Understanding the genetic basis of adaptation to high altitude life is of paramount importance for preserving and managing genetic diversity in highland animals. This objective has been addressed mainly in terrestrial fauna but rarely in aquatic animals. Tibetan Schizothoracinae fish is the ideal model system in evolutionary biology, carrying key insights into evolutionary genetics of speciation and adaptation at high altitude. Gymnocypris przewalskii is the newly formed Schizothoracinae fish species in the Tibetan Plateau, inhabits chronic cold, extreme saline and alkaline aquatic environment in Lake Qinghai, thus evolving the unique genomic signatures to adapt extremely severe environments. To characterize its genomic features, we assembled de novo transcriptome of G. przewalskii from Lake Qinghai. Intriguingly, by comparative genomic analyses of G. przewalskii and 8 other fish species, we identified potential expansions in gene families related to energy metabolism, transport and developmental functions, possibly underlying the adaptation to these environmental stresses. Through comprehensive molecular evolution analyses, we found that sets of genes controlling mitochondrion, ion homoeostasis, acid-base balance and innate immunity show significant signals of positive selection. Compared to previous studies on highland fishes, we failed to identify any positively selected genes related to hypoxia response. Our findings provide comprehensive insights into the genetic basis of teleost fish that underlie their adaptation to extreme high altitude aquatic life on the Tibetan Plateau.

Domestication and artificial selection during production-oriented breeding have greatly shaped the level of genomic variability in sheep. However, the genetic variation associated with increased reproduction remains elusive. Here, two groups of samples from consecutively monotocous and polytocous sheep were collected for genome-wide association, transcriptomic, proteomic, and metabolomic analyses to explore the genetic variation in fecundity in Tibetan sheep. Genome-wide association study revealed strong associations between BMPR1B (p.Q249R) and litter size, as well as between PAPPA and lambing interval; these findings were validated in 1,130 individuals. Furthermore, we constructed the first single-cell atlas of Tibetan sheep ovary tissues and identified a specific mural granulosa cell subtype with PAPPA-specific expression and differential expression of BMPR1B between the two groups. Bulk RNA-seq indicated that BMPR1B and PAPPA expressions were similar between the two groups of sheep. 3D protein structure prediction and coimmunoprecipitation analysis indicated that mutation and mutually exclusive exons of BMPR1B are the main mechanisms for prolific Tibetan sheep. We propose that PAPPA is a key gene for stimulating ovarian follicular growth and development, and steroidogenesis. Our work reveals the genetic variation in reproductive performance in Tibetan sheep, providing insights and valuable genetic resources for the discovery of genes and regulatory mechanisms that improve reproductive success.

Genome-wide studies on highland adaptation mechanism in terrestrial animal have been widely reported with few available for aquatic animals. Tibetan Schizothoracinae species are ideal model systems to study speciation and adaptation of fish. The Schizothoracine fish, Gymnocypris przewalskii ganzihonensis had underwent the ecological niche shift from salt water to freshwater, and also experienced a recent split from Gymnocypris przewalskii przewalskii. In addition, G. p. ganzihonensis inhabited harsh aquatic environment including low temperature and hypoxia as well as other Schizothoracinae species, its genetic mechanism of highland adaptation have yet to be determined. Our study used comparative genomic analysis based on the transcriptomic data of G. p. ganzihonensis and other four fish genome datasets to investigate the genetic basis of highland adaptation in Schizothoracine fish. We found that Schizothoracine fish lineage on the terminal branch had an elevated dN/dS ratio than its ancestral branch. A total of 202 gene ontology (GO) categories involved into transport, energy metabolism and immune response had accelerated evolutionary rates than zebrafish. Interestingly, we also identified 162 genes showing signature of positive selection (PSG) involved into energy metabolism, transport and immune response in G. p. ganzihonesis. While, we failed to find any PSG related to hypoxia response as previous studies. Comparative genomic analysis based on G. p. ganzihonensis transcriptome data revealed significant genomic signature of accelerated evolution ongoing within Tibetan Schizothoracinae species lineage. Molecular evolution analysis suggested that genes involved in energy metabolism, transport and immune response functions in Schizothoracine fish underwent positive selection, especially in innate immunity including toll-like receptor signaling pathway genes. Taken together, our result as a case study in Schizothoracinae species provides novel insights in understanding the aquatic animal adaptation to extreme environment on the Tibetan Plateau, and also provides valuable genomic resource for further functional verification studies.

The Tibetan highland fish, Gymnocypris przewalskii, migrates from Lake Qinghai to its spawning grounds every summer. This seasonal reproduction is critically regulated by intrinsic and extrinsic signals. However, the molecular mechanisms that process environmental oscillations to initiate the seasonal mating are largely unknown. A transcriptomic analysis was conducted on the brain and gonad of male and female G. przewalskii in reproductive and nonreproductive seasons. We obtained 2034, 760, 1158 and 17,856 differentially expressed genes between the reproductively active and dormant female brain, male brain, ovary and testis. Among these genes, DIO2 was upregulated in the reproductively active brain and gonad of both males and females. Neuroactive ligand-receptor genes were activated in male and female brain. Functional enrichment analysis suggested that retinol metabolism was uniquely stimulated in reproductively active males. Genes involved in GnRH signaling and sex hormone synthesis exhibited higher expression levels in brain and gonad during the reproductive season. A co-expression network classified all the genes into 9 modules. The network pinpointed CDC42 as the hub gene that connected the pathways in responsible for modulating reproduction in G. przewalskii. Meanwhile, the sex pheromone receptor gene prostaglandin receptor was identified to link to multiple endocrine receptors, such as GnRHR2 in the network. The current study profiled transcriptomic variations between reproductively active and dormant fish, highlighting the potential regulatory mechanisms of seasonal reproduction in G. przewalskii. Our data suggested that the seasonal regulation of reproduction in G. przewalskii was controlled by the external stimulation of photoperiodic variations. The activated transcription of neuroendocrine and sex hormone synthesis genes contributed to seasonal reproduction regulation in G. przewalskii, which was presumably influenced by the increased day-length during the breeding season.

The identification of candidate genes and genetic variations associated with growth traits is important for sheep breeding. Insulin like growth factor 1 (IGF1) and insulin like growth factor 1 receptor (IGF1R) are well-accepted candidate genes that affect animal growth and development. The current study attempted to assess the association between IGF1 and IGF1R genetic polymorphisms and growth traits in Hulun Buir sheep. To achieve this goal, we first identified three and ten single nucleotide polymorphisms (SNPs) in exons of IGF1 and IGF1R in Hulun Buir sheep and then constructed six haplotypes of IGF1R based on linkage disequilibrium, respectively. Association studies were performed between SNPs and haplotypes of IGF1 and IGF1R with twelve growth traits in a population encompassing 229 Hulun Buir sheep using a general linear model. Our result indicated three SNPs in IGF1 were significantly associated with four growth traits (p < 0.05). In IGF1R, three SNPs and two haplotype blocks were significantly associated with twelve growth traits (p < 0.05). The combined haplotype H5H5 and H5H6 in IGF1R showed the strong association with 12 superior growth traits in Hulun Buir sheep (p < 0.05). In conclusion, we identified SNPs and haplotype combinations associated with the growth traits, which provided genetic resources for marker-assisted selection (MAS) in Hulun Buir sheep breeding.

Teleost fish have evolved various adaptations that allow them to tolerate cold water conditions. However, the underlying mechanism of this adaptation is poorly understood in Tibetan Plateau fish. RNA-seq combined with liquid chromatography‒mass spectrometry (LC‒MS/MS) metabolomics was used to investigate the physiological responses of a Tibetan Plateau-specific teleost, Gymnocypris przewalskii, under cold conditions. The 8-month G. przewalskii juvenile fish were exposed to cold (4 ℃, cold acclimation, CA) and warm (17 ℃, normal temperature, NT) temperature water for 15 days. Then, the transcript profiles of eight tissues, including the brain, gill, heart, intestine, hepatopancreas, kidney, muscle, and skin, were evaluated by transcriptome sequencing. The metabolites of the intestine, hepatopancreas, and muscle were identified by LC‒MS/MS. A total of 5,745 differentially expressed genes (DEGs) were obtained in the CA group. The key DEGs were annotated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. The DEGs from the eight tissues were significantly enriched in spliceosome pathways, indicating that activated alternative splicing is a critical biological process that occurs in the tissues to help fish cope with cold stress. Additionally, 82, 97, and 66 differentially expressed metabolites were identified in the intestine, hepatopancreas, and muscle, respectively. Glutathione metabolism was the only overlapping significant pathway between the transcriptome and metabolome analyses in these three tissues, indicating that an activated antioxidative process was triggered during cold stress. In combination with the multitissue transcriptome and metabolome, we established a physiology-gene‒metabolite interaction network related to energy metabolism during cold stress and found that gluconeogenesis and long-chain fatty acid metabolism played critical roles in glucose homeostasis and energy supply.