated with the evaluated litter traits were reported in more than one population or for more than one trait. Based on this evaluation, the most interesting candidate gene is PRKD1, which has an association with SB and TNB traits. Based on GO term analysis, PRKD1 was shown to be involved in angiogenesis as well. As a result of the MA, two new genomic regions, which have not been previously reported, were found to be associated with the TNB trait. One SNP was located on Sus scrofa chromosome (SSC) 14 in the intron of the FAM13C gene. The second SNP was located on SSC9 within the intron of the AGMO gene. Functional analysis revealed a strong candidate causal gene underlying the QTL on SSC9. The third best hit and the most promising candidate gene for litter size was found within the SOSTDC1 gene, associated with lower male fertility in rats. We showed that litter traits studied across pig populations have only a few genomic regions in common based on candidate gene comparison. PRKD1 could be an interesting candidate gene with a wider association with fertility. The MA identified new genomic regions on SSC9 and SSC14 associated with TNB. Further functional analysis indicated the most promising gene was SOSTDC1, which was confirmed to affect male fertility in other m

The aim of this study was to perform genome-wide association analyses for backfat thickness and osteochondrosis in Landrace pigs and to fine map pleiotropic genomic regions. In order to characterise genomic regions, phenotypic information of 5,000 animals with osteochondrosis scored from CT images and 40,000 animals with backfat thickness scored from CT or ultrasound images were analysed. All animals were genotyped with a medium density SNP chip and a subset of them were genotyped with a high-density SNP chip as well, allowing for imputation. Two genomic loci were found in common for osteochondrosis and backfat thickness, one on chromosome 5 and one on chromosome 14. For both regions, an antagonistic relationship was found. Fine mapping using an impact score approach identified the CCND2 gene as the most likely causal gene on chromosome 5, whereas a mutation in CRTAC1 had the highest impact score in the chromosome 14 region.

Genomics related research in animal breeding is usually performed by comparing genomic information to an existing reference genome. However, even if the reference genome is of high quality, the use of a single reference genome has clear drawbacks. Therefore, the breeding community is shifting towards the construction of a pan-genome for important agricultural species. In this study we produced a pig pangenome based on four different breeds (Landrace, Large White, Synthetic, Duroc) using the nanopore long read sequencing technology. We produced chromosome arm level assemblies comparable to the current Sus scrofa 11.1 reference genome. We identified between breed structural variation, which gives a unique insight in the genomic structural variation that define and differentiates breeds. The pig pan-genome will facilitate the discovery of novel variation providing a unique fundamental insight into breed genomic characteristics, which can subsequently be utilized for breeding.

The aim of this work was to identify functional variants potentially associated to complex phenotypes, to improve genomic selection. In this study, we combined genomics, transcriptomics and epigenomics data. First, we genotyped 100 pigs with the high-density genotyping array (with 660K markers). Then, we generated RNA-seq data on these animals in 4 biologically divergent tissues: liver, spleen, lung and muscle. We performed an expression GWAS (eGWAS) resulting in the identification of over 150,000 significant associations in each of the tissues. We were able to detect over 1,300 genes that showed a significant eGWAS associations. We also found an enrichment of eGWAS hits within regulatory elements such as enhancers and promoters, which indicates their pivotal role as drivers of phenotypic variation and thus are of highly relevance towards understanding the genetics of complex traits.

Accuracy of genomic prediction is key for genetic progress in breeding programs and including genome functional annotation may help. Here we report on the added value of Combined Annotation Dependent Depletion (CADD) scores to weigh SNPs in GBLUP analyses. Multiple transformations of CADD scores were considered and empirically validated on a layer dataset including 5 traits and 18K animals with 27K SNP genotypes. This initial analysis revealed that the use of (squared) CADD scores yielded marginally higher accuracy for 3 traits. We anticipate that the added value of CADD scores will increase by using a higher number of SNPs and ultimately whole genome sequence SNPs.

Several QTL regions affecting number of teats have been detected in commercial pig lines. In this study we follow the indirect effects of index selection on different QTL regions for number of teats in two maternal lines in 40K animals with imputed 555K SNP. In total, 3 QTL regions overlap between the two populations. For a QTL on Sus scrofa chromosome 7 (SSC7), the underlying functional variation affecting number of thoracic vertebrae located in the vertnin gene has also been genotyped showing an allelic substitution effect of nearly 0.4 teats in both lines. However, allele frequencies show an opposite trend at the vertnin gene locus in the two lines. Moreover, epistatic effects between two QTL regions on SSC7 and SSC12 are investigated at the molecular and phenotypic level.

Cell lines are useful for investigating traits of interest e.g. intestinal absorption, feed efficiency and immunity in farm animals. We earlier investigated an intestinal cell line in pig and a fibroblast cell line in chicken and found chromosomal abnormalities by whole genome sequence (WGS) data analysis. Results from RNAseq allele-specific expression analysis (ASE) in 4 cell lines showed aneuploidy in some chromosomes. In this paper we show that RNA-seq can be used to detect whole/partial chromosomal abnormalities based on ASE analysis.

The growth of animals is a complex trait, in chicken resulting in a diverse variety of forms, caused by a heterogeneous genetic basis. Bantam chicken, known as an exquisite form of dwarfism, has been used for crossbreeding to create corresponding dwarf counterparts for native fowls in the Dutch populations. Here, we demonstrate the heterogeneity of the bantam trait in Dutch chickens and reveal the underlying genetic causes, using whole‐genome sequence data from matching pairs of bantam and normal‐sized breeds. During the bantam‐oriented crossbreeding, various bantam origins were used to introduce the bantam phenotype, and three major bantam sources were identified and clustered. The genome‐wide association studies revealed multiple genetic variants and genes associated with bantam phenotype, including HMGA2 and PRDM16, genes involved in body growth and stature. The comparison of associated variants among studies illustrated differences related to divergent bantam origins, suggesting a clear heterogeneity among bantam breeds. We show that in neo‐bantam breeds, the bantam‐related regions underwent a strong haplotype introgression from the bantam source, outcompeting haplotypes from the normal‐sized counterpart. The bantam heterogeneity is further confirmed by the presence of multiple haplotypes comprising associated alleles, which suggests the selection of the bantam phenotype is likely subject to a convergent direction across populations. Our study demonstrates that the diverse history of human‐mediated crossbreeding has contributed to the complexity and heterogeneity of the bantam phenotype.

The pig breeding system provides a unique framework to study recessive defects and the consequence on the phenotype. We examined a commercial synthetic Duroc population for recessive defects and identified a haplotype on chromosome 9 significantly affecting pre-weaning mortality. To identify the causal variant underlying the mortality, we examined sequence data of four carrier animals and 21 non-carrier animals from the same population. The results yield a strong candidate causal stop-gained variant (NM_001099928.1:c.541C>T) affecting the MYO7A gene in complete linkage disequilibrium with the lethal haplotype. The variant leads to an impaired (p.Gln181*) MYO7A protein that truncates 2032 amino acids from the protein. We examined a litter from a carrier sow inseminated by a carrier boar. From the resulting piglets, two confirmed homozygous piglets suffered from severe balance difficulties and the inability to walk properly. The variant segregates at a carrier frequency of 8.2% in the evaluated population and will be gradually purged from the population, improving animal welfare. Finally, this 'natural knockout' will increase our understanding of the functioning of the MYO7A gene and provides a potential model for Usher syndrome in humans.

Repository for the study: Martijn F.L. Derks, Barbara Harlizius, Marcos S. Lopes, Sylvia W.M. Greijdanus-van der Putten, Bert Dibbits, Kimberley Laport, Hendrik-Jan Megens, Martien A.M. Groenen. Detection of a frameshift deletion in the SPTBN4 gene leads to prevention of severe myopathy and postnatal mortality in pigs. 2019. Published in Frontiers in Genetics, doi: 10.3389/fgene.2019.01226

Our work use the WGS data present the molecular genetic evidence for a novel mutation potentially underlying autosomal dwarfism in chicken. The identification of the adw mutation provides the basis for future studies towards dwarf status in different species, as well as the functional role of TMEM263 in growth and developmental pathways. Autosomal dwarfism (adw) in chicken is known as a growth deficiency caused by a recessive mutation. The features of autosomal dwarfism are known as a proportionally 30% growth reduction with short shank length. The adw variant was first recognized in the Cornell K-strain of White Leghorns but the genetic causal variant remained unknown. To detect the underlying causal variant underlying the trait of adw, fine mapping was conducted based on previous linkage research on chromosome 1. We found a nonsense mutation in the transmembrane protein 263 gene (TMEM263) that is completely associated with the autosomal dwarf phenotype. Variants were detected by comparing whole-genome sequencing data from white leghorns vs adw chicken. Many potential variants were identified but after filtering for the known variant with variant databases, only one potential variant remained associated with autosomal dwarfism. A stop gain variant in TMEM263 is found unique in dwarf chicken and absent in normal-sized controls. In human, TMEM263 is associated with bone mineral density and the protein interacts with growth hormone 1. Therefore the nonsense mutation in TMEM263 likely leads to a protein truncation and therefore affects its function., Autosomal dwarfism (adw) in chicken is known as a growth deficiency caused by a recessive mutation. The features of autosomal dwarfism are known as a proportionally 30% growth reduction with short shank length. The adw variant was first recognized in the Cornell K-strain of White Leghorns but the genetic causal variant remained unknown. To detect the underlying causal variant underlying the trait of adw, fine mapping was conducted based on previous linkage research on chromosome 1. We found a nonsense mutation in the transmembrane protein 263 gene (TMEM263) that is completely associated with the autosomal dwarf phenotype. Variants were detected by comparing whole-genome sequencing data from white leghorns vs adw chicken. Many potential variants were identified but after filtering for the known variant with variant databases, only one potential variant remained associated with autosomal dwarfism. A stop gain variant in TMEM263 is found unique in dwarf chicken and absent in normal-sized controls. In human, TMEM263 is associated with bone mineral density and the protein interacts with growth hormone 1. Therefore the nonsense mutation in TMEM263 likely leads to a protein truncation and therefore affects its function.

Grain legume improvement is currently impeded by a lack of genomic resources. The paucity of genome information for faba bean can be attributed to the intrinsic difficulties of assembling/annotating its giant (~13 Gb) genome. In order to address this challenge, RNA-sequencing analysis was performed on faba bean (cv. Wizard) leaves. Read alignment to the faba bean reference transcriptome identified 16 300 high quality unigenes. In addition, Illumina paired-end sequencing was used to establish a baseline for genomic information assembly. Genomic reads were assembled de novo into contigs with a size range of 50–5000 bp. Over 85% of sequences did not align to known genes, of which ~10% could be aligned to known repetitive genetic elements. Over 26 000 of the reference transcriptome unigenes could be aligned to DNA-sequencing (DNA-seq) reads with high confidence. Moreover, this comparison identified 56 668 potential splice points in all identified unigenes. Sequence length data were extended at 461 putative loci through alignment of DNA-seq contigs to full-length, publicly available linkage marker sequences. Reads also yielded coverages of 3466× and 650× for the chloroplast and mitochondrial genomes, respectively. Inter- and intraspecies organelle genome comparisons established core legume organelle gene sets, and revealed polymorphic regions of faba bean organelle genomes.

The rate of directional environmental change may have profound consequences for evolutionary dynamics and outcomes. Yet, most evolution experiments impose a sudden large change in the environment, after which the environment is kept constant. We previously cultured replicate Saccharomyces cerevisiae populations for 500 generations in the presence of either gradually increasing or constant high concentrations of the heavy metals cadmium, nickel, and zinc. Here, we investigate how each of these treatments affected genomic evolution. Whole genome sequencing of evolved clones revealed that adaptation occurred via a combination of SNPs, small indels, and whole genome duplications and other large-scale structural changes. In contrast to some theoretical predictions, gradual and abrupt environmental change caused similar numbers of genomic changes. For cadmium, which is toxic already at comparatively low concentrations, mutations in the same genes were used for adaptation to both gradual and abrupt increase in concentration. Conversely, for nickel and zinc, which are toxic at high concentrations only, mutations in different genes were used for adaptation depending on the rate of change. Moreover, evolution was more repeatable following a sudden change in the environment, particularly for nickel and zinc. Our results show that the rate of environmental change and the nature of the selection pressure are important drivers of evolutionary dynamics and outcomes, which has implications for a better understanding of societal problems such as climate change and pollution.

This study is part of the FAANG project., "This study is part of the FAANG project, promoting rapid prepublication of data to support the research community. These data are released under Fort Lauderdale principles, as confirmed in the Toronto Statement (Toronto International Data Release Workshop. Birney et al. 2009. Pre-publication data sharing. Nature 461:168-170). Any use of this dataset must abide by the FAANG data sharing principles. Data producers reserve the right to make the first publication of a global analysis of this data. If you are unsure if you are allowed to publish on this dataset, please contact faang@iastate.edu to enquire. The full guidelines can be found at http://www.faang.org/data-share-principle”

We present the first genome sequence for a strain of the main mycetoma causative agent, Madurella mycetomatis. This 36.7-Mb genome sequence will offer new insights into the pathogenesis of mycetoma, and it will contribute to the development of better therapies for this neglected tropical disease.

Background: Studies on vertebrate DNA methylomes have revealed a regulatory role of tissue specific DNAmethylation in relation to gene expression. However, it is not well known how tissue-specific methylation variesbetween different functional and structural components of genes and genomes. Using whole-genome bisulfitesequencing data we here describe both CpG and non-CpG methylation profiles of whole blood and brain tissue inrelation to gene features, CpG-islands (CGIs), transposable elements (TE), and their functional roles in an ecologicalmodel species, the great tit (Parus major).Results: We show that hypomethylation at the transcription start site (TSS) is enriched in genes with functionalclasses that relate directly to processes specific to each tissue type. We find that 6877 (~21 %) of the CGIs aredifferentially methylated between blood and brain, of which 1186 and 2055 are annotated to promoter andintragenic regions, respectively. We observe that CGI methylation in promoter regions is more conserved betweentissues compared to CGI methylation in intra and inter-genic regions. Differentially methylated CGIs in promoterand intragenic regions are overrepresented in genomic loci linked to development, suggesting a distinct role forCGI methylation in regulating expression during development. Additionally, we find significant non-CpGmethylation in brain but not in blood with a strong preference for methylation at CpA dinucleotide sites. Finally,CpG hypermethylation of TEs is significantly stronger in brain compared to blood, but does not correlate with TEactivity. Surprisingly, TEs showed significant hypomethylation in non-CpG contexts which was negatively correlatedwith TE expression.Conclusion: The discovery that TSS methylation levels are directly linked to functional classes related to each tissueprovides new insights in the regulatory role of DNA-methylation patterns. The dominant sequence motifs for brainnon-CpG methylation, similar to those found in mammals

We present the first genome sequence for a strain of the main mycetoma causative agent, Madurella mycetomatis. This 36.7-Mb genome sequence will offer new insights into the pathogenesis of mycetoma, and it will contribute to the development of better therapies for this neglected tropical disease.

Collembola (springtails) are detritivorous hexapods that inhabit the soil and its litter layer. The ecology of the springtail Orchesella cincta is extensively studied in the context of adaptation to anthropogenically disturbed areas. Here, we present a draft genome of an O. cincta reference strain with an estimated size of 286.8 Mbp, containing 20,249 genes. In total, 446 gene families are expanded and 1,169 gene families evolved specific to this lineage. Besides these gene families involved in general biological processes, we observe gene clusters participating in xenobiotic biotransformation. Furthermore, we identified 253 cases of horizontal gene transfer (HGT). Although the largest percentage of them originated from bacteria (37.5%), we observe an unusually high percentage (30.4%) of such genes of fungal origin. The majority of foreign genes are involved in carbohydrate metabolism and cellulose degradation. Moreover, some foreign genes (e.g., bacillopeptidases) expanded after HGT. We hypothesize that horizontally transferred genes could be advantageous for food processing in a soil environment that is full of decaying organic material. Finally, we identified several lineage-specific genes, expanded gene families, and horizontally transferred genes, associated with altered gene expression as a consequence of genetic adaptation to metal stress. This suggests that these genome features may be preadaptations allowing natural selection to act on. In conclusion, this genome study provides a solid foundation for further analysis of evolutionary mechanisms of adaptation to environmental stressors.

Operophtera brumata genome reference project, Operophtera brumata genome reference project

The goal of this project is to create an annotated reference genome for the Winter Moth (Operophtera brumata), which is studied for its adaptation to climate change., The goal of this project is to create an annotated reference genome for the Winter Moth (Operophtera brumata), which is studied for its adaptation to climate change.

Whole genome sequencing of Wolbachia endosymbiont of Operophtera brumata, Whole genome sequencing of Wolbachia endosymbiont of Operophtera brumata

The winter moth (Operophtera brumata) belongs to one of the most species-rich families in Lepidoptera, the Geometridae (approximately 23,000 species). This family is of great economic importance as most species are herbivorous and capable of defoliating trees. Genome assembly of the winter moth allows the study of genes and gene families, such as the cytochrome P450 gene family, which is known to be vital in plant secondary metabolite detoxification and host-plant selection. It also enables exploration of the genomic basis for female brachyptery (wing reduction), a feature of sexual dimorphism in winter moth, and for seasonal timing, a trait extensively studied in this species. Here we present a reference genome for the winter moth, the first geometrid and largest sequenced Lepidopteran genome to date (638 Mb) including a set of 16,912 predicted protein-coding genes. This allowed us to assess the dynamics of evolution on a genome-wide scale using the P450 gene family. We also identified an expanded gene family potentially linked to female brachyptery, and annotated the genes involved in the circadian clock mechanism as main candidates for involvement in seasonal timing. The genome will contribute to Lepidopteran genomic resources and comparative genomics. In addition, the genome enhances our ability to understand the genetic and molecular basis of insect seasonal timing and thereby provides a reference for future evolutionary and population studies on the winter moth.