Daoliang Lan

Zebularine and Scriptaid Significantly Improve Epigenetic Reprogramming of Yak Fibroblasts and Cloning Efficiency

Abnormal epigenetic reprogramming of the donor nucleus after somatic cell nuclear transfer (SCNT) is thought to be the main cause of low cloning efficiency. Following SCNT, the donor nucleus often fails to express early embryonic genes and establish a normal embryonic pattern of chromatin modification. Therefore, in this study, we have attempted to improve epigenetic reprogramming of the donor nucleus and cloned embryos with Zebularine and Scriptaid. Yak fibroblasts were treated with 20 μM Zebularine alone or 20 μM Zebularine plus 0.5 μM Scriptaid for 24 h, whereas yak cloned embryos were treated exclusively with 0.5 μM Scriptaid for 12 h. There was no effect on cellular viability and proliferation after drug treatment. The treatment of fibroblasts with Zebularine or Zebularine plus Scriptaid increased histone acetylation of histone 3 lysine 9 (H3K9), but decreased the level of DNA methylation of Oct-4 and Sox-2 promoter regions. When donor cells were used after Zebularine plus Scriptaid treatment to reconstruct cloned embryos and then treated with Scriptaid, the developmental competence and cryosurvival of embryos were improved significantly. In addition, the relative expression of Oct-4 and Sox-2 were increased significantly. The expression levels of Dnmt-1 and Hdac-1 were significantly decreased when fibroblasts and cloned embryos were treated with Zebularine or Scriptaid. This work provides functional evidence that treatment with Zebularine and Scriptaid modifies the epigenetic status of yak fibroblasts, subsequently enhancing in vitro developmental potential and the quality of yak cloned embryos.

RNA-Seq analysis of yak ovary: improving yak gene structure information and mining reproduction-related genes

RNA-Seq, a high-throughput (HT) sequencing technique, has been used effectively in large-scale transcriptomic studies, and is particularly useful for improving gene structure information and mining of new genes. In this study, RNA-Seq HT technology was employed to analyze the transcriptome of yak ovary. After Illumina-Solexa deep sequencing, 26826516 clean reads with a total of 4828772880 bp were obtained from the ovary library. Alignment analysis showed that 16992 yak genes mapped to the yak genome and 3734 of these genes were involved in alternative splicing. Gene structure refinement analysis showed that 7340 genes that were annotated in the yak genome could be extended at the 5′ or 3′ ends based on the alignments been the transcripts and the genome sequence. Novel transcript prediction analysis identified 6321 new transcripts with lengths ranging from 180 to 14884 bp, and 2267 of them were predicted to code proteins. BLAST analysis of the new transcripts showed that 1200?4933 mapped to the non-redundant (nr), nucleotide (nt) and/or SwissProt sequence databases. Comparative statistical analysis of the new mapped transcripts showed that the majority of them were similar to genes in Bos taurus (41.4%), Bos grunniens mutus (33.0%), Ovis aries (6.3%), Homo sapiens (2.8%), Mus musculus (1.6%) and other species. Functional analysis showed that these expressed genes were involved in various Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes pathways. GO analysis of the new transcripts found that the largest proportion of them was associated with reproduction. The results of this study will provide a basis for describing the normal transcriptome map of yak ovary and for future studies on yak breeding performance. Moreover, the results confirmed that RNA-Seq HT technology is highly advantageous in improving gene structure information and mining of new genes, as well as in providing valuable data to expand the yak genome information.

Establishment of mammary gland model in vitro: culture and evaluation of a yak mammary epithelial cell line.

This study aimed to establish yak mammary epithelial cells (YMECs) for an in vitro model of yak mammary gland biology. The primary culture of YMECs was obtained from mammary gland tissues of lactating yak and then characterized using immunocytochemistry, RT-PCR, and western blot analysis. Whether foreign genes could be transfected into the YMECs were examined by transfecting the EGFP gene into the cells. Finally, the effect of Staphylococcus aureus infection on YMECs was determined. The established YMECs retained the mammary epithelial cell characteristics. A spontaneously immortalized yak mammary epithelial cell line was established and could be continuously subcultured for more than 60 passages without senescence. The EGFP gene was successfully transferred into the YMECs, and the transfected cells could be maintained for a long duration in the culture by continuous subculturing. The cells expressed more antimicrobial peptides upon S.aureus invasion. Therefore, the established cell line could be considered a model system to understand yak mammary gland biology.

Toward Understanding the Genetic Basis of Yak Ovary Reproduction: A Characterization and Comparative Analyses of Estrus Ovary Transcriptiome in Yak and Cattle

Background Yaks (Bos grunniens) are endemic species that can adapt well to thin air, cold temperatures, and high altitude. These species can survive in harsh plateau environments and are major source of animal production for local residents, being an important breed in the Qinghai–Tibet Plateau. However, compared with ordinary cattle that live in the plains, yaks generally have lower fertility. Investigating the basic physiological molecular features of yak ovary and identifying the biological events underlying the differences between the ovaries of yak and plain cattle is necessary to understand the specificity of yak reproduction. Therefore, RNA-seq technology was applied to analyze transcriptome data comparatively between the yak and plain cattle estrous ovaries. Results After deep sequencing, 3,653,032 clean reads with a total of 4,828,772,880 base pairs were obtained from yak ovary library. Alignment analysis showed that 16992 yak genes mapped to the yak genome, among which, 12,731 and 14,631 genes were assigned to Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Furthermore, comparison of yak and cattle ovary transcriptome data revealed that 1307 genes were significantly and differentially expressed between the two libraries, wherein 661 genes were upregulated and 646 genes were downregulated in yak ovary. Functional analysis showed that the differentially expressed genes were involved in various Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. GO annotations indicated that the genes related to “cell adhesion,” “hormonal” biological processes, and “calcium ion binding,” “cation transmembrane transport” molecular events were significantly active. KEGG pathway analysis showed that the “complement and coagulation cascade” pathway was the most enriched in yak ovary transcriptome data, followed by the “cytochrome P450” related and “ECM–receptor interaction” pathways. Moreover, several novel pathways, such as “circadian rhythm,” were significantly enriched despite having no evident associations with the reproductive function. Conclusion Our findings provide a molecular resource for further investigation of the general molecular mechanism of yak ovary and offer new insights to understand comprehensively the specificity of yak reproduction.

Molecular characterization and tissue distribution of estrogen receptor genes in domestic yak.

Estrogen and its receptors are essential hormones for normal reproductive function in males and females during developmental stage. To better understand the effect of estrogen receptor (ER) gene in yak (Bos grunniens), reverse transcription-polymerase chain reaction (PCR) was carried out to clone ERα and ERβ genes. Bioinformatics methods were used to analyze the evolutionary relationship between yaks and other species, and real-time PCR was performed to identify the mRNA expression of ERα and ERβ. Sequence analysis showed that the ER open reading frames (ORFs) encoded 596 and 527 amino acid proteins. The yak ERα and ERβ shared 45.3% to 99.5% and 53.9% to 99.1% protein sequence identities with other species homologs, respectively. Real-time PCR analysis revealed that ERα and ERβ were expressed in a variety of tissues, but the expression level of ERα was higher than that of ERβ in all tissues, except testis. The mRNA expression of ERα was highest in the mammary gland, followed by uterus, oviduct, and ovary, and lowest in the liver, kidney, lung, testis, spleen, and heart. The ERβ mRNA level was highest in the ovary; intermediary in the uterus and oviduct; and lowest in the heart, liver, spleen, lung, kidney, mammary gland, and testis. The identification and tissue distribution of ER genes in yaks provides a foundation for the further study on their biological functions.

Identification of candidate miRNAs and expression profile of yak oocytes before and after in vitro maturation by high-throughput sequencing.

Small RNA represents several unique non-coding RNA classes that have important function in a wide range of biological processes including development of germ cells and early embryonic, cell differentiation, cell proliferation and apoptosis in diverse organisms. However, little is known about their expression profiles and effects in yak oocytes maturation and early development. To investigate the function of small RNAs in the maturation process of yak oocyte and early development, two small RNA libraries of oocytes were constructed from germinal vesicle stage (GV) and maturation in vitro to metaphase II-arrested stage (M II) and then sequenced using small RNA high-throughput sequencing technology. A total of 9,742,592 and 12,168,523 clean reads were obtained from GV and M II oocytes, respectively. In total, 801 and 1,018 known miRNAs were acquired from GV and M II oocytes, and 75 miRNAs were found to be significantly differentially expressed: 47 miRNAs were upregulated and 28 miRNAs were downregulated in the M II oocytes compared to the GV stage. Among the upregulated miRNAs, miR-342 has the largest fold change (9.25-fold). Six highly expressed miRNAs (let-7i, miR-10b, miR-10c, miR-143, miR-146b and miR-148) were validated by real-time quantitative PCR (RT-qPCR) and consistent with the sequencing results. Furthermore, the expression patterns of two miRNAs and their potential targets were analysed in different developmental stages of oocytes and early embryos. This study provides the first miRNA profile in the mature process of yak oocyte. Seventy-five miRNAs are expressed differentially in GV and M II oocytes as well as among different development stages of early embryos, suggesting miRNAs involved in regulating oocyte maturation and early development of yak. These results showed specific miRNAs in yak oocytes had dynamic changes during meiosis. Further functional and mechanistic studies on the miRNAs during meiosis may beneficial to understanding the role of miRNAs on meiotic division.

Author Correction: Correlations between gut microbiota community structures of Tibetans and geography

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Transcriptome profile and unique genetic evolution of positively selected genes in yak lungs

The yak (Bos grunniens), which is a unique bovine breed that is distributed mainly in the Qinghai-Tibetan Plateau, is considered a good model for studying plateau adaptability in mammals. The lungs are important functional organs that enable animals to adapt to their external environment. However, the genetic mechanism underlying the adaptability of yak lungs to harsh plateau environments remains unknown. To explore the unique evolutionary process and genetic mechanism of yak adaptation to plateau environments, we performed transcriptome sequencing of yak and cattle (Bos taurus) lungs using RNA-Seq technology and a subsequent comparison analysis to identify the positively selected genes in the yak. After deep sequencing, a normal transcriptome profile of yak lung that containing a total of 16,815 expressed genes was obtained, and the characteristics of yak lungs transcriptome was described by functional analysis. Furthermore, Ka/Ks comparison statistics result showed that 39 strong positively selected genes are identified from yak lungs. Further GO and KEGG analysis was conducted for the functional annotation of these genes. The results of this study provide valuable data for further explorations of the unique evolutionary process of high-altitude hypoxia adaptation in yaks in the Tibetan Plateau and the genetic mechanism at the molecular level.

Genetic Diversity, Molecular Phylogeny and Selection Evidence of Jinchuan Yak Revealed by Whole-Genome Resequencing

Jinchuan yak, a newly discovered yak breed, not only possesses a large proportion of multi-ribs but also exhibits many good characteristics, such as high meat production, milk yield, and reproductive performance. However, there is limited information about its overall genetic structure, relationship with yaks in other areas, and possible origins and evolutionary processes. In this study, 7,693,689 high-quality single-nucleotide polymorphisms were identified by resequencing the genome of Jinchuan yak. Principal component and population genetic structure analyses showed that Jinchuan yak could be distinguished as an independent population among the domestic yak population. Linkage disequilibrium analysis showed that the decay rate of Jinchuan yak was the lowest of the domestic yak breeds, indicating that the degree of domestication and selection intensity of Jinchuan yak were higher than those of other yak breeds. Combined with archaeological data, we speculated that the origin of domestication of Jinchuan yak was ∼6000 yr ago (4000–10,000 yr ago). The quantitative dynamics of population growth history in Jinchuan yak was similar to that of other breeds of domestic and wild yaks, but was closer to that of the wild yak. No significant gene exchange between Jinchuan and other domestic yaks occurred. Compared with other domestic yaks, Jinchuan yak possessed 339 significantly and positively selected genes, several of which relate to physiological rhythm, histones, and the breed’s excellent production characteristics. Our results provide a basis for the discovery of the evolution, molecular origin, and unique traits of Jinchuan yak.

Correlations between gut microbiota community structures of Tibetans and geography

Microbial communities of human gut directly influence health and bear adaptive potential to different geography environment and lifestyles. However, knowledge about the influences of altitude and geography on the gut microbiota of Tibetans is currently limited. In this study, fecal microbiota from 208 Tibetans across six different locations were analyzed by MiSeq sequencing; these locations included Gannan, Gangcha, Tianzhu, Hongyuan, Lhasa and Nagqu, with altitudes above sea level ranging from 2800 m to 4500 m across the Tibetan plateau. Significant differences were observed in microbial diversity and richness in different locations. At the phylum level, gut populations of Tibetans comprised Bacteroidetes (60.00%), Firmicutes (29.04%), Proteobacteria (5.40%), and Actinobacteria (3.85%) and were marked by a low ratio (0.48) of Firmicutes to Bacteroidetes. Analysis based on operational taxonomic unit level revealed that core microbiotas included Prevotella, Faecalibacterium, and Blautia, whereas Prevotella predominated all locations, except Gangcha. Four community state types were detected in all samples, and they mainly belong to Prevotella, Bacteroides, and Ruminococcaceae. Principal component analysis and related correspondence analysis results revealed that bacterial profiles in Tibetan guts varied significantly with increasing altitude, BMI, and age, and facultative anaerobes were rich in Tibetan guts. Gut microbiota may play important roles in regulating high-altitude and geographical adaptations.