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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.

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.

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.

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.

Over the last decades major advances in the use of sequence data to improve pig breeding have been achieved. A key achievement was the publication of the pig reference genome and subsequent design of selection single-nucleotide polymorphism (SNP) chips to implement genomic selection. This chapter outlines major advances and future use of genome sequencing for improved pig breeding. It discusses that the breeding community will shift from the use of a single reference genome to a more comprehensive pig pangenome that better covers structural variation. In addition, the impact of deleterious variation and subsequent purging strategies is discussed. It is predicted that the functional genomic information published by the Functional Annotation of Animal Genomes (FAANG) consortium will aid in the discovery of functionally important (regulatory) variation. More specifically, regulatory variants can be discovered with large-scale gene expression data sets to discover expression quantitative trait loci (eQTLs). The annotation can be further improved by mapping regulatory regions in the genome by using-large scale epigenomic data sets that assess chromatin modifications and methylation states. The discovered functional variation can subsequently be used to improve pig breeding by adding functional markers to the selection chips, significantly improving prediction accuracies. Together, the chapter provides a comprehensive overview of the current status and future steps in the use of genome sequencing for pig breeding.

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.

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.

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.

Most angiosperms produce seeds that are desiccated on dispersal with the ability to retain viability in storage facilities for prolonged periods. However, some species produce desiccation sensitive seeds which rapidly lose viability in storage, precluding ex situ conservation. Current consensus is that desiccation sensitive seeds either lack or do not express mechanisms necessary for the acquisition of desiccation tolerance. We sequenced the genome of Castanospermum australe, a legume species producing desiccation sensitive seeds, and characterized its seed developmental physiology and - transcriptomes. C. australe has a low rate of evolution, likely due to its perennial life-cycle and long generation times. The genome is syntenic with itself, with several orthologs of genes from desiccation tolerant legume seeds, from gamma whole-genome duplication events being retained. Changes in gene expression during development of C. australe seeds, as compared to desiccation tolerant Medicago truncatula seeds, suggest they remain metabolically active, prepared for immediate germination. Our data indicates that the phenotype of C. australe seeds arose through few changes in specific signalling pathways, precluding or bypassing activation of mechanisms necessary for acquisition of desiccation tolerance. Such changes have been perpetuated as the habitat in which dispersal occurs is favourable for prompt germination.

The level of deleterious genetic variation in highly managed domestic populations is influenced by a variety of factors including mutation rate, effective population size, and artificial selection. Small populations enhance the risk of inbreeding depression, which negatively impacts individual fitness and population viability. Inbreeding depression has largely been attributed to the accumulation of recessive harmful mutations in the genome: inbreeding increases the probability of these mutations to become homozygous. Past population bottlenecks, including domestication, have been indicated as major driver of genetic load in populations. While lethal variants can quickly be purged from small populations, the frequency of slightly deleterious mutations is expected to rise due to less effective natural selection. Because of genetic hitch-hiking, mildly harmful mutations are thought to be over-represented in regions of the genome under selection. Recent advances in genome sequencing have opened exciting possibilities to actually measure the amount of harmful mutations in genomes. Using re-sequence data from 421 individuals, we provide an overview of the occurrence of deleterious mutations in individual pig genomes. An alternative approach to identify lethal variants in the genome, is offered by the vast amount of genotyped pedigreed individuals due to the widespread use of genomic selection in current breeding programs. This allows the identification of recessive deleterious variants by testing for statistical depletion, or even the absence, of specific haplotypes in homozygous state. We have used a combination of whole genome sequencing and 60K genotyping (23,800 individuals) to identify and characterize lethal variants segregating in three commercial pig breeding lines. Keywords: genome sequencing, lethal recessive, loss of function mutation, selective sweeps

Genome sequences nowadays play a central role in molecular biology and bioinformatics. These sequences are shared with the scientific community through sequence databases. The sequence repositories of the International Nucleotide Sequence Database Collaboration (INSDC, comprising GenBank, ENA and DDBJ) are the largest in the world. Preparing an annotated sequence in such a way that it will be accepted by the database is challenging because many validation criteria apply. In our opinion, it is an undesirable situation that researchers who want to submit their sequence need either a lot of experience or help from partners to get the job done. To save valuable time and money, we list a number of recommendations for people who want to submit an annotated genome to a sequence database, as well as for tool developers, who could help to ease the process