Molecular microevolution and epigenetic patterns of the long non-coding gene H19 show its potential function in pig domestication and breed divergence.
BACKGROUND
The domestic pig Sus scrofa domesticus originated from the wild boar S. scrofa about 10,000 years ago. During domestication, drastic morphological, physiological, and behavioral changes developed between domestic pigs and wild boars through artificial and natural selection. The long non-coding RNA (lncRNA) H19, which is located within the imprinting gene cluster H19-IGF2, plays an important role in regulating muscle development in humans and mice. This study systematically analyzed the molecular evolution of H19 and its possible epigenetic changes during pig domestication and breeding to explore the genetic and epigenetic contributions of H19 to pig domestication.


RESULTS
The molecular evolution of H19 was initially analyzed on a large phylogenetic scale. Results showed that the gene was highly conserved within a broad range, especially in the 5' terminal sequence. The molecular evolution of the gene was then analyzed using published re-sequencing data of 30 wild boars from Tibet, 3 wild boars from Sichuan, and 15 native pigs from other regions in China. Eight polymorphic sites were identified, and the nucleotide diversity (π) value within the H19 gene body was significantly higher (Z-test, P < 0.05) in domesticated pigs than in wild pigs. However, no significant divergence occurred between domesticated and wild pigs. Single nucleotide polymorphisms in the 3' terminal sequence were surveyed in other Chinese local breeds and foreign pig breeds. We observed a consistently higher diversity in domesticated pigs than in wild pigs. The methylation pattern of the H19 gene in pigs was subsequently analyzed using published methylated DNA immunoprecipitation data and an unpublished single-base resolution liver methylome. Analysis results showed distinct methylation levels in some tissues. Among the samples surveyed, Landrace showed the lowest methylation level, followed by the Guizhou wild boar, whereas the Enshi pig exhibited the highest methylation level in the 2 kb upstream region of the H19 gene. Liver transcriptome data suggested that Landrace harbored the highest expression of the H19 gene, followed by the Guizhou wild boar, whereas the Enshi pig harbored the lowest expression of the gene. Differential methylation sites (DMSs) among the three breeds were mainly identified in the 2 kb upstream region of the H19 gene. In the Enshi pig, we detected allele-specific methylation (ASM) regions in the 2 kb upstream region of the H19 gene. Most of the DMSs in the upstream 2 kb region of the gene were also located in the ASM region in this breed.


CONCLUSIONS
Molecular analyses suggest that the H19 gene was highly conserved during large-scale evolution and exhibited genotype differentiation during domestication and breed differentiation. The drastic diversity pattern between domestic and wild pigs in the H19 gene body, which was highly conserved during large-scale evolution, suggests that this gene might have played roles in the breed differentiation of domestic pigs. Methylation analysis indicates an opposite epigenetic regulation direction between Chinese and European pig (EU) domestication, which resulted in opposite expression changes in this gene between the two domesticated groups. Our preliminary analyses on DMSs among different pig breeds and ASM imply that imprinting was associated with methylation differences. This study systematically demonstrates the genetic and epigenetic patterns of H19 during pig domestication and provide valuable cues and basis for further research on the function of H19 in pig domestication.
Key Lab of Agriculture Animal Genetics, Breeding, and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People's Republic of China.
Cencen
C
Li
State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 East Jiaochang Road, Kunming, Yunnan Province, 65022, China.|Graduate School of the Chinese Academy of Sciences, Beijing, 100049, China.
Xiao
X
Wang
Department of Computer Science, City University of Hong Kong, Hong Kong, 999077, China.|BGI Co Ltd, Shenzhen, 518083, China.
Huimin
H
Cai
Key Lab of Agriculture Animal Genetics, Breeding, and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People's Republic of China.
Yuhua
Y
Fu
Key Lab of Agriculture Animal Genetics, Breeding, and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People's Republic of China.
Yu
Y
Luan
State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 East Jiaochang Road, Kunming, Yunnan Province, 65022, China.|Graduate School of the Chinese Academy of Sciences, Beijing, 100049, China.
Wen
W
Wang
State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 East Jiaochang Road, Kunming, Yunnan Province, 65022, China. xiangh@mail.kiz.ac.cn.|Graduate School of the Chinese Academy of Sciences, Beijing, 100049, China. xiangh@mail.kiz.ac.cn.|South China Normal University, Guangzhou, 510631, China. xiangh@mail.kiz.ac.cn.
Hui
H
Xiang
Key Lab of Agriculture Animal Genetics, Breeding, and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People's Republic of China. lichangchun@mail.hzau.edu.cn.
Changchun
C
Li
D000818:Animals; D000829:Animals, Domestic / Q000235:genetics; D001483:Base Sequence; D001947:Breeding; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D019143:Evolution, Molecular; D005260:Female; D005838:Genotype; D008297:Male; D010802:Phylogeny; D062085:RNA, Long Noncoding / Q000235:genetics*; D012641:Selection, Genetic; D017423:Sequence Analysis, RNA; D013045:Species Specificity; D034421:Sus scrofa / Q000235:genetics*
C094335:H19 long non-coding RNA; D062085:RNA, Long Noncoding
Breed divergence; Domestication; H19; Long non-coding gene; Methylation; Molecular evolution; Pig