Xiao Gou

The local origin of the Tibetan pig and additional insights into the origin of Asian pigs.

Background The domestic pig currently indigenous to the Tibetan highlands is supposed to have been introduced during a continuous period of colonization by the ancestors of modern Tibetans. However, there is no direct genetic evidence of either the local origin or exotic migration of the Tibetan pig. Methods and Findings We analyzed mtDNA hypervariable segment I (HVI) variation of 218 individuals from seven Tibetan pig populations and 1,737 reported mtDNA sequences from domestic pigs and wild boars across Asia. The Bayesian consensus tree revealed a main haplogroup M and twelve minor haplogroups, which suggested a large number of small scale in situ domestication episodes. In particular, haplogroups D1 and D6 represented two highly divergent lineages in the Tibetan highlands and Island Southeastern Asia, respectively. Network analysis of haplogroup M further revealed one main subhaplogroup M1 and two minor subhaplogroups M2 and M3. Intriguingly, M2 was mainly distributed in Southeastern Asia, suggesting for a local origin. Similar with haplogroup D6, M3 was mainly restricted in Island Southeastern Asia. This pattern suggested that Island Southeastern Asia, but not Southeastern Asia, might be the center of domestication of the so-called Pacific clade (M3 and D6 here) described in previous studies. Diversity gradient analysis of major subhaplogroup M1 suggested three local origins in Southeastern Asia, the middle and downstream regions of the Yangtze River, and the Tibetan highlands, respectively. Conclusions We identified two new origin centers for domestic pigs in the Tibetan highlands and in the Island Southeastern Asian region.

Polymorphisms of the insulin-like growth factor-binding protein 3 gene (IGFBP3) in gayal (Bos frontalis).

The gene coding for insulin-like growth factor-binding protein 3 (IGFBP3) is important for regulation of growth, development and metabolism in mammals. The present investigation was conducted to study nucleotide polymorphism of the IGFBP3 in gayal (Bos frontalis) and to compare the variations with those which occur in other ruminants. A fragment of 645 base pairs of the IGFBP3 covering a part of exon 2, the complete intron 2 and exon 3 and a part of intron 3 was amplified, sequenced (n=46) and digested (n=79) with HaeIII restriction enzyme from 125 collected gayal samples. Nine single nucleotide polymorphisms (SNPs) [C14T, A122C, C137T, G144C, C155T, G213A, C279A, G334A and G460A] were identified and located in intron 2, revealing high genetic variability. The alignment of nucleotide sequences was found to be very similar to those for other bovid species. Sequencing and HaeIII digestion showed that frequency of alleles C and A [consisting of fragments of sizes 56, 64, 228, 264, 282, 298 and 497 bp (CC genotype)] was 0.96 and 0.04 for the SNP C279A. Moreover, the genotype frequency of the SNP C279A in gayal was compared with that in other ruminants and it appears that this polymorphism may be associated with low fat content and rapid growth in this rare species.

Genetic variability of the coding region for the prion protein gene (PRNP) in gayal (Bos frontalis)

The gayal (Bos frontalis) is a rare semi-wild bovid species in which bovine spongiform encephalopathy (BSE) has not been reported. Polymorphisms of the prion protein gene (PRNP) have been correlated significantly with resistance to BSE. In this study, the coding region of PRNP was cloned and characterized in samples from 125 gayal. A total of ten single nucleotide polymorphisms (SNPs), including six silent mutations (C60T, G75A, A108T, G126A, C357T and C678T) and four mis-sense mutations (C8A, G145A, G461A and C756G), corresponding to amino acids T3K, G49S9, N154S and I252M were identified, revealing high genetic diversity. Three novel SNPs including C60T, G145A and C756G, which have not been reported previously in bovid species, were retrieved. There also was one insertion–deletion (187Del24) at the N-terminal octapeptide repeat region. Alignment of nucleotide and amino acid sequences showed a high degree of similarity with other bovid species. Using phylogenetic analyses it was revealed that gayal has a close genetic relationship with Zebu cattle. In short, preliminary information is provided about genotypes of the PRNP in gayal. This could assist with the study of the pathogenesis of transmissible spongiform encephalopathies and cross species transmission as well as a molecular breeding project for gayal in China.

Gene Co-Expression Network Analysis Unraveling Transcriptional Regulation of High-Altitude Adaptation of Tibetan Pig.

Tibetan pigs have survived at high altitude for millennia and they have a suite of adaptive features to tolerate the hypoxic environment. However, the molecular mechanisms underlying the regulation of hypoxia-adaptive phenotypes have not been completely elucidated. In this study, we analyzed differentially expressed genes (DEGs), biological pathways and constructed co-expression regulation networks using whole-transcriptome microarrays from lung tissues of Tibetan and Duroc pigs both at high and low altitude. A total of 3,066 DEGs were identified and this list was over-represented for the ontology terms including metabolic process, catalytic activity, and KEGG pathway including metabolic pathway and PI3K-Akt signaling pathway. The regulatory (RIF) and phenotypic (PIF) impact factor analysis identified several known and several potentially novel regulators of hypoxia adaption, including: IKBKG, KLF6 and RBPJ (RIF1), SF3B1, EFEMP1, HOXB6 and ATF6 (RIF2). These findings provide new details of the regulatory architecture of hypoxia-adaptive genes and also insight into which genes may undergo epigenetic modification for further study in the high-altitude adaptation.

Screening and characterization of a novel ruminal cellulase gene （Umcel-1） from a metagenomic library of gayal （Bos frontalis）

Abstract Gayal is a rare semi-wild bovine species found in the Indo-China. They can graze grasses, including bamboo leaves, as well as reeds and other plant species, and grow to higher mature live weights than Yunnan Yellow cattle maintained in similar harsh environments. The aim of this study was to identify specific cellulase in the gayal rumen. A metagenomic fosmid library was constructed using genomic DNA isolated from the ruminal contents of four adult gayals. This library contained 38 400 clones with an average insert size of 35.5 kb. The Umcel-1 gene was isolated from this library. Investigation of the cellulase activity of 24 random clones led to the identification of the Umcel-1 gene , which exhibited the most potent cellulase activity. Sequencing the Umcel-1 gene revealed that it contained an open reading frame of 942 base pairs that encoded a product of 313 amino acids. The putative gene Umcel-1 product belonged to the glycosyl hydrolase family 5 and showed the highest homology to the cellulase (GenBank accession no. YP_004310852.1) from Clostridium lentocellum DSM 5427, with 44% identity and 62% similarity. The Umcel-1 gene was heterologously expressed in Escherichia coli BL21, and recombinant Umcel-1 was purified. The activity of purified recombinant Umcel-1 was assessed, and the results revealed that it hydrolyzed carboxymethyl cellulose with optimal activity at pH 5.5 and 45°C. To our knowledge, this study provides the first evidence for a cellulase produced by bacteria in gayal rumen.

Molecular Cloning and Characteristics of the PSPH, snrpa1 and TPM1 Genes in Black-Boned Sheep (Ovis Aries)

ABSTRACT The first animal with black traits to be studied was the Black-boned chicken. In China, we identified a flock of sheep with black traits. In the present study, the complete coding sequences of three genes, PSPH, SNRPA1 and TPM1, of Black-boned sheep (Ovis aries) were amplified using the reverse transcription polymerase chain reaction, according to the conserved sequence information for cattle and other mammals and known highly homologous sheep ESTs. The results showed that the Black-boned sheep PSPH gene encodes a protein of 225 amino acids, which has high homology with the PSPH proteins of seven species: cattle (99%), humans (94%), rat (93%), mouse (93%), African clawed frog (77%), Atlantic salmon (70%) and zebrafish (68%). The Black-boned sheep SNRPA1 gene encodes a protein of 255 amino acids, which has high identity with the SNRPA1 proteins of four species: cattle (99%), humans (99%), mouse (97%) and chicken (91%). The Black-boned sheep TPM1 gene encodes a protein of 284 amino acids, which has high homology with the TPM1 proteins of four species: cattle (82%), rabbit (82%), mouse (82%) and humans (81%). Using phylogenetic analysis, it was shown that the Black-boned sheep PSPH, SNRPA1 and TPM1 proteins are closely related to their cattle counterparts. The tissue expression analyses revealed that the Black-boned sheep PSPH, SNRPA1 and TPM1 genes were expressed in a range of tissues, including leg muscles, the kidneys, the skin, the longissimus dorsi muscle, the spleen, the heart and the liver. These data serve as a foundation for further insight into these three genes in this rare sheep breed.

Natural selection on TMPRSS6 associated with the blunted erythropoiesis and improved blood viscosity in Tibetan Pigs

Tibetan pigs, indigenous to Tibetan plateau, are well adapted to hypoxia. So far, there have been not any definitively described genes and functional sites responsible for hypoxia adaptation for the Tibetan pig. Here we conducted resequencing of the nearly entire genomic region (40.1 kb) of the candidate gene TMPRSS6 (Transmembrane protease, serine 6) associated with hemoglobin concentration (HGB) and red blood cell count (RBC) in 40 domestic pigs and 40 wild boars from five altitudes along the Tea-horse ancient road and identified 708 SNPs, in addition to an indel (CGTG/----) in the intron 10. Both the CGTG deletion frequency and the pairwise r2 linkage disequilibrium showed an increase with elevated altitudes in 838 domestic pigs from five altitudes, suggesting that TMPRSS6 has been under Darwinian positive selection. As the conserved core sequence of hypoxia-response elements (HREs), the deletion of CGTG in Tibetan pigs decreased the expression levels of TMPRSS6 mRNA and protein in the liver revealed by real-time quantitative PCR and western blot, respectively. To explore whether reduced TMPRSS6 expression level could improve blood viscosity, the relationship between CGTG indel and hematologic and hemorheologic parameters in 482 domestic pigs from continuous altitudes was detected and dissected a genetic effect on reducing HGB by 13.25g/L in Gongbo’gyamda Tibetan pigs and decreasing MCV by 4.79 fl in Diqing Tibetan pigs. In conclusion, the CGTG deletion of TMPRSS6 resulted in lower HGB and smaller MCV, thereby blunting erythropoiesis and improving blood viscosity as well as erythrocyte deformability.

Haplotype diversity in mitochondrial DNA reveals the multiple origins of Tibetan horse

The Tibetan horse is a species endemic to the Tibetan plateau, with considerable economic value in the region. However, we currently have little genetic evidence to verify whether the breed originated in Tibet or if it entered the area via an ancient migratory route. In the present study, we analyzed the hypervariable segment I sequences of mitochondrial DNA (mtDNA) in 2,050 horses, including 290 individuals from five Tibetan populations and 1,760 from other areas across Asia. Network analysis revealed multiple maternal lineages in the Tibetan horse. Component analysis of sub-lineage F3 indicated that it decreased in frequency from east to west, a trend reflected both southward and northward from Inner Mongolia. Analysis of population genetics showed that the Deqen horse of eastern Tibet was more closely related to the Ningqiang horse of northern China than to other Tibetan horses or the Yunnan horse. These results indicated that the Tibetan horse migrated first from Central Asia to Mongolia, moved south to eastern Tibet (near Deqen), then finally westward to other regions of Tibet. We also identified a novel lineage K that mainly comprises Tibetan and Yunnan horses, suggesting autochthonous domesticated origin for some Tibetan horse breeds from local wild horses. In conclusion, our study demonstrated that modern Tibetan horse breeds originated from the introgression of local wild horses with exotic domesticated populations outside China.