Genome-wide Detection Of High-altitude Related Genes And DNA Methylation Regulation Of Gene Expression In Tibetan Pigs

Tibetan pigs are mainly distributed in the Tibetan highlands, the largest continuous high elevation ecosystem in the world with an average elevation of more than 4000 m. And it’s the unique mini pig breed that lives in the highaltitude region of China. Now most of the researches are focused on the phenotypic and physiological area on Tibetan pigs, lacking of studies about the underlying mechanism of the high-altitude adaptation, which limited the utilization of this pig breed. In current study, we aim to uncover the genetic determination of high-altitude adaptation using next generation sequencing technology. We performed mitochondrial DNA analysis, comparative genomic analysis, transcriptome analysis, trying to demonstrate the molecular mechanism for adaptation in Tibetan pigs. We also design the "resettlement" experiment between Tibetan pigs. We dermined the whole genome single base resolution DNA methylome for longissimus doris muscle in Tibetan pigs. We also identify tremendous genes that showed different methylation between the highland and lowland pigs. The main points are indicated as follows:(1) We obtained the complete mtDNA sequence for each of the six Tibetan pig which located in different geographic region. In most of the regions in the mitogenome, the Tibetan pigs showed higher nucleotide diversity than the domestic pigs.(2) The lower diversity of 16 s rRNA in Tibetan pigs suggested that this gene has undergone purifying selection. In addition, the relative higher non-synonymous nucleotide substitution rate of the ATP6 gene provided clues to the existence of adaptive selection in Tibetan pigs.(3) All the sequencing was performed on the Illumina HiSeq 2000 platform, and generated a total of 659.4 Gb of paired-end DNA sequence.8,390,501 SNPs were detected in Tibetan pigs and 4,909,564 were shared with the domestic pigs, which was more than number of the shared SNPs among Tibetan pis, Chinese domestic pigs and European domestic pigs.(4) The phylogeny analysis indicated that Tibetan pigs and Chinese domestic pigs are genetically indistinguishable at an overall genomic level. And Chinese pigs were separated into southwest group and southeast group.(5) The genetic structure analysis showed that Tibetan pigs in Ganzi and Diqing regions exhibited more pure blood than the other region.(6) There are twice as many genomic regions with strong selective sweep signals in Chinese domestic pig (75.58 Mb, or 3.12% of the genome and containing 516 genes) as there are in Tibetan pigs (34.41 Mb, or 1.42% of the genome and containing 268 genes). The total of 268 Tibetan pig genes embedded in selected regions belong predominantly to categories that are related to adaptation to high altitudes, such as maintaining genomic stability against UV radiation and molecular adaptation under hypoxia.516 genes embedded in selected regions for domestic pigs were related mainly to olfactory reception, immune response and growth.(7) We find that ALB, SPTLC2, GLDC, ECE1, GNG2 and PIK3C2G showed strong signaling of positive selection. These genes could metabolically activate the form of vitamin B6 and aids in the synthesis of hemoglobin and enhances oxygen binding.(8) Transcripome analysis of seven tissues between Tibetan pigs and wild boars showed that lung harbor more different expression genes. And more gene showed lung-specific expression. These results demonstrated the function of lung may play a more important role in high-altitude adaptation in Tibetan pigs than other tissues.(9) We constructed the whole genome single base resolution DNA methylome for longissimus doris muscle in Tibetan pigs for the first time, and found the methylation occurred more frequently in the CpG dinucleotide than other contexts.(10) We found genes that showed different methylation between highland and lowland Tibetan pigs were significantly enriched in metabolic process, transport, phosphorylation, and ATP binding, which related to energy metabolism.(11) Genes that showed different expression between highland and lowland Tibetan pigs were significantly enriched in "positive regulation of cellular biosynthetic process", "positive regulation of gene expression" and "response to hypoxia". And we found that EGLN3 and ALB may play an important role in high-altitude adaptation of Tibetan pigs.