Huaming Mao

Whole-genome sequencing of six dog breeds from continuous altitudes reveals adaptation to high-altitude hypoxia

The hypoxic environment imposes severe selective pressure on species living at high altitude. To understand the genetic bases of adaptation to high altitude in dogs, we performed whole-genome sequencing of 60 dogs including five breeds living at continuous altitudes along the Tibetan Plateau from 800 to 5100 m as well as one European breed. More than 150× sequencing coverage for each breed provides us with a comprehensive assessment of the genetic polymorphisms of the dogs, including Tibetan Mastiffs. Comparison of the breeds from different altitudes reveals strong signals of population differentiation at the locus of hypoxia-related genes including endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1) and beta hemoglobin cluster. Notably, four novel nonsynonymous mutations specific to high-altitude dogs are identified at EPAS1, one of which occurred at a quite conserved site in the PAS domain. The association testing between EPAS1 genotypes and blood-related phenotypes on additional high-altitude dogs reveals that the homozygous mutation is associated with decreased blood flow resistance, which may help to improve hemorheologic fitness. Interestingly, EPAS1 was also identified as a selective target in Tibetan highlanders, though no amino acid changes were found. Thus, our results not only indicate parallel evolution of humans and dogs in adaptation to high-altitude hypoxia, but also provide a new opportunity to study the role of EPAS1 in the adaptive processes.

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.

Genetic diversity and origin of Gayal and cattle in Yunnan revealed by mtDNA control region and SRY gene sequence variation.

There are hump, humpless cattle and gayal distributed in Yunnan province, south-west China, but their genetic background remains unclear. To determine the origin and genetic diversity of Yunnan gayal and cattle (Diqing, Nujiang and Wenshan cattle), we analysed mtDNA control region sequences of 71 samples and SRY gene sequences of 39 samples, together with the available sequences in GenBank. The neighbour-joining phylogeny and the reduced median network analysis showed that Yunnan gayal originated from the hybridization between male Bos frontalis and female Bos taurus or Bos indicus, and that Yunnan cattle mostly originated from B. indicus, also containing some hybrids of male B. indicus and female B. taurus. The phylogenetic pattern of Yunnan cattle was consistent with the recently described cattle matrilineal pool from China and indicated more contribution to the Yunnan cattle from B. indicus than from B. taurus.

Dominant bacterial communities in the rumen of Gayals (Bos frontalis), Yaks (Bos grunniens) and Yunnan Yellow Cattle (Bos taurs) revealed by denaturing gradient gel electrophoresis

The dominant rumen bacteria in Gayals, Yaks and Yunnan Yellow Cattle were investigated using PCR-DGGE approach. The analysis of DGGE profiles, identification of dominant bands and phylogenetic analysis 16S rDNA sequences in DGGE profiles were combined to reveal the dominant bacterial communities and compared the differences between those cattle species. DGGE profiles revealed that Gayals had the most abundant dominant bacteria and the lowest similarity of intraspecies between individuals than other two cattle species. A total of 45 sequences were examined and sequence similarity analysis revealed that Gayals had the most sequences appeared to uncultured bacteria, accounting for 85.0% of the total sequences, Yaks and Yunnan Yellow Cattle had 44.4 and 68.8% uncultured bacterial sequences, respectively. According to phylogenetic analysis, the rumen dominant bacteria of Gayals were mainly phylogenetically placed within phyla firmicutes and bacteroidetes, and the known bacteria were mainly belonged to the genera Lachnospiraceae bacterium, Ruminococcus flavefaciens and Clostridium celerecrescens. Moreover, the dominant bacteria of Yaks were also mainly belonged to phyla firmicutes and bacteroidetes, and the known dominant bacteria were including Ruminococcus flavefaciens, Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis, Schwartzia succinivorans and Clostridiales bacterium, most of them are common rumen bacteria. In addition, the dominant bacteria in Yunnan Yellow Cattle were belonged to phyla firmicutes, bacteroidetes and Actinobacteria, and the known dominant bacteria containing Prevotella sp., Staphylococcilentus, Staphylococcus xylosus and Corynebacterium casei. Present study first detected Staphylococcus lentus and Staphylococcus xylosus in the rumen of cattle.

Assessment of protozoa in Yunnan Yellow Cattle rumen based on the 18S rRNA sequences

Library of ruminal protozoal 18S rRNA of Yunnan Yellow Cattle has been constructed in the present study. Phylogenic analysis of sequences was meanwhile employed to reveal the diversity of protozoa in the rumen of Yunnan Yellow Cattle. One Yellow Cattle was fed malt meal (YCRPB) and the other was fed wheat straw (YCRPS). A protozoa-specific primer (P-SSU-342f) and a eukarya-specific primer (Medlin B) were used to amplify a 1,360-bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from rumen fluid. The results showed as follows: A total of 121 clones were obtained and fell into four genera identified as Entodinium (66.9%), Dasytricha (5.8%), Isotricha (9.1%), and Diplodinium (18.2%). Within the genus Entidinium, 48 of the YCRPB sequences and 33 of the YCRPS sequences clustered with the Entodinium caudatum. 7 of the YCRPB sequences were phylogenetically placed within the genus Dasytricha. 11 of the YCRPB sequences were related with high confidence to Isotricha intestinalis. 22 of the YCRPS sequences were phylogenetically placed within the genus Diplodinium. The predominant protozoal genus identified in the rumen fluid belonged to the Entodinium group, and the divergences between two cattle may due to diet and individual differences.

Molecular diversity of bacteria in Yunnan yellow cattle (Bos taurs) from Nujiang region, China

The rumen content of four Yunnan Yellow Cattle (Bos taurs) were collected to determine the bacteria diversity by using 16S rRNA gene sequence analysis. A total of 129 sequences were examined and the sequences were referred as 107 OTU (Operational Taxonomy Unit) according to the similarity level of 97% in gene sequence. Similarity analysis revealed that Yunnan Yellow Cattle had 12 sequences (10 OTU) shared 97% or greater similarity with cultured rumen bacteria Butyrivibrio fibrisolvens, Succiniclasticum ruminis, Ruminococcus bromii, Clostridium proteoclasticum, Ruminococcus flavefaciens, Pseudobutyrivibrio ruminis, Jeotgalicoccus psychrophilus, and Prevotella ruminicola, which accounting for 9.3% of the total clones (9.2% of the total OTU). The further 12 sequences (9 OTU) shared 90–97% similarity with cultured bacteria Clostridium aminobutyricum, butyrate-producing bacterium, Schwartzia succinivorans, Prevotella ruminicola, Eubacterium ruminantium, Ruminococcus albus, and Clostridium termitidis, also accounting for 9.3% of the total sequences (8.3% of the total OTU). The remaining 105 sequences (90 OTU) shared less than 90% similarity with cultured bacteria, accounting for 81.4% of the total sequences (82.5% of the total OTU). According to the phylogenetic analysis, all sequences were phylogenetically placed within phyla of low G+C subdivision (accounting for 72.1 and 72.5% of the total clones and OTU, respectively) and CFB subdivision (Cytophaga-Flexibacter-Bacteroides; accounting for 27.9 and 27.5% of the total clones and OTU, respectively). Among the examined clones, rare bacteria Jeotgalicoccus psychrophilus was detected in the rumen of cattle.

Molecular cloning, sequence characterization and tissue transcription profile analyses of two novel genes: LCK and CDK2 from the Black-boned sheep (Ovis aries).

The complete coding sequences of two sheep genes—LCK and CDK2 were amplified using the rapid amplification of cDNA ends method based on three sheep EST sequences whose translated amino acids contain the domain PTKc_Lck_BIk and S_TKc domain, respectively. The sequence analyses of these two genes revealed that the sheep LCK gene encodes a protein of 509 amino acids which has high homology with the lymphocyte-specific protein tyrosine kinase (LCK) of eight species: bovine (99%), human (96%), dog (96%), Aotus nancymaae (95%), mouse (94%), rat (91%), horse (91%) and chicken (81%). The sheep CDK2 gene encodes a protein of 298 amino acids which has high homology with the cyclin-dependent kinase 2 (CDK2) of ten species: bovine (100%), goat (100%), rat (99%), mouse (99%), Chinese hamster (99%), dog (98%), golden hamster (98%), human (98%), horse (98%) and rhesus monkey (98%). The tissue transcription profile analyses indicated that that the Black-boned sheep LCK and CDK2 genes are generally but differentially expressed in the detected tissues including in tissues including spleen, muscle, skin, kidney, lung, liver and heart. These data serve as a foundation for further insight into these two genes.

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.

Molecular cloning, sequence identification and tissue expression profile of three novel genes Sfxn1, Snai2 and Cno from Black-boned sheep (Ovis aries)

The complete coding sequences of three of Black-boned sheep (Ovis aries) genes Sfxn1, Snai2 and Cno were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) according to the conserved sequence information of the cattle or other mammals and known highly homologous sheep ESTs. Black-boned sheep Sfxn1 gene encodes a protein of 322 amino acids which has high homology with the Sfxn1 proteins of five species—cattle 98%, pig 95%, human 95%, rat 93%, and mouse 93%. Black-boned sheep Snai2 gene encodes a protein of 268 amino acids that has high identity with the Snai2 proteins of six species—cattle 99%, pig 94%, human 93%, dog 93%, rat 91%, and mouse 90%. Black-boned sheep Cno gene encodes a protein of 214 amino acids that has high homology with the Cno proteins of four species—cattle 97%, human 75%, mouse 67%, and rat 65%. The phylogenetic tree analysis demonstrated that Black-boned sheep Sfxn1, Snai2 and Cno proteins have close relationship with cattle Sfxn1, Snai2 and Cno proteins. The tissue expression analysis indicated that Black-boned sheep Sfxn1, Snai2 and Cno genes were expressed in a range of tissues including leg muscle, kidney, skin, longissimus dorsi muscle, spleen, heart and liver. Our experiment is the first to provide the primary foundation for further insight into these three sheep genes.

Isolation, nucleotide identification and tissue expression of three novel ovine genes—SLC25A4, SLC25A5 and SLC25A6

The complete coding sequences of three of sheep genes SLC25A4, SLC25A5 and SLC25A6 were firstly amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) according to the conserved sequence information of the cattle or other mammals and known highly homologous sheep ESTs. Sheep SLC25A4, SLC25A5 and SLC25A6 genes encode three corresponding proteins of 298 amino acids which contain the identically conserved putative mitochondrial carrier protein domain. Sheep SLC25A4 protein has high homology with the SLC25A4 proteins of six species—cattle (99%), human (95%), rat (95%), mouse (94%), dog (94%) and chicken (89%). Sheep SLC25A5 protein has high identity with the SLC25A5 proteins of five species—cattle (100%), dog (99%), mouse (98%), rat (98%) and human (98%). Sheep SLC25A6 protein also has high homology with the SLC25A6 proteins of four species—cattle (99%), human (97%), pig (97%) and chicken (93%). The phylogenetic tree analysis demonstrated that sheep SLC25A4, SLC25A5 and SLC25A6 proteins share a common ancestor. Moreover, SLC25A4, SLC25A5 and SLC25A6 proteins present stronger interaction each other. The tissue expression analysis indicated that sheep SLC25A4, SLC25A5 and SLC25A6 genes were expressed in a range of tissues including leg muscle, kidney, skin, longissimus dorsi muscle, spleen, heart and liver. Our experiment is the first to provide the primary foundation for further insight into these three sheep genes.