OrthReg: a tool to predict cis-regulatory elements based on cross-species orthologous sequence conservation.

Cis-regulatory elements play an important role in the development of traits and disease in organisms (Ma et al., 2020; Woolfe et al., 2005) and their annotation could facilitate genetic studies. The Encyclopedia of DNA Elements (ENCODE) (Davis et al., 2018) and Functional Annotation of Animal Genomes (FAANG) (FAANG Consortium et al., 2015) offer pioneering data on regulatory elements in several species. Currently, however, regulatory element annotation data remain limited for most organisms. In this study, we developed a tool (OrthReg) for annotating conserved orthologous cis-regulatory elements in targeted genomes using an annotated reference genome. Cross-species validation of this annotation tool using human and mouse ENCODE data confirmed the robustness of this strategy. To explore the efficiency of the tool, we annotated the pig genome and identified more than 28 million regulatory annotation records using the reference human ENCODE data. With this regulatory annotation, some putative regulatory non-coding variants were identified within domestication sweeps in European and East Asian pigs. Thus, this tool can utilize data produced by ENCODE, FAANG, and similar projects, and can be easily extended to customized experimental data. The extensive application of this tool will help to identify informative single nucleotide polymorphisms (SNPs) in post-genome-wide association studies and resequencing analysis of organisms with limited regulatory annotation data.

Whole genome resequencing reveals an association of ABCC4 variants with preaxial polydactyly in pigs.

BACKGROUND: Polydactyly is one of the most common congenital limb dysplasia in many animal species. Although preaxial polydactyly (PPD) has been comprehensively studied in humans as a common abnormality, the genetic variations in other animal species have not been fully understood. Herein, we focused on the pig, as an even-toed ungulate mammal model with its unique advantages in medical and genetic researches, two PPD families consisting of four affected and 20 normal individuals were sequenced. RESULTS: Our results showed that the PPD in the sampled pigs were not related to previously reported variants. A strong association was identified at ABCC4 and it encodes a transmembrane protein involved in ciliogenesis. We found that the affected and normal individuals were highly differentiated at ABCC4, and all the PPD individuals shared long haplotype stretches as compared with the unaffected individuals. A highly differentiated missense mutation (I85T) in ABCC4 was observed at a residue from a transmembrane domain highly conserved among a variety of organisms. CONCLUSIONS: This study reports ABCC4 as a new candidate gene and identifies a missense mutation for PPD in pigs. Our results illustrate a putative role of ciliogenesis process in PPD, coinciding with an earlier observation of ciliogenesis abnormality resulting in pseudo-thumb development in pandas. These results expand our knowledge on the genetic variations underlying PPD in animals.

Population Genomics Analysis Revealed Origin and High-altitude Adaptation of Tibetan Pigs.

Tibetan pig is native to the Qinghai-Tibet Plateau and has adapted to the high-altitude environmental condition such as hypoxia. However, its origin and genetic mechanisms underlying high-altitude adaptation still remain controversial and enigmatic. Herein, we analyze 229 genomes of wild and domestic pigs from Eurasia, including 63 Tibetan pigs, and detect 49.6 million high-quality variants. Phylogenomic and structure analyses show that Tibetan pigs have a close relationship with low-land domestic pigs in China, implying a common domestication origin. Positively selected genes in Tibetan pigs involved in high-altitude physiology, such as hypoxia, cardiovascular systems, UV damage, DNA repair. Three of loci with strong signals of selection are associated with EPAS1, CYP4F2, and THSD7A genes, related to hypoxia and circulation. We validated four non-coding mutations nearby EPAS1 and CYP4F2 showing reduced transcriptional activity in Tibetan pigs. A high-frequency missense mutation is found in THSD7A (Lys561Arg) in Tibetan pigs. The selective sweeps in Tibetan pigs was found in association with selection against non-coding variants, indicating an important role of regulatory mutations in Tibetan pig evolution. This study is important in understanding the evolution of Tibetan pigs and advancing our knowledge on animal adaptation to high-altitude environments.

Genetic variations associated with six-white-point coat pigmentation in Diannan small-ear pigs.

A common phenotypic difference among domestic animals is variation in coat color. Six-white-point is a pigmentation pattern observed in varying pig breeds, which seems to have evolved through several different mechanistic pathways. Herein, we re-sequenced whole genomes of 31 Diannan small-ear pigs from China and found that the six-white-point coat color in Diannan small-ear pigs is likely regulated by polygenic loci, rather than by the MC1R locus. Strong associations were observed at three loci (EDNRB, CNTLN, and PINK1), which explain about 20 percent of the total coat color variance in the Diannan small-ear pigs. We found a mutation that is highly differentiated between six-white-point and black Diannan small-ear pigs, which is located in a conserved noncoding sequence upstream of the EDNRB gene and is a putative binding site of the CEBPB protein. This study advances our understanding of coat color evolution in Diannan small-ear pigs and expands our traditional knowledge of coat color being a monogenic trait.

DNA methylation signatures of long intergenic noncoding RNAs in porcine adipose and muscle tissues.

Long intergenic noncoding RNAs (lincRNAs) are one of the major unexplored components of genomes. Here we re-analyzed a published methylated DNA immunoprecipitation sequencing (MeDIP-seq) dataset to characterize the DNA methylation pattern of pig lincRNA genes in adipose and muscle tissues. Our study showed that the methylation level of lincRNA genes was higher than that of mRNA genes, with similar trends observed in comparisons of the promoter, exon or intron regions. Different methylation pattern were observed across the transcription start sites (TSS) of lincRNA and protein-coding genes. Furthermore, an overlap was observed between many lincRNA genes and differentially methylated regions (DMRs) identified among different breeds of pigs, which show different fat contents, sexes and anatomic locations of tissues. We identify a lincRNA gene, linc-sscg3623, that displayed differential methylation levels in backfat between Min and Large White pigs at 60 and 120 days of age. We found that a demethylation process occurred between days 150 and 180 in the Min and Large White pigs, which was followed by remethylation between days 180 and 210. These results contribute to our understanding of the domestication of domestic animals and identify lincRNA genes involved in adipogenesis and muscle development.