Single-nucleus RNA and ATAC sequencing uncovers the molecular and cellular characteristics in the musk gland of Chinese forest musk deer (Moschus berezovskii).

The Chinese forest musk deer (FMD; Moschus berezovskii) is an endangered artiodactyl mammal. Musk secreted by the musk gland of male has extremely high economic and medicinal value. However, the molecular and cellular characteristics of the musk gland have not been studied. Here, we investigated the diversity and transcriptional composition of musk gland cell types and the effect of cell type-specific chromatin accessibility on gene expression using single-nucleus RNA sequencing (snRNA-seq) and single-nucleus ATAC sequencing (snATAC-seq) association analysis. Based on uniform manifold approximation and projection (UMAP) analysis, we identified 13 cell types from the musk gland, which included two different acinar cells (cluster 0 and cluster 10). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that many pathways related to musk secretion were enriched in acinar cells. Our analysis also revealed acinar cell core transcription factors and core target genes, and further constructed acinar cell-specific regulatory networks. In cluster 0, 11 core target genes (Nedd4l, Adcy9, Akr1c1, Vapb, Me1, Acsl1, Acss3, Srd5a1, Scnn1a, Acadm, and Nceh1) possibly related to musk secretion were regulated by 24 core transcription factors (SP3, NFIC, NR6A1, EHF, RUNX1, TFAP2A, RREB1, GRHL2, NFIB, ELF1, MAX, KLF5, REL, HES1, POU2F3, TFDP1, NR2C1, ATF7, MEIS1, NR4A2, NFIA, PBX1, ZNF652, and NFKB1). In cluster 10, four core target genes (Akr1c1, Pcca, Atp1b1, and Sgk1) possibly related to musk secretion were regulated by 10 core transcription factors (BARX2, EHF, PBX1, RUNX1, NFIB, FOXP1, KLF3, KLF6, ETV6, and NR3C2). Moreover, the credibility of snRNA-seq and snATAC-seq data was verified by fluorescence in situ hybridization and immunohistochemistry. Finally, cell communication analysis demonstrated that the two types of acinar cells mainly have communications in musk secretion-related processes. In conclusion, we provided important insights and invaluable resources for the molecular and cellular characteristics of the musk gland, which will lay a foundation for the study of musk secretion mechanism in the future.

Exploring the genomic resources and analysing the genetic diversity and population structure of Chinese indigenous rabbit breeds by RAD-seq.

BACKGROUND: Chinese indigenous rabbits have distinct characteristics, such as roughage resistance, stress resistance and environmental adaptability, which are of great significance to the sustainable development of the rabbit industry in China. Therefore, it is necessary to study the genetic diversity and population structure of this species and develop genomic resources. RESULTS: In this study, we used restriction site-associated DNA sequencing (RAD-seq) to obtain 1,006,496 SNP markers from six Chinese indigenous rabbit breeds and two imported rabbit breeds. Jiuyishan and Fujian Yellow rabbits showed the highest nucleotide diversity (π) and decay of linkage disequilibrium (LD), as well as higher observed heterozygosity (Ho) and expected heterozygosity (He), indicating higher genetic diversity than other rabbits. The inbreeding coefficient (FIS) of New Zealand rabbits and Belgian rabbits was higher than that of other rabbits. The neighbour-joining (NJ) tree, principal component analysis (PCA), and population structure analysis of autosomes and Y chromosomes showed that Belgian, New Zealand, Wanzai, Sichuan White, and Minxinan Black rabbits clustered separately, and Fujian Yellow, Yunnan Colourful, and Jiuyishan rabbits clustered together. Wanzai rabbits were clearly separated from other populations (K = 3), which was consistent with the population differentiation index (FST) analysis. The selection signature analysis was performed in two populations with contrasting coat colours. With Sichuan White and New Zealand rabbits as the reference populations and Minxinan Black and Wanzai rabbits as the target populations, 408, 454, 418, and 518 genes with a selection signature, respectively, were obtained. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on the genes with a selection signature. The results showed that the genes with a selection signature were enriched in the melanogenesis pathway in all four sets of selection signature analyses. CONCLUSIONS: Our study provides the first insights into the genetics and genomics of Chinese indigenous rabbit breeds and serves as a valuable resource for the further effective utilization of the species.

Single-nucleus multiomics unravels the genetic mechanisms underlying musk secretion in Chinese forest musk deer (Moschus berezovskii).

Musk secreted by the musk glands in male forest musk deer (FMD; Moschus berezovskii) is highly valued for its pharmaceutical and perfumery applications. However, the regulatory mechanisms underlying musk secretion are not well understood. This study aimed to investigate the genes and transcription factors involved in musk secretion across different periods and ages. We analyzed the musk glands of adult male FMD during the non-secretory and secretory periods, as well as juvenile and adult male FMD during the secretory period, using single-cell multiome ATAC+gene expression technique. Our analysis identified 13 cell types, including acinar cells of Types 1 and 2. Chromatin accessibility analysis and gene expression data confirmed that the genes Map3k2, Hsd17b12, and Jun are critical for musk secretion. Additionally, EHF, NR4A2, and FOXO1 proteins play crucial regulatory roles. Weighted gene co-expression network analysis (WGCNA) highlighted the importance of GnRH signaling pathway in musk secretion. Gene set enrichment analysis (GSEA) showed that the steroid hormone biosynthesis pathway is notably enriched in acinar cells. Furthermore, intercellular communication appears to influence both the initiation and maintenance of musk secretion. These findings provide valuable insights into the molecular pathways of musk secretion in FMD, offering potential avenues for increasing musk production and developing treatment for inflammation and tumors.

Single-nucleus transcriptomics and chromatin accessibility analysis of musk gland development in Chinese forest musk deer (Moschus berezovskii).

Musk secreted by male forest musk deer (Moschus berezovskii) musk glands is an invaluable component of medicine and perfume. Musk secretion depends on musk gland maturation; however, the mechanism of its development remains elusive. Herein, using single cell multiome ATAC + gene expression coupled with several bioinformatic analyses, a dynamic transcriptional cell atlas of musk gland development was revealed, and key genes and transcription factors affecting its development were determined. Twelve cell types, including two different types of acinar cells (Clusters 0 and 10) were identified. Single-nucleus RNA and single-nucleus ATAC sequencing analyses revealed that seven core target genes associated with musk secretion (Hsd17b2, Acacb, Lss, Vapa, Aldh16a1, Aldh7a1, and Sqle) were regulated by 12 core transcription factors (FOXO1, CUX2, RORA, RUNX1, KLF6, MGA, NFIC, FOXO3, ETV5, NR3C1, HSF4, and MITF) during the development of Cluster 0 acinar cells. Kyoto Encyclopedia of Genes and Genomes enrichment showed significant changes in the pathways associated with musk secretion during acinar cell development. Gene set variation analysis also revealed that certain pathways associated with musk secretion were enriched in 6-year-old acinar cells. A gene co-expression network was constructed during acinar cell development to provide a precise understanding of the connections between transcription factors, genes, and pathways. Finally, intercellular communication analysis showed that intercellular communication is involved in musk gland development. This study provides crucial insights into the changes and key factors underlying musk gland development, which serve as valuable resources for studying musk secretion mechanisms and promoting the protection of this endangered species.

Coupling of soil methane emissions at different depths under typical coastal wetland vegetation types.

As an important source of atmospheric methane, methane emissions from coastal wetlands are affected by many factors. However, the methane emission process and interrelated coupling mechanisms in coastal wetland soils of a variety of environments remain unclear owing to complex interactions between intensified anthropogenic activities and climate change in recent years. In this study, we investigated methane cycling processes and the response mechanisms of environmental and microbial factors in soils at different depths under four typical coastal wetland vegetation types of the Yellow River Delta, China, using laboratory culture and molecular biology techniques. Our results show that methane generation pathways differed among the different soil layers, and that the methane emission process has a special response to soil N compounds (NO3-, NH4+). We found that nitrogen can indirectly affect methane emission by impacting key physicochemical properties (pH, oxidation reduction potential, etc.) and some functional communities (mcrA, ANME-2d, sulfate-reducing bacteria (SRB), narG, nosZII). Methane production processes in shallow soils compete closely with sulfate reduction processes, while methane emissions facilitated in deeper soils due to denitrification processes. We believe that our results provide a reference for future research and wetland management practices that seek to mitigate the global greenhouse effect and climate change.

Soil nitrogen content and key functional microorganisms influence the response of wetland anaerobic oxidation of methane to trivalent iron input.

Trivalent iron (Fe3+)-dependent anaerobic oxidation of methane (Fe-AOM), which is mediated by metal-reducing bacteria, is widely recognized as a major sink for the greenhouse gas methane (CH4), and is a key driver of the carbon (C) biogeochemical cycle. However, the effect of Fe3+ addition on AOM in the present investigation is still ambiguous, and the mechanism is vague. In this study, we investigated the mechanism of changes in AOM response to Fe3+ input at different wetlands by using laboratory incubation methods combined with molecular biology techniques. Results indicated that Fe3+ input did not always lead to promoted AOM rates, which may be mediated by complex environmental factors, while lower soil total nitrogen (TN) had a positive effect on the response of AOM subjected to Fe3+ input. Notably, the promoted response of AOM was regulated by higher soil microbial diversity, of which the Shannon index was a key indicator leading to variation in the AOM response. Additionally, several biomarkers, including Planctomycetota and Burkholderiaceae, were key microorganisms responsible for alterations in AOM response. Our results suggest that the capacity of Fe3+ cycling-mediated AOM may gradually decrease in light of increasing anthropogenic N and Fe inputs to global estuarine wetlands, while its reaction processes will become more complex and more strongly coupled with multiple environmental factors. This finding contributes to the enhanced understanding and prediction of the wetland CH4-related C with Fe cycles, as well as provides theoretical support for the underlying mechanisms.

Exploring the genomic resources of seven domestic Bactrian camel populations in China through restriction site-associated DNA sequencing.

The domestic Bactrian camel is a valuable livestock resource in arid desert areas. Therefore, it is essential to understand the roles of important genes responsible for its characteristics. We used restriction site-associated DNA sequencing (RAD-seq) to detect single nucleotide polymorphism (SNP) markers in seven domestic Bactrian camel populations. In total, 482,786 SNPs were genotyped. The pool of all remaining others were selected as the reference population, and the Nanjiang, Sunite, Alashan, Dongjiang, Beijiang, Qinghai, and Hexi camels were the target populations for selection signature analysis. We obtained 603, 494, 622, 624, 444, 588, and 762 selected genes, respectively, from members of the seven target populations. Gene Ontology classifications and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed, and the functions of these genes were further studied using Genecards to identify genes potentially related to the unique characteristics of the camel population, such as heat resistance and stress resistance. Across all populations, cellular process, single-organism process, and metabolic process were the most abundant biological process subcategories, whereas cell, cell part, and organelle were the most abundant cellular component subcategories. Binding and catalytic activity represented the main molecular functions. The selected genes in Alashan camels were mainly enriched in ubiquitin mediated proteolysis pathways, the selected genes in Beijiang camels were mainly enriched in MAPK signaling pathways, the selected genes in Dongjiang camels were mainly enriched in RNA transport pathways, the selected genes in Hexi camels were mainly enriched in endocytosis pathways, the selected genes in Nanjiang camels were mainly enriched in insulin signaling pathways, while the selected genes in Qinghai camels were mainly enriched in focal adhesion pathways; these selected genes in Sunite camels were mainly enriched in ribosome pathways. We also found that Nanjiang (HSPA4L and INTU), and Alashan camels (INO80E) harbored genes related to the environment and characteristics. These findings provide useful insights into the genes related to the unique characteristics of domestic Bactrian camels in China, and a basis for genomic resource development in this species.

Development of Genomic Resources and Identification of Genetic Diversity and Genetic Structure of the Domestic Bactrian Camel in China by RAD Sequencing.

The domestic Bactrian camel is indispensable to agricultural production in the desertification area of China owning to its endurance to hunger and thirst, cold resistance, drought resistance, and good long-distance transportation. Therefore, it is necessary to investigate the genetic diversity, genetic structure, and genes with important roles in the evolution of this species. In this study, 1,568,087 SNPs were identified in 47 domestic Bactrian camels inhabiting four regions of China, namely Inner Mongolia, Gansu, Qinghai, and Xinjiang, by restriction site associated DNA sequencing (RAD-seq). The SNP data were used for nucleotide diversity analysis (π) and linkage disequilibrium (LD) attenuation analysis to elucidate the genetic diversity of the domestic Bactrian camel in the four regions studied. Results showed that Xinjiang camels had the highest nucleotide diversity and the fastest decay rate of the LD coefficient; therefore, Xinjiang camels had the highest genetic diversity. Structure analysis, principal component analysis (PCA), and phylogenetic tree construction by the neighbor-joining (NJ) method showed that Qinghai camels clustered separately, at a larger phylogenetic distance from camels in the other regions. Through analyses of selection signals, it was found that the number of selected genes shared by Inner Mongolia camels, Qinghai camels, Xinjiang camels, and Gansu camels was 7, 24, 25, and 113, respectively. The shared selected genes of the domestic Bactrian camel in the four regions were further analyzed, and three shared genes (GRIA3, XIAP, and THOC2) of the domestic Bactrian camel in China were identified. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on the shared selected genes of the domestic Bactrian camel in all four regions studied. Across all regions, genes involved in the cellular process were the most abundant subcategory under biological process. Cell and cell part represented the main proportion of genes under cellular component. Binding represented the main molecular function. In addition, the shared selected genes of the domestic Bactrian camel in the four regions of China were significantly enriched in the long-term depression pathway. The research should enable further study of the genetic resources of the domestic Bactrian camel, as well as the conservation of these resources.

Population genetic analysis of the domestic Bactrian camel in China by RAD-seq.

Restriction site-associated DNA sequencing (RAD-seq) is one of the most effective high-throughput sequencing technologies for SNP development and utilization and has been applied to studying the origin and evolution of various species. The domestic Bactrian camels play an important role in economic trade and cultural construction. They are precious species resources and indispensable animals in China's agricultural production. Recently, the rapid development of modern transportation and agriculture, and the deterioration of the environment have led to a sharp decline in the number of camels. Although there have been some reports on the evolution history of the domestic Bactrian camel in China, the origin, evolutionary relationship, and genetic diversity of the camels are unclear due to the limitations of sample size and sequencing technology. Therefore, 47 samples of seven domestic Bactrian camel species from four regions (Inner Mongolia, Gansu, Qinghai, and Xinjiang) were prepared for RAD-seq analysis to study the evolutionary relationship and genetic diversity. In addition, seven domestic Bactrian camel species are located in different ecological zones, forming different characteristics and having potential development value. A total of 6,487,849 SNPs were genotyped. On the one hand, the filtered SNP information was used to conduct polymorphism mapping construction, LD attenuation analysis, and nucleotide diversity analysis. The results showed that the number of SNPs in Dongjiang camel was the highest, the LD coefficient decayed the fastest, and the nucleotide diversity was the highest. It indicates that Dongjiang camel has the highest genetic diversity. On the other hand, the filtered SNPs information was used to construct the phylogenetic tree, and F ST analysis, inbreeding coefficient analysis, principal component analysis, and population structure analysis were carried out. The results showed that Nanjiang camel and Beijiang camels grouped together, and the other five Bactrian camel populations gathered into another branch. It may be because the mountains in the northern part of Xinjiang and the desert in the middle isolate the two groups from the other five groups.