Simultaneous testing of rule- and model-based approaches for runs of homozygosity detection opens up a window into genomic footprints of selection in pigs.

BACKGROUND: Past selection events left footprints in the genome of domestic animals, which can be traced back by stretches of homozygous genotypes, designated as runs of homozygosity (ROHs). The analysis of common ROH regions within groups or populations displaying potential signatures of selection requires high-quality SNP data as well as carefully adjusted ROH-defining parameters. In this study, we used a simultaneous testing of rule- and model-based approaches to perform strategic ROH calling in genomic data from different pig populations to detect genomic regions under selection for specific phenotypes. RESULTS: Our ROH analysis using a rule-based approach offered by PLINK, as well as a model-based approach run by RZooRoH demonstrated a high efficiency of both methods. It underlined the importance of providing a high-quality SNP set as input as well as adjusting parameters based on dataset and population for ROH calling. Particularly, ROHs ≤ 20 kb were called in a high frequency by both tools, but to some extent covered different gene sets in subsequent analysis of ROH regions common for investigated pig groups. Phenotype associated ROH analysis resulted in regions under potential selection characterizing heritage pig breeds, known to harbour a long-established breeding history. In particular, the selection focus on fitness-related traits was underlined by various ROHs harbouring disease resistance or tolerance-associated genes. Moreover, we identified potential selection signatures associated with ear morphology, which confirmed known candidate genes as well as uncovered a missense mutation in the ABCA6 gene potentially supporting ear cartilage formation. CONCLUSIONS: The results of this study highlight the strengths and unique features of rule- and model-based approaches as well as demonstrate their potential for ROH analysis in animal populations. We provide a workflow for ROH detection, evaluating the major steps from filtering for high-quality SNP sets to intersecting ROH regions. Formula-based estimations defining ROHs for rule-based method show its limits, particularly for efficient detection of smaller ROHs. Moreover, we emphasize the role of ROH detection for the identification of potential footprints of selection in pigs, displaying their breed-specific characteristics or favourable phenotypes.

Positive selection signatures in Anqing six-end-white pig population based on reduced-representation genome sequencing data.

Anqing six-end-white (AQ) pig performs well on resistance to coarse fodder and disease, reproduction and meat quality, offering high potential for exploitation. Environmental conditions and strict selections from local farmers have cultivated the AQ pig to be an outstanding and unique local pig breed. Thus we aim to detect genetic positive selection signatures within the AQ pig population to explore underlying genetic mechanisms. A relative extended haplotype homozygosity (REHH) test was performed in the population of 79 AQ pigs to seek evidence demonstrating that selective actions have left an imprint on the whole genome. In total, 430 500 REHH tests were performed on 53 067 core regions with average REHH tests of 8.11, average lengths of 11.50 kb and an overall length of 610.38 Mb which accounted for 26.94% of the whole genome. Finally, a total of 1819 core haplotypes (P < 0.01) and 586 candidate genes were obtained. These genes were mainly related to meat quality (MYOG, SNX19), resistance to disease (CRISPLD2, CD14) and reproduction traits (ERBB2, NRP2). A panel of genes within the 30 top significant REHH tests was mainly categorized to traits of meat quality and disease resistance. Among 13 KEGG pathways, MAPK, GnRH and Oxytocin signaling pathways, associated with the biological processes of crucial economic traits, were noteworthy. The excellent characteristics of the AQ pig benefited from the combination of natural and human factors. We provide a sketch map that shows the distribution of selection footprints on the whole genome of AQ pig and found potential genes for future studies.

The genetic diversity and population structures of indigenous pig breeds in Zhejiang Province revealed by GGRS sequencing.

Chinese indigenous pigs in Zhejiang Province are well known for their high fecundity. In order to verify breed subdivision at the genomic level, we investigated genetic diversity and population structure of seven breeds and made comparisons with three Western pig breeds using next-generation sequencing data. Parameters obtained from allelic richness and proportion of polymorphic markers indicated that the genetic diversity of the Chinese indigenous pigs was higher than that of the Western pigs, with the highest and lowest values found in the Chaluand and the Landrace pigs respectively. Both neighbor-joining tree and principal components analysis could distinguish breeds from one another and structure analysis showed less differentiation among Western pigs than among the Chinese pigs. The average linkage disequilibrium decay over distance was significantly less in the Chinese pigs compared with the Western pigs, ranging from 188.2 to 280.6 kb for the Chinese pigs and 680.3 to 752.8 kb for the Western breeds and showing an average r2 threshold value of 0.3. Results obtained from high-density SNP comparison over the whole genome on genetic diversity and population structure were in agreement with the current breed classification of the pigs in Zhejiang Province. More importantly, the results presented here advances our current understanding of the genomic biology of Chinese indigenous pigs in Zhejiang Province and allows for implementation of conservation strategies in additional breeds.

Genetic diversity and population structure of six Chinese indigenous pig breeds in the Taihu Lake region revealed by sequencing data.

The Chinese indigenous pig breeds in the Taihu Lake region are the most prolific pig breeds in the world. In this study, we investigated the genetic diversity and population structure of six breeds, including Meishan, Erhualian, Mi, Fengjing, Shawutou and Jiaxing Black, in this region using whole-genome SNP data. A high SNP with proportions of polymorphic markers ranging from 0.925 to 0.995 was exhibited by the Chinese indigenous pigs in the Taihu Lake region. The allelic richness and expected heterozygosity also were calculated and indicated that the genetic diversity of the Meishan breed was the greatest, whereas that of the Fengjing breed was the lowest. The genetic differentiation, as indicated by the fixation index, exhibited an overall mean of 0.149. Both neighbor-joining tree and principal components analysis were able to distinguish the breeds from each other, but structure analysis indicated that the Mi and Erhualian breeds exhibited similar major signals of admixture. With this genome-wide comprehensive survey of the genetic diversity and population structure of the indigenous Chinese pigs in the Taihu Lake region, we confirmed the rationality of the current breed classification of the pigs in this region.

A genome-wide scan for selection signatures in Yorkshire and Landrace pigs based on sequencing data.

Pigs have experienced dramatic selection due to domestication, which has led to many different phenotypes when compared to their wild counterparts, especially in the last several decades. Currently, genome-wide scans in both cattle and humans showing positive selection footprints have been investigated. However, few studies have focused on porcine selection footprints, particularly on a genome-wide scale. Surveying for selection footprints across porcine genomes can be quite valuable for revealing the genetic mechanisms of phenotypic diversity. Here, we employed a medium sequencing depth (5-20x/site per individual, on average) approach called genotyping by genome reducing and sequencing (GGRS) to detect genome-wide selection signatures of two domestic pig breeds (Yorkshire and Landrace) that have been under intensive selection for traits of muscle development, growth and behavior. The relative extended haplotype homozygosity test, which identifies selection signatures by measuring the characteristics of haplotypes' frequency distribution within a single population, was also applied to identify potential positively selected regions. As a result, signatures of positive selection were found in each breed. However, most selection signatures were population specific and related to genomic regions containing genes for biological categories including brain development, metabolism, growth and olfaction. Furthermore, the result of the gene set enrichment analysis indicated that selected regions of the two breeds presented a different over-representation of genes in the Gene Ontology annotations and Kyoto Encyclopedia of Genes and Genomes pathways. Our results revealed a genome-wide map of selection footprints in pigs and may help us better understand the mechanisms of selection in pig breeding.

Retrotransposon Insertion Polymorphisms (RIPs) in Pig Reproductive Candidate Genes.

Retrotransposons account for more than one-third of the pig reference genome. On account of the genome variability in different breeds, structural variation (SV) caused by retrotranspos-on-generated deletion or insertion (indel) may have a function in the genome. Litter size is one of the most important reproductive traits and significantly impacts profitability in terms of pig production. We used the method of bioinformatics, genetics, and molecular biology to make an analysis among different pig genomes. Predicted 100 SVs were annotated as retrotransposon indel in 20 genes related to reproductive performance. The PCR detection based on these predicted SVs revealed 20 RIPs in 20 genes, that most RIPs (12) were generated by SINE indel, and eight RIPs were generated by the ERV indel. We selected 12 RIPs to make the second round PCR detection in 24 individuals among nine pig breeds. The PCR detection results revealed that the RIP-A1CF-4 insertion in the breed of Bama, Large White, and Meishan only had the homozygous genotype but low to moderately polymorphisms were present in other breeds. We found that RIP-CWH43-9, RIP-IDO2-9, RIP-PRLR-6, RIP-VMP1-12, and RIP-OPN-1 had a rich polymorphism in the breed of Large White pigs. The statistical analysis revealed that RIP-CWH43-9 had a SINE insertion profitable to the reproductive traits of TNB and NBA but was significantly affected (p < 0.01) and (p < 0.05) in the reproductive traits of litter birthweight (LW) in Large White. On the other hand, the SINE insertion in IDO2-9 may be a disadvantage to the reproductive traits of LW, which was significantly affected (p < 0.05) in Large White. These two RIPs are significant in pig genome research and could be useful molecular markers in the breeding system.

Genomic Signatures Reveal Breeding Effects of Lulai Pigs.

In Chinese pig populations in which crossbreeding is used, these animals show a level of weakness compared with their original purebred ancestors. For instance, in the Lulai pig, a newly developed Chinese breed that is raised on the basis of the Laiwu pig (a Chinese indigenous breed with exceptionally high intramuscular fat content) and the Yorkshire pig using a method of systematic crossbreeding, both their market acceptance and performance are inferior. To reveal the practical role of these admixed breeds and traditional systematic crossbreeding methods at the genomic level, we explored population structure, genetic signatures, and introgression. We conducted this study based on the SNP chip data of 381 Lulai pigs, 182 Laiwu pigs, and 127 Yorkshires, which showed deficient genome coverage during our study. Therefore, we further selected the Genotyping by Genome Reducing and Sequencing (GGRS) method, which has a high density and suitable genome coverage as a supplement. We applied the GGRS data of 38 Lulai pigs, 75 Laiwu pigs, and 75 Yorkshires. In terms of the SNP chip data, by Fst analysis, we detected 782 significantly different genes between Lulai pigs and Yorkshires, including 3 major genes associated with growth (LEPR) and meat quality (SCD and TBC1D1), and we detected 426 significantly different genes between Lulai pigs and Laiwu pigs. With rIBD, we detected 12 genomic regions that included 182 genes that Yorkshires introgressed to Lulai pigs, and we detected 27 genomic regions that included 229 genes with a major gene (SCD) that Laiwu pigs introgressed to Lulai pigs. Regarding the GGRS data, we detected 601 significantly different genes between Lulai pigs and Yorkshires by Fst analysis, including 3 major genes associated with growth and fat deposits (IGF2 and FTO) and with hair color (KIT), and we detected 634 significantly different genes between Lulai pigs and Laiwu pigs, including 3 major genes related to their body composition (MYPN), hair color (KIT), and ear size (PPARD). By rIBD, we detected 94 deep sections that included 363 genes that Yorkshires introgressed to Lulai pigs, and we detected 149 deep sections that included 727 genes with a major gene (ESR1) that Laiwu pigs introgressed to Lulai pigs. Altogether, this study provides both insight into the molecular background of synthesized breeds of Lulai pigs and a reference for the evaluation of systematic crossbreeding in China.

The Organization of the Pig T-Cell Receptor γ (TRG) Locus Provides Insights into the Evolutionary Patterns of the TRG Genes across Cetartiodactyla.

The domestic pig (Sus scrofa) is a species representative of the Suina, one of the four suborders within Cetartiodactyla. In this paper, we reported our analysis of the pig TRG locus in comparison with the loci of species representative of the Ruminantia, Tylopoda, and Cetacea suborders. The pig TRG genomic structure reiterates the peculiarity of the organization of Cetartiodactyla loci in TRGC "cassettes", each containing the basic V-J-J-C unit. Eighteen genes arranged in four TRGC cassettes, form the pig TRG locus. All the functional TRG genes were expressed, and the TRGV genes preferentially rearrange with the TRGJ genes within their own cassette, which correlates the diversity of the γ-chain repertoire with the number of cassettes. Among them, the TRGC5, located at the 5' end of the locus, is the only cassette that retains a marked homology with the corresponding TRGC cassettes of all the analyzed species. The preservation of the TRGC5 cassette for such a long evolutionary time presumes a highly specialized function of its genes, which could be essential for the survival of species. Therefore, the maintenance of this cassette in pigs confirms that it is the most evolutionarily ancient within Cetartiodactyla, and it has undergone a process of duplication to give rise to the other TRGC cassettes in the different artiodactyl species in a lineage-specific manner.

Genome-Wide PERV Inactivation in Pigs Using CRISPR/Cas9.

The shortage of organs for transplantation is probably the biggest unmet medical need. A potential problem with the clinical use of porcine xenografts is the risk that porcine endogenous retroviruses (PERVs) could infect human cells. In the past, we determined the PERV copy number in the porcine kidney epithelial cell line PK15 and in primary fibroblasts. Using CRISPR-Cas9, we disrupted the catalytic center of pol, which is essential for virus replication. Next, we isolated cells in which 100% of the PERV elements had been inactivated. This method enables the possibility of eradicating PERVs in vitro for application to pig-to-human xenotransplantation. Here we describe the methodological bases of this work.

Retrotransposons evolution and impact on lncRNA and protein coding genes in pigs.

BACKGROUND: Retrotransposons are the major determinants of genome sizes and they have shaped both genes and genomes in mammalian organisms, but their overall activity, diversity, and evolution dynamics, particularly their impact on protein coding and lncRNA genes in pigs remain largely unknown. RESULTS: In the present study, we performed de novo detection of retrotransposons in pigs by using multiple pipelines, four distinct families of pig-specific L1 s classified into 51 distinct subfamilies and representing four evolution models and three expansion waves of pig-specific SINEs represented by three distinct families were identified. ERVs were classified into 18 families and found two most "modern" subfamilies in the pig genome. The transposition activity of pig L1 was verified by experiment, the sense and antisense promoter activities of young L1 5'UTRs and ERV LTRs and expression profiles of young retrotransposons in multiple tissues and cell lines were also validated. Furthermore, retrotransposons had an extensive impact on lncRNA and protein coding genes at both the genomic and transcriptomic levels. Most protein coding and lncRNA (> 80%) genes contained retrotransposon insertions, and about half of protein coding genes (44.30%) and one-fourth (24.13%) of lncRNA genes contained the youngest retrotransposon insertions. Nearly half of protein coding genes (43.78%) could generate chimeric transcripts with retrotransposons. Significant distribution bias of retrotransposon composition, location, and orientation in lncRNA and protein coding genes, and their transcripts, were observed. CONCLUSIONS: In the current study, we characterized the classification and evolution profile of retrotransposons in pigs, experimentally proved the transposition activity of the young pig L1 subfamily, characterized the sense and antisense expression profiles and promoter activities of young retrotransposons, and investigated their impact on lncRNA and protein coding genes by defining the mobilome landscapes at the genomic and transcriptomic levels. These findings help provide a better understanding of retrotransposon evolution in mammal and their impact on the genome and transcriptome.

Generation of B cell-deficient pigs by highly efficient CRISPR/Cas9-mediated gene targeting.

Generating B cell-deficient mutant is the first step to produce human antibody repertoires in large animal models. In this study, we applied the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to target the JH region of the pig IgM heavy chain gene which is crucial for B cell development and differentiation. Transfection of IgM-targeting Cas9 plasmid in primary porcine fetal fibroblasts (PFFs) enabled inducing gene knock out (KO) in up to 53.3% of colonies analyzed, a quarter of which harbored biallelic modification, which was much higher than that of the traditional homologous recombination (HR). With the aid of somatic cell nuclear transfer (SCNT) technology, three piglets with the biallelic IgM heavy chain gene mutation were produced. The piglets showed no antibody-producing B cells which indicated that the biallelic mutation of the IgM heavy chain gene effectively knocked out the function of the IgM and resulted in a B cell-deficient phenotype. Our study suggests that the CRISPR/Cas9 system combined with SCNT technology is an efficient genome-editing approach in pigs.