Genetic structure analysis of yak breeds and their response to adaptive evolution.

Yaks are crucial genetic resources in the Tibetan Plateau and surrounding regions. Throughout the long process of domestication, natural and artificial selection pressures have enabled yaks to demonstrate adaptive characteristics to the environment in terms of physiological structure and genetic molecules, but no systematic cell analysis has been carried out on this phenomenon of yaks. Here, the population structure and genetic diversity of yak were studied by WGRS, and the genes related to yak adaptability were excavated. Combined with scRNA-seq method, the transcription map of yak lung tissue and skin tissue was constructed, which provided a new comprehensive insight into yak adaptability. The analysis of yak population structure showed that there was obvious genetic differentiation between TZ _ yak and other seven yak populations, while there was significant genetic exchange between PL _ yak and SB _ yak at high altitude. WGRS and scRNA-seq analysis revealed that the gene HIF1A related to high altitude adaptation was expressed in various cell types, while EPAS1 was predominantly expressed in epithelial and endothelial cells of yak lung tissue. Endothelial cells play a critical role in hypoxia-adapted VEGF signaling, which correlates closely with the high expression of KDR and VEGFA genes in endothelial cells and monocytes. Furthermore, in the selection signal of High _ yak vs Low _ yak, 19.8 % of the genes overlapped with the genes screened by skin scRNA-seq, including genes related to coat color such as RORA, BNC2, and KIT. Notably, BNC2 is a gene associated with melanin deposition and shows high expression levels in HS cells. Additionally, GRN in melanocytes and SORT1 in IRS play an important role in cell communication between melanocytes and IRS. These findings offer new insights into the natural polymorphism of yaks and provide a valuable reference for future research on high-altitude mammals, and potentially even human genetics.

A comprehensive study on the longissius dorsi muscle of Ashdan yaks under different feeding regimes based on transcriptomic and metabolomic analyses.

Yak is an important dominant livestock species at high altitude, and the growth performance of yak has obvious differences under different feeding methods. This experiment was conducted to compare the effects of different feeding practices on growth performance and meat quality of yaks through combined transcriptomic and metabolomic analyses. In terms of yak growth performance, compared with traditional grazing, in-house feeding can significantly improve the average daily weight gain, carcass weight and net meat weight of yaks; in terms of yak meat quality, in-house feeding can effectively improve the quality of yak meat. A combined transcriptomic and metabolomic analysis revealed 31 co-enriched pathways, among which arginine metabolism, proline metabolism and glycerophospholipid metabolism may be involved in the development of the longissimus dorsi muscle of yak and the regulation of meat quality-related traits. The experimental results increased our understanding of yak meat quality and provided data materials for subsequent deep excavation of the mechanism of yak meat quality.

An HD-ZIP transcription factor, MxHB13, integrates auxin-regulated and juvenility-determined control of adventitious rooting in Malus xiaojinensis.

Adventitious root (AR) formation is a critical factor in the vegetative propagation of forestry and horticultural plants. Competence for AR formation declines in many species during the miR156/SPL-mediated vegetative phase change. Auxin also plays a regulatory role in AR formation. In apple rootstock, both high miR156 expression and exogenous auxin application are prerequisites for AR formation. However, the mechanism by which the miR156/SPL module interacts with auxin in controlling AR formation is unclear. In this paper, leafy cuttings of juvenile (Mx-J) and adult (Mx-A) phase Malus xiaojinensis were used in an RNA-sequencing experiment. The results revealed that numerous genes involved in phytohormone signaling, carbohydrate metabolism, cell dedifferentiation, and reactivation were downregulated in Mx-A cuttings in response to indole butyric acid treatment. Among the differentially expressed genes, an HD-ZIP transcription factor gene, MxHB13, was found to be under negative regulation of MdSPL26 by directly binding to MxHB13 promoter. MxTIFY9 interacts with MxSPL26 and may play a role in co-repressing the expression of MxHB13. The expression of MxTIFY9 was induced by exogenous indole butyric acid. MxHB13 binds to the promoter of MxABCB19-2 and positively affects the expression. A model is proposed in which MxHB13 links juvenility-limited and auxin-limited AR recalcitrance mechanisms in Mx-A.

Construction of transcriptome atlas of white yak hair follicle during anagen and catagen using single-cell RNA sequencing.

BACKGROUND: As the direct organ of villus, hair follicles have obvious seasonal cycles. The hair follicle cycle is orchestrated by multiple cell types that together direct cell renewal and differentiation. But the regulation property of hair follicle cells from anagen to catagen in yak is still unknown. RESULTS: In this study, single-cell RNA sequencing was performed on 24,124 single cells of the scapular skin from white yak. Based on tSNE cluster analysis, the cell types of IFE-DC, epidermal cell lines, fibroblasts, keratinocytes, IRS, DS, INFU, and other cells in yak hair follicles during anagen and catagen were successfully identified, and the gene expression profiles were described. The GO enrichment analysis indicated the different cells characteristic genes to be mainly enriched in the epidermal development, epithelial cell differentiation and wound healing pathways. The pseudotime trajectory analysis described the differentiation trajectory of the epidermal lineage and dermal lineage of the hair follicle during anagen and catagen. Moreover, the dynamic changes of the genes like LHX2, KRT25, and KRT71 were found to be highly expressed in HS and IRS, but not in the IFE-DC, INFU, and keratinocyte during differentiation. CONCLUSIONS: Our results analyzed the time-varying process of gene expression in the dermal cell lineage and epidermal cell lineage of hair follicles during anagen and catagen during fate differentiation was expounded at the single cell level, revealing the law of fate specialization of different types of cells. In addition, based on the enrichment analysis, the transcriptional regulatory factors involved in the different cell fates were also revealed. These results will help to enhance our understanding of yak hair follicle cycle and promote the development and utilization of yak villus.

The Idesia polycarpa genome provides insights into its evolution and oil biosynthesis.

The deciduous tree Idesia polycarpa can provide premium edible oil with high polyunsaturated fatty acid contents. Here, we generate its high-quality reference genome, which is ∼1.21 Gb, comprising 21 pseudochromosomes and 42,086 protein-coding genes. Phylogenetic and genomic synteny analyses show that it diverged with Populus trichocarpa about 16.28 million years ago. Notably, most fatty acid biosynthesis genes are not only increased in number in its genome but are also highly expressed in the fruits. Moreover, we identify, through genome-wide association analysis and RNA sequencing, the I. polycarpa SUGAR TRANSPORTER 5 (IpSTP5) gene as a positive regulator of high oil accumulation in the fruits. Silencing of IpSTP5 by virus-induced gene silencing causes a significant reduction of oil content in the fruits, suggesting it has the potential to be used as a molecular marker to breed the high-oil-content cultivars. Our results collectively lay the foundation for breeding the elite cultivars of I. polycarpa.

The Novel Structural Variation in the GHR Gene Is Associated with Growth Traits in Yaks (Bos grunniens).

The growth hormone receptor (GHR) is a member of the cytokine/hematopoietic factor receptor superfamily, which plays an important role in the growth and development, immunity, and metabolism of animals. This study identified a 246 bp deletion variant in the intronic region of the GHR gene, and three genotypes, including type II, type ID, and type DD, were observed. Genotype analysis of structural variation (SV) was performed on 585 individuals from 14 yak breeds, and it was found that 246 bp deletion was present in each breed. The II genotype was dominant in all yak breeds except for SB yak. The association analysis of gene polymorphisms and growth traits in the ASD yak population showed that the 246 bp SV was significantly associated with body length at 6 months (p < 0.05). GHR messenger RNA (mRNA) was expressed in all the tested tissues, with significantly higher levels in the liver, muscle, and fat than in other organs. The results of transcription activity showed that the luciferase activity of the pGL4.10-DD vector was significantly higher than that of the pGL4.10-II vector (p < 0.05). Additionally, the transcription-factor binding prediction results showed that the SV in the runt-related transcription factor 1 (Runx1) transcription-factor binding site may affect the transcriptional activity of the GHR gene, regulating yak growth and development. This study showed that the novel SV of the GHR gene could be used as a candidate molecular marker for the selection of the early growth trait in ASD yak.

Interpretation of the Yak Skin Single-Cell Transcriptome Landscape.

The morphogenesis of hair follicle structure is accompanied by the differentiation of skin tissue. Mammalian coats are produced by hair follicles. The formation of hair follicles requires signal transmission between the epidermis and dermis. However, knowledge of the transcriptional regulatory mechanism is still lacking. We used single-cell RNA sequencing to obtain 26,573 single cells from the scapular skin of yaks at hair follicle telogen and anagen stages. With the help of known reference marker genes, 11 main cell types were identified. In addition, we further analyzed the DP cell and dermal fibroblast lineages, drew a single-cell map of the DP cell and dermal fibroblast lineages, and elaborated the key genes, signals, and functions involved in cell fate decision making. The results of this study provide a very valuable resource for the analysis of the heterogeneity of DP cells and dermal fibroblasts in the skin and provide a powerful theoretical reference for further exploring the diversity of hair follicle cell types and hair follicle morphogenesis.

MdWOX4-2 modulated MdLBD41 functioning in adventitious shoot of apple (Malus domestica).

Apple (Malus domestica Borkh.) is not only an important fruit crop distributed worldwide, but also a common model plant. However, the lack of efficient genetic transformation procedures for apples limits the in-depth studies of their gene functions. Although leaf-regenerated adventitious shoots (LRAS) are a prerequisite for successful genetic transformation of apple, little is known about the underlying molecular mechanism of LRAS. Here, we identified the WUSCHEL-related homeobox (WOX) transcription factor in apple, MdWOX4-2, which was a transcriptional activator. Gene expression as well as morphological and histological observations revealed that MdWOX4-2 is involved in the development of LRAS. Overexpression of MdWOX4-2 conferred higher regenerative capacity in transgenic tobacco (Nicotiana tabacum) as compared to the wild type (WT). The combined results of the yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), dual luciferase assays, and transient transactivation assay, revealed that MdWOX4-2 directly bound to and activated the MdLBD41 promoter. Moreover, transgenic experiments further demonstrated that MdLBD41 could significantly enhance the formation of adventitious shoot in transgenic tobacco. Collectively, our findings demonstrate that MdWOX4-2 is important for regulating the LRAS development by activating MdLBD41.

Comparative Analysis of mRNA and miRNA Expression between Dermal Papilla Cells and Hair Matrix Cells of Hair Follicles in Yak.

The interaction between the dermal papilla cells (DPCs) and epidermal hair matrix cells (HMCs) of hair follicles (HFs) is crucial for the growth and development of HFs, but the molecular mechanism is complex and remains unclear. MicroRNAs (miRNAs) are the key signaling molecules for cellular communication. In this study, the DPCs and HMCs of yak were isolated and cultured, and the differentially expressed mRNA and miRNA were characterized to analyze the molecular basis of the interaction between DPCs and HMCs during hair follicle (HF) development in yak. The mRNA differential expression and functional enrichment analysis revealed that there were significant differences between DPCs and HMCs, and they showed the molecular functional characteristics of dermal cells and epidermal cells, respectively. Multiple KEGG pathways related to HF development were enriched in the highly expressed genes in DPCs, while the pathways associated with microbiota and immunity were significantly enriched in the highly expressed genes in HMCs. By combining analysis with our previous 10× genomics single-cell transcriptome data, 39 marker genes of DPCs of yak were identified. A total of 123 relatively specifically expressed miRNAs were screened; among these, the miRNAs associated with HF development such as miR-143, miR-214, miR-125b, miR-31, and miR-200 were presented. In conclusion, the large changes in yak DPCs and HMCs for both mRNA and miRNA expression were revealed, and numerous specifically expressed mRNAs and miRNAs in DPCs or HMCs were identified, which may contribute to the interaction and cellular communication between DPCs and HMCs during HF development in yak.

Understanding Mammalian Hair Follicle Ecosystems by Single-Cell RNA Sequencing.

Single-cell sequencing technology can fully reflect the heterogeneity of cell populations at the single cell level, making it possible for us to re-recognize various tissues and organs. At present, the sequencing study of hair follicles is transiting from the traditional ordinary transcriptome level to the single cell level, which will provide diverse insights into the function of hair follicle cells. This review focuses on research advances in the hair follicle microenvironment obtained from scRNA-seq studies of major cell types in hair follicle development, with a special emphasis on the discovery of new subpopulations of hair follicles by single-cell techniques. We also discuss the problems and current solutions in scRNA-seq observation and look forward to its prospects.

Comprehensive Genome-Wide Identification and Transcript Profiling of GABA Pathway Gene Family in Apple (Malus domestica).

γ-Aminobutyric Acid (GABA), a four-carbon non-protein amino acid, is a significant component of the free amino acid pool in most prokaryotic and eukaryotic organisms. GABA is involved in pH regulation, maintaining C/N balance, plant development and defence, as well as a compatible osmolyte and an alternative pathway for glutamate utilization via anion flux. Glutamate decarboxylase (GAD, EC 4.1.1.15) and GABA transaminase (GABA-T, EC 2.6.1.19) are two key enzymes involved in the synthesis and metabolism of GABA. Recently, GABA transporters (GATs), protein and aluminium-activated malate transporter (ALMT) proteins which function as GABA receptors, have been shown to be involved in GABA regulation. However, there is no report on the characterization of apple GABA pathway genes. In this study, we performed a genome-wide analysis and expression profiling of the GABA pathway gene family in the apple genome. A total of 24 genes were identified including five GAD genes (namely MdGAD 1-5), two GABA-T genes (namely MdGABA-T 1,2), 10 GAT genes (namely GAT 1-10) and seven ALMT genes (namely MdALMT1-7). These genes were randomly distributed on 12 chromosomes. Phylogenetic analyses grouped GABA shunt genes into three clusters-cluster I, cluster II, and cluster III-which had three, four, and five genes, respectively. The expression profile analysis revealed significant MdGAD4 expression levels in both fruit and flower organs, except pollen. However, there were no significant differences in the expression of other GABA shunt genes in different tissues. This work provides the first characterization of the GABA shunt gene family in apple and suggests their importance in apple response to abiotic stress. These results can serve as a guide for future studies on the understanding and functional characterization of these gene families.

MicroRNA156 (miR156) Negatively Impacts Mg-Protoporphyrin IX (Mg-Proto IX) Biosynthesis and Its Plastid-Nucleus Retrograde Signaling in Apple.

Plastid-nucleus retrograde signaling (PNRS) play essential roles in regulating nuclear gene expression during plant growth and development. Excessive reactive oxygen species can trigger PNRS. We previously reported that in apple (Malus domestica Borkh.) seedlings, the expression of microRNA156 (miR156) was significantly low in the adult phase, which was accompanied by high levels of hydrogen peroxide (H2O2) accumulation in chloroplasts. However, it was unclear whether adult-phase-specific chloroplast H2O2 may induce PNRS and affect miR156 expression, or miR156 triggers adult phase PNRS during the ontogenesis. In this paper, we examined the relationship between miR156 levels and six PNRS components in juvenile and adult phase leaves from 'Zisai Pearl'×'Red Fuji' hybrids. We found that PNRS generated by singlet oxygen (1O2), the photosynthetic redox state, methylerythritol cyclodiphosphate (MEcPP), SAL1-3-phosphoadenosine 5-phosphate (PAP) and WHIRLY1 were not involved. The accumulation of Mg-protoporphyrin IX (Mg-Proto IX), the expression of the synthetic genes MdGUN5 and MdGUN6, and Mg-Proto IX PNRS related nuclear genes increased with ontogenesis. These changes were negatively correlated with miR156 expression. Manipulating Mg-Proto IX synthesis with 5-aminolevulinic acid (ALA) or gabaculine did not affect miR156 expression in vitro shoots. In contrast, modulating miR156 expression via MdGGT1 or MdMIR156a6 transgenesis led to changes in Mg-Proto IX contents and the corresponding gene expressions. It was concluded that the Mg-Proto IX PNRS was regulated downstream of miR156 regardless of adult-phase-specific plastid H2O2 accumulation. The findings may facilitate the understanding of the mechanism of ontogenesis in higher plants.

MdGGT1 Impacts Apple miR156 Precursor Levels via Ontogenetic Changes in Subcellular Glutathione Homeostasis.

The vegetative phase change in flowering plants is controlled by microRNA156 (miR156) under transcriptional regulation. However, the developmental signals upstream of miR156 are not well understood. The glutathione/glutathione disulfide (GSH/GSSG) ratios and GSH levels decline significantly during phase change, which is consistent with miR156 expression in apple (Malus domestica Borkh.). Here, we found that the content of protein conjugated glutathione was remarkably higher in chloroplasts and nuclei of adult than juvenile phase apple hybrids. The decrease in miR156 expression was most relevant to the activities of serine acetyltransferase (SAT) and soluble γ-glutamyl transpeptidase (GGT), and the expressions of MdGGT1 or MdSATs. Transgenic apples over-expressing MdMIR156 or miR156-mimetic (MIM156) did not alter MdGGT1 expression or the soluble GGT activity. Inhibition of GGT activity with serine-borate complex or acivicin led to significant reduction in GSH content, the GSH/GSSG ratio, and the expressions of MdMIR156a5, MdMIR156a12, and miR156. Depletion of GSH with diethyl maleate without altering GGT activity caused a dramatic decrease in the expression of MdMIR156a5, MdMIR156a12, and miR156. Manipulating GGT activity and GSH homeostasis by transgenic over-expressing or RNAi MdGGT1 increased or decreased MdMIR156a5 and MdMIR156a12 levels, respectively. These data provided novel evidence that MdGGT1 participates in transcriptional level of transcription regulation of miR156 precursors during ontogenesis. HIGHLIGHTS: - MdGGT1 affects thiol redox status and indirectly participates in the regulation of miR156 expression during vegetative phase change.