Uncovering genetic variation through resequencing is limited by the fact that only sequences with similarity to the reference genome are examined. Reference genomes are often incomplete and cannot represent the full range of genetic diversity as a result of geographical divergence and independent demographic events. To more comprehensively characterize genetic variation of pigs (Sus scrofa), we generated de novo assemblies of nine geographically and phenotypically representative pigs from Eurasia. By comparing them to the reference pig assembly, we uncovered a substantial number of novel SNPs and structural variants, as well as 137.02-Mb sequences harboring 1737 protein-coding genes that were absent in the reference assembly, revealing variants left by selection. Our results illustrate the power of whole-genome de novo sequencing relative to resequencing and provide valuable genetic resources that enable effective use of pigs in both agricultural production and biomedical research.

Obesity is a major driver of metabolic diseases such as nonalcoholic fatty liver disease, certain cancers, and insulin resistance. However, there are no effective drugs to treat obesity. Betaine is a nontoxic, chemically stable and naturally occurring molecule. This study shows that dietary betaine supplementation significantly inhibits the white fat production in a high-fat diet (HFD)-induced obese mice. This might be due to betaine preventing the formation of new white fat (WAT), and guiding the original WAT to burn through stimulated mitochondrial biogenesis and promoting browning of WAT. Furthermore, dietary betaine supplementation decreases intramyocellular lipid accumulation in HFD-induced obese mice. Further analysis shows that betaine supplementation reduced intramyocellular lipid accumulation might be associated with increasing polyunsaturated fatty acids (PUFA), fatty acid oxidation, and the inhibition of fatty acid synthesis in muscle. Notably, by performing insulin-tolerance tests (ITTs) and glucose-tolerance tests (GTTs), dietary betaine supplementation could be observed for improvement of obesity and non-obesity induced insulin resistance. Together, these findings could suggest that inhibiting WAT production, intramyocellular lipid accumulation and inflammation, betaine supplementation limits HFD-induced obesity and improves insulin resistance.

Age-related physiological, biochemical and functional changes in mammalian skeletal muscle have been shown to begin at the mid-point of the lifespan. However, the underlying changes in DNA methylation that occur during this turning point of the muscle aging process have not been clarified. To explore age-related genomic methylation changes in skeletal muscle, we employed young (0.5 years old) and middle-aged (7 years old) pigs as models to survey genome-wide DNA methylation in the longissimus dorsi muscle using a methylated DNA immunoprecipitation sequencing approach. We observed a tendency toward a global loss of DNA methylation in the gene-body region of the skeletal muscle of the middle-aged pigs compared with the young group. We determined the genome-wide gene expression pattern in the longissimus dorsi muscle using microarray analysis and performed a correlation analysis using DMR (differentially methylated region)-mRNA pairs, and we found a significant negative correlation between the changes in methylation levels within gene bodies and gene expression. Furthermore, we identified numerous genes that show age-related methylation changes that are potentially involved in the aging process. The methylation status of these genes was confirmed using bisulfite sequencing PCR. The genes that exhibited a hypomethylated gene body in middle-aged pigs were over-represented in various proteolysis and protein catabolic processes, suggesting an important role for these genes in age-related muscle atrophy. In addition, genes associated with tumorigenesis exhibited aged-related differences in methylation and expression levels, suggesting an increased risk of disease associated with increased age. This study provides a comprehensive analysis of genome-wide DNA methylation patterns in aging pig skeletal muscle. Our findings will serve as a valuable resource in aging studies, promoting the pig as a model organism for human aging research and accelerating the development of comparative animal models in aging research. The online version of this article (doi:10.1186/1471-2164-15-653) contains supplementary material, which is available to authorized users.

Intramuscular fat (IMF) content and composition are considered crucial indicators of porcine meat quality. However, the molecular mechanism of porcine IMF development is still mostly unclear. Recently, new evidence suggested that microRNA (miRNAs) play important roles in porcine intramuscular adipogenesis. Previously, microRNA-125a-5p (miR-125a-5p) was identified as an important regulator of adipogenesis. In the present study, we found that the expression of miR-125a-5p is dynamically regulated during porcine intramuscular preadipocytes differentiation and that its expression levels in different porcine muscle tissues were negatively involved with IMF content. To investigate the potential function role of miR-125a-5p in IMF development, porcine intramuscular preadipocytes were collected and transfected with miR-125a-5p mimics, inhibitors, or a negative control (NC), respectively. The results showed that overexpression of miR-125a-5p promoted proliferation and inhibited differentiation of porcine intramuscular preadipocytes while inhibition of miR-125a-5p had the opposite effects. Furthermore, a luciferase reporter assay demonstrated that porcine kruppel like factor 3 (KLF13) is a target gene of miR-125a-5p during porcine intramuscular preadipocytes differentiation. Interestingly, porcine ELOVL fatty acid elongase 6 (ELOVL6), a regulator of fatty acid composition, was also identified as a target gene of miR-125a-5p during porcine intramuscular adipogenesis. Further studies show that miR-125a-5p overexpression reduced total saturated fatty acids (SFA) content and monounsaturated fatty acids (MUFA)/SFA ratios while having no significant impact on polyunsaturated fatty acids (PUFA)/SFA and n-6/n-3 ratios. Taken together, our results identified that miR-125a-5p may be a novel regulator of porcine intramuscular adipogenesis and the fatty acid composition of porcine IMF.

Genesis of novel gene regulatory modules is largely responsible for morphological and functional evolution. De novo generation of novel cis-regulatory elements (CREs) is much rarer than genomic events that alter existing CREs such as transposition, promoter switching or co-option. Only one case of de novo generation has been reported to date, in fish and without involvement of phenotype alteration. Yet, this event likely occurs in other animals and helps drive genetic/phenotypic variation. Using a porcine model of spontaneous hearing loss not previously characterized we performed gene mapping and mutation screening to determine the genetic foundation of the phenotype. We identified a mutation in the non-regulatory region of the melanocyte-specific promoter of microphthalmia-associated transcription factor (MITF) gene that generated a novel silencer. The consequent elimination of expression of the MITF-M isoform led to early degeneration of the intermediate cells of the cochlear stria vascularis and profound hearing loss, as well as depigmentation, all of which resemble the typical phenotype of Waardenburg syndrome in humans. The mutation exclusively affected MITF-M and no other isoforms. The essential function of Mitf-m in hearing development was further validated using a knock-out mouse model. Elimination of the MITF-M isoform alone is sufficient to cause deafness and depigmentation. To our knowledge, this study provides the first evidence of a de novo CRE in mammals that produces a systemic functional effect.

TRNA-derived fragments (tRFs) are a new class of non-coding RNA that play an important role in regulating cellular RNA processing and protein translation. However, there is currently no study reporting the influence of tRFs on myocardial hypertrophy. In this study, we used an isoproterenol (ISO)-induced myocardial hypertrophy rat model. Small RNA (<40 nts) transcriptome sequencing was used to select differentially expressed tRFs. We also compared the tRFs expression pattern in F0 sperm and the hearts of F1 offspring between the myocardial hypertrophy group (Hyp) and the control group (Con). Isoproterenol successfully induced a typical cardiac hypertrophy model in our study. Small RNA-seq revealed that tRFs were extremely enriched (84%) in the Hyp heart. Overexpression of tRFs1 and tRFs2 both enlarged the surface area of cardiac cells and increased expression of hypertrophic markers (ANF, BNP, and β-MHC). Luciferase reporter assay identified that tRFs1 directly target 3′UTR of Timp3. tRFs1, tRFs2, tRFs3, and tRFs4 were also highly expressed in Hyp F0 sperm and in Hyp F1 offspring hearts, but there was no differential expression of tRFs7, tRFs9, and tRFs10. Compared to Con F1 offspring, Hyp F1 offspring had elevated expression levels of β-MHC and ANP genes, and they had increased fibrosis and apoptosis in their hearts. These results demonstrated that tRFs are involved in regulating the response of myocardial hypertrophy. Besides, tRFs might serve as novel epigenetic factors that contribute to the intergenerational inheritance of cardiac hypertrophy.