Search

The adaptation and diversity of animals to the extreme environments of the Qinghai-Tibet Plateau (QTP) are typical materials to study adaptive evolution. The recently discovered Jinchuan yak population has many individuals with multiple ribs. However, little is known about this yak's origin, evolution, and the genetic mechanisms that formed its unique multirib trait. Here, we report a valuable population genome resource of the Jinchuan yak by resequencing the whole genome of 150 individuals. Population genetic polymorphism and structure analysis reveal that Jinchuan yak can be differentiated as a unique and original yak population among the domestic yak. Combined with geological change, the Jinchuan yak's evolutionary origin is speculated to be about 6290 years ago, which may be related to the unique geographical environment of the eastern edge of the QTP during this period. Compared with other domestic yaks, this new population has 280 positively selected genes. The genes related to skeletal function hold a considerable and remarkable proportion, suggesting that the specific skeletal characteristics have been enhanced in the adaptive evolution of Jinchuan yak in the extreme plateau environment. The genome-wide association study has revealed that TUBA8 and TUBA4A, the genes that regulate the cytoskeleton, are potential genes associated with the multirib trait. Our findings provide a basis to further understand the generation mechanism of the adaptive evolution of this new population in high-altitude extreme environments and the multivertebrate trait of domestic animals.

The male infertility of cattleyak resulted from spermatogenic arrest has greatly restricted the effective utilization of the heterosis from crossbreeding of cattle and yak. Based on our previous studies, the significant divergences of the transcriptomic and proteomic sequencing between yak and cattleyak prompt us to investigate the critical roles of microRNAs in post-transcriptional regulation of gene expression during spermatogenesis. TUNEL-POD analysis presented sharply decreased spermatogenic cell types and the increased apoptotic spermatogonia in cattleyak. The STA-PUT velocity sedimentation was employed to obtain spermatogonia and spermatocytes from cattle, yak and cattleyak and these spermatogenic cells were verified by the morphological and phenotypic identification. MicroRNA microarray showed that 27 differentially expressed miRNAs were simultaneously identified both in cattleyak vs cattle and in cattleyak vs yak comparisons. Further analysis revealed that the down-regulation of bta-let-7 families, bta-miR-125 and bta-miR-23a might impair the RA-induced differentiation of spermatogonia. Target gene analysis for differentially expressed miRNAs revealed that miRNAs targeted major players involved in vesicle-mediated transport, regulation of protein kinase activity and Pathways in cancer. In addition, spermatogonia transfection analysis revealed that the down-regulation of bta-miR-449a in the cattleyak might block the transition of male germ cells from the mitotic cycle to the meiotic program. The present study provided valuable information for future elucidating the regulatory roles of miRNAs involved in spermatogenic arrest of cattleyak.

Cattleyaks are the crossbred offspring between cattle and yaks, exhibiting the prominent adaptability to the harsh environment as yaks and much higher growth performances than yaks around Qinghai-Tibet plateau. Unfortunately, cattleyak cannot be effectively used in yak breeding due to its male infertility resulted from spermatogenic arrest. In this study, we performed RNA sequencing (RNA-seq) and bioinformatics analysis to determine the expression profiles of long noncoding RNA (lncRNA) from cattleyak and yak testis. A total of 604 differentially expressed (DE) lncRNAs (135 upregulated and 469 downregulated) were identified in cattleyak with respect to yak. Through gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, we identified several DE lncRNAs regulating the mitotic cell cycle processes by targeting the genes significantly associated with the mitotic cell cycle checkpoint and DNA damage checkpoint term and also significantly involved in p53 signaling pathway, mismatch repair and homologous recombination pathway (P 0.05). The reverse transcription PCR (RT-PCR) and quantitative Real-Time PCR (qRT-PCR) analysis of the randomly selected fourteen DE lncRNAs and the seven target genes validated the RNA-seq data and their true expressions during spermatogenesis in vivo. Molecular cloning and sequencing indicated that the testis lncRNAs NONBTAT012170 and NONBTAT010258 presented higher similarity among different cattleyak and yak individuals. The downregulation of these target genes in cattleyak contributed to the abnormal DNA replication and spermatogenic arrest during the S phase of mitotic cell cycle. This study provided a novel insight into lncRNA expression profile changes associated with spermatogenic arrest of cattleyak.

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.

Cattleyak exhibit equivalent adaptability to harsh environment as yak and much higher performances than yak. However, male infertility of cattleyak due to spermatogenic arrest greatly restricts their effective utilization in yak breeding. Although much work has been done to investigate the mechanisms of spermatogenic arrest, there is little information available in regard to the differences in transcriptomic profiling between cattleyak and yak testes. In this work, histologic observation indicated that spermatogonia were the main type of germ cells present in cattleyak testis, whereas all types of germ cells in differentiation were present in yak testis. Transcriptomic profiling identified 2960 differentially expressed genes (DEGs) in which 679 were upregulated and 2281 were downregulated in cattleyak. Significantly enriched gene ontology terms comprised a large number of DEGs associated with male infertility of cattleyak. The upregulation of STRA8 and NLRP14 may be associated with the accumulation of undifferentiated spermatogonial cells and serious cellular apoptosis in cattleyak. However, downregulated SPP1, SPIN2B, and PIWIL1 were associated with cell cycle progression and spermatogonial genome integrity, whereas CDKN2C, CYP26A1, OVOL1, GGN, MAK, INSL6, RNF212, TSSK1B, TSSK2, and TSSK6 were involved in meiosis. Furthermore, scores of genes associated with sperm components were also downregulated in cattleyak. Wnt/β-catenin signaling pathway was involved in the top-listed three significantly enriched pathways, and the downregulation of Wnt3a, PP2A, and TCF/LEF-1 may have contributed to the arrest of spermatogonial differentiation in cattleyak. The data suggest that spermatogenic arrest of cattleyak might occur at the stage of spermatogonial differentiation and get aggravated during meiosis, which results in minimal number of sperms with morphologic abnormalities and structural deficiency lacking fertilization ability.

Cattleyak (hybrid of cattle and yak) exhibit higher capability in adaptability and production than cattle and yak, while the infertility of F1 males greatly restricts the effective utilization of this hybrid and little progress has been made on investigating the mechanisms of the cattleyak infertility. Cattleyak individuals at three development stages (10, 12 and 14-month old) were sampled in this work and the isobaric tag for relative and absolute quantification method was employed to identify differences between their testicular proteomes. The proteomic analysis identified 318 proteins differentially expressed with significance at 12-month stage and 327 at 14-month compared with 10-month stage, respectively. Compared with the testicular proteome from 10-month cattleyak, the gene ontology (GO) annotations of the differentially expressed proteins at 12 months did not indicate significant differences from those at 14 months, which confirmed the histological observation that germ cell reduction was more obvious and spermatogenic arrest may become more serious in 12-month-old cattleyak. On the other hand, 56 differentially expressed proteins were coexpressed at 12 and 14-month stage compared with 10-month stage, in which 32 proteins were upregulated and 24 downregulated. GO analysis revealed that most of the differently expressed proteins were involved in the molecular function of catalytic activity, transporter activity, oxidoreductase activity and protein binding. Further analysis indicated that the differently expressed proteins including testis-expressed protein 101 precursor, RNA-binding motif protein, X chromosome, putative RNA-binding protein 3, heparin-binding proteins, tudor domain-containing protein 1, glutathione S-transferases (GSTA2, GSTP1), heat shock-related 70 kDa protein 2, estradiol 17-β-dehydrogenase11, 2,4-dienoyl-CoA reductase and peroxiredoxin-2 were possibly associated with testis development and spermatogenesis, which could be selected as candidate proteins in future study to examine the mechanisms of cattleyak infertility.

Male infertility of cattleyak due to spermatogenic arrest greatly restricts their effective utilization in yak breeding. Although much work has been done to investigate the mechanisms of spermatogenic arrest, there is no information regarding the differences of protein composition between cattleyak and yak testis. Comparative investigation of testis proteomes between cattleyak and yak using iTRAQ proteomics identified 256 differentially abundant proteins with fold change values higher than ± 1.5. Most of the differentially abundant proteins were involved in extracellular matrix organization, response to stimulus, metabolic and cellular process, in which a large number of the cattleyak predominant proteins were associated with various stresses, cell adhesion and germ cell migration. Such upregulated proteins as integrins and their ligands in the extracellular matrix involved in ECM-receptor interaction pathway may help germ cells to endure pulling forces and impede their migration. In contrast, down-regulated proteins in cattleyak were associated with defects in various metabolic processes and cellular processes during spermatogenesis. Such Downregulated proteins as the subunits of mitochondrial cytochrome Bc1 complex involved in the Alzheimer's disease and oxidative phosphorylation pathways may lead to mitochondrial dysfunction and cell death in cattleyak testis. Biological significance Spermatogenic arrest of cattleyak involves defects of both germ cells and their micro-environment in testis. In this study, Dozens of proteins possibly associated with spermatogenic arrest of cattleyak were identified by comparative iTRAQ proteomics, in which a large number of the cattleyak predominant proteins may act in response to various stresses (especially inflammatory stresses), enhance cell adhesion and impeded germ cell migration, while down-regulated proteins in cattleyak were associated with defects in various metabolic processes and cellular processes during spermatogenesis. Therefore, germ cells in testis of cattleyak may be constantly in a stress state (similar to inflammation) due to accumulation of some toxic intermediate products resulted from metabolic disturbances. Metabolic defects of germ cells and their deleterious micro-environment in testis of cattleyak may be the root of all other problems during spermatogenesis.

Background Cattleyak are the hybrid offspring between cattle and yak and combine yak hardiness with cattle productivity. Much attempt has been made to examine the mechanisms of male sterility caused by spermatogenic arrest, but yet there is no research systematically and precisely elucidated testis gene expression profiling between cattleyak and yak. Methods To explore the higher resolution comparative transcriptome map between the testes of yak and cattleyak, and further analyze the mRNA expression dynamics of spermatogenic arrest in cattleyak. We characterized the comparative transcriptome profile from the testes of yak and cattleyak using high-throughput sequencing. Then we used quantitative analysis to validate several differentially expressed genes (DEGs) in testicular tissue and spermatogenic cells. Results Testis transcriptome profiling identified 6477 DEGs (2919 upregulated and 3558 downregulated) between cattleyak and yak. Further analysis revealed that the marker genes and apoptosis regulatory genes for undifferentiated spermatogonia were upregulated, while the genes for differentiation maintenance were downregulated in cattleyak. A majority of DEGs associated with mitotic checkpoint, and cell cycle progression were downregulated in cattleyak during spermatogonial mitosis. Furthermore, almost all DEGs related to synaptonemal complex assembly, and meiotic progression presented no sign of expression in cattleyak. Even worse, dozens of genes involved in acrosome formation, and flagellar development were dominantly downregulated in cattleyak. Conclusion DEGs indicated that spermatogenic arrest of cattleyak may originate from the differentiation stage of spermatogonial stem cells and be aggravated during spermatogonial mitosis and spermatocyte meiosis, which contributes to the scarcely presented sperms in cattleyak.

Cattleyak are interspecific hybrids between cattle and yak, exhibiting the same prominent adaptability as yak and much higher performances than yak. However, male infertility of cattleyak resulted from spermatogenic arrest has greatly restricted their effective utilization in yak breeding. In past decades, much work has been done to investigate the mechanisms of spermatogenic arrest, but little is known about the differences of the post-transcriptional regulators between cattleyak and yak, which may contribute to the impaired spermatogenesis. MiRNAs, a class of endogenous non-coding small RNA, were revealed to play crucial roles in regulating gene expression at post-transcriptional level. In the present study, we identified 50 differentially expressed (DE) known miRNAs and 11 novel miRNAs by using Illumina HISeq and bioinformatic analysis. A total of 50 putative target sites for the 13 DE known miRNAs and 30 for the 6 DE novel miRNAs were identified, respectively. GO and KEGG analyses were performed to reveal the functions of target genes for DE miRNAs. In addition, RT-qPCR was performed to validate the expression of the DE miRNAs and its targets. The identification of these miRNAs may provide valuable information for a better understanding of spermatogenic arrest in cattleyak.

The researches on yak genetics and breeding were extremely restricted due to lacking of reliable DNA molecular markers. The microsatellites with repeat motif (AC)n/(GT)n in yak genome were enriched by Dynal magnetic beads and the gene libraries containing (AC)n/(GT)n were constructed. Among the 92 identified and sequenced positive clones, 40 contained perfect repeats (43.48%), 41 contained imperfect repeats (44.57%) and 11 contained compound repeats (11.96%). As compared with the percentage of perfect repeats, no significant increases of imperfect repeats were observed in yak genome, which indicated that the level of adaptive evolution of the ability to repair damaged genomic DNA for yaks were high enough to endure the natural pressure of nucleotide substitution resulted from ultraviolet irradiation in high-altitude areas. Totally 19 polymorphic microsatellite loci were screened and genotyped on the basis of electropherograms on an ABI 310 Genetic Analyzer. All the loci exhibited moderate to high-level polymorphisms in a test population of Bos grunniens and the polymorphic information content ranged from 0.299 to 0.861 (mean 0.678). The newly isolated (AC)n/(GT)n repeats from yak genome will display their potential values in examining intra-population genetic structure and inter-population relationships, and also in investigating molecular markers for production and adaptive traits of individual/population.

MC4R gene was proved to play important roles in body weight regulation in many mammals and exhibit higher homology among different species. The mutations MC4R significantly correlated to the restricted feeding weight, fat deposition and energy balance. In this work, ORF sequences of MC4R gene of Bos grunniens were cloned and phylogenetic relationships of yak and other mammals were analyzed on the basis of MC4R genes. Totally 290 variable sites were examined in 25 sequences from 22 different mammals, and 23 haplotypes were defined with a haplotype diversity of 0.9900. All the sequences were clustered into phylogenetic clades representing different orders or families. The individuals of Bos grunniens, Bos taurus and Ovis aries which belonged to the family of Bovidae were more divergent from the other orders or families and bovid animals may have branched out from the phylogenetic tree earlier than other mammals analyzed during 450 million years of vertebrate evolution. Amino acid sequences inferred from MC4R genes exhibited 54 variable sites, while high conservation of MC4R was observed within the same order or family. We concluded that coding region of MC4R gene displayed abundant variations among different mammal phylogenetic clades, whereas, the conservation of MC4R within order or family could be explained that MC4R gene may have been subjected to substantial constraints or strong purifying selection during several million years of mammal evolution.

MC4R (melanocortin 4 receptor) is expressed in the appetite-regulating areas of the brain and takes part in leptin signaling pathways. Sequencing of the coding region of the MC4R gene for 354 yaks identified the following five single nucleotide polymorphisms (SNPs): SNP1 (273C>T), SNP2 (321 G>T), SNP3 (864 C>A), SNP4 (1069G>C) and SNP5 (1206 G>C). SNP1, SNP2 and SNP3 were synonymous mutations, whereas SNP4 and SNP5 were missense mutations resulting in amino acid substitutions (V286L and R331S). Pairwise linkage disequilibrium (LD) analysis indicated that two pairs of SNPs, SNP2 and SNP5 (r(2)=0.81027) and SNP4 and SNP5 (r(2)=0.53816), exhibited higher degrees of LD. CC genotype of SNP4, CGACG and CTCCC haplotypes for all SNPs were associated with increased BW of animals that were 18 months old and with the average daily gain. The secondary structure and transmembrane region prediction of the yak MC4R protein suggested that SNP4 was correlated with influential changes in the seventh transmembrane domain of the MC4R protein and with the functional deterioration or even incapacitation of MC4R, which may contribute to the increased feed intake, BW and average daily gain of the yaks with CC genotypes. The data from this study suggested that 1069G>C SNP of the MC4R gene could be used in marker-assisted selection of growth traits in the Maiwa yak breed.