MicroRNAs (miRNAs) are endogenous ∼23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

MicroRNAs: Target Recognition and Regulatory Functions

Background The forest musk deer, Moschus berezovskii, is one of seven musk deer (Moschus spp.) and is distributed in Southwest China. Akin to other musk deer, the forest musk deer has been traditionally and is currently hunted for its musk (i.e., global perfume industry). Considerable hunting pressure and habitat loss have caused significant population declines. Consequently, the Chinese government commenced captive breeding programs for musk harvesting in the 1950s. However, the prevalence of fatal diseases is considerably restricting population increases. Disease severity and extent are exacerbated by inbreeding and genetic diversity declines in captive musk deer populations. It is essential that knowledge of captive and wild forest musk deer populations' immune system and genome be gained in order to improve their physical and genetic health. We have thus sequenced the whole genome of the forest musk deer, completed the genomic assembly and annotation, and performed preliminary bioinformatic analyses. Findings A total of 407 Gb raw reads from whole-genome sequencing were generated using the Illumina HiSeq 4000 platform. The final genome assembly is around 2.72 Gb, with a contig N50 length of 22.6 kb and a scaffold N50 length of 2.85 Mb. We identified 24,352 genes and found that 42.05% of the genome is composed of repetitive elements. We also detected 1,236 olfactory receptor genes. The genome-wide phylogenetic tree indicated that the forest musk deer was within the order Artiodactyla, and it appeared as the sister clade of four members of Bovidae. In total, 576 genes were under positive selection in the forest musk deer lineage. Conclusions We provide the first genome sequence and gene annotation for the forest musk deer. The availability of these resources will be very useful for the conservation and captive breeding of this endangered and economically important species and for reconstructing the evolutionary history of the order Artiodactyla.

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The draft genome sequence of forest musk deer (Moschus berezovskii).

The Chinese forest musk deer (Moschus berezovskii; FMD) is an artiodactyl mammal and is both economically valuable and highly endangered To investigate the genetic mechanisms of musk secretion and adaptive immunity in FMD, we compared its genome to nine other artiodactyl genomes Comparative genomics demonstrated that eight positively selected genes (PSGs) in FMD were annotated in three KEGG pathways that were related to metabolic and synthetic activity of musk, similar to previous transcriptome studies Functional enrichment analysis indicated that many PSGs were involved in the regulation of immune system processes, implying important reorganization of the immune system in FMD FMD-specific missense mutations were found in two PSGs (MHC class II antigen DRA and ADA) that were classified as deleterious by PolyPhen-2, possibly contributing to immune adaptation to infectious diseases Functional assessment showed that the FMD-specific mutation enhanced the ADA activity, which was likely to strengthen the immune defense against pathogenic invasion Single nucleotide polymorphism-based inference showed the recent demographic trajectory for FMD Our data and findings provide valuable genomic resources not only for studying the genetic mechanisms of musk secretion and adaptive immunity, but also for facilitating more effective management of the captive breeding programs for this endangered species

/pdf/comparative-genomics-reveals-the-genetic-mechanisms-of-musk-1eamfhb0mu.pdf

Comparative Genomics Reveals the Genetic Mechanisms of Musk Secretion and Adaptive Immunity in Chinese Forest Musk Deer.

Animal gut microbiota begins to colonize after birth and is functionally indispensable for maintaining the health of the host. It has been reported that gender and age influence the composition of the intestinal microbiome. However, the effects of gender and age on the intestinal microorganism of forest musk deer (FMD) remain unclear. The aim of this study was to establish the relationship between the structure and composition of fecal microbiota of male and female forest musk deer with age. Here, Illumina Miseq 300PE sequencing platform targeting 16S rRNA V3–V4 hypervariable region applied to define the fecal microbiota of male and female FMD with two age groups, juvenile (age 1–2 years) and adult (age 4–10 years). Alpha diversity index did not show significant difference in bacterial diversity between the males and females or among age groups. The intestinal microbiota of FMD was dominated by three phyla, the Firmicutes, Proteobacteria and Bacteroidetes regardless of gender and different ages. Higher proportions of Proteobacteria were found in adult male and juvenile female individuals. The composition of Bacteroidetes was stable with the gender and age of FMD. Interestingly, the relative abundance of genera Clostridiales and Bacteroidales were higher in the juvenile FMD. Conversely, proportions of Pseudomonas and Lachnospiraceae were abundant in the adult FMD. Higher proportions of Ruminococcaceae, Dore, and 5-7N15 were found in the juvenile male groups. They may reflect the different immune resistance of male and female individuals at different stages of development. This study explored the fecal microbiota composition of forest musk deer in relation to gender and age, which may provide an effective strategy for developing intestinal microecological preparations and potential musk deer breeding.

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Gut Microbiome of Chinese Forest Musk Deer Examined across Gender and Age.

Accumulating evidence has shown that follicle-stimulating hormone (FSH) and luteinizing hormone (LH) may influence the functions of nongonadal tissues in addition to their classic target gonads. Our previous studies revealed that the scented glands of male muskrats expressed prolactin receptor, steroidogenic enzymes, and inhibin/activin subunits. To further seek the evidence of the activities of pituitary gonadotropins in scented glands, we investigated the seasonal expression patterns of FSH receptor (FSHR) and LH/choriogonadotropin receptor (LHCGR). The weight and size of scented glands during the breeding season were significantly higher than those during the nonbreeding season. Immunohistochemical studies showed that FSHR was present in the serous cells of scented glands, whereas LHCGR was present in the interstitial cells. The protein and mRNA expression levels of FSHR and LHCGR were significantly higher in the scented glands during the breeding season than those during the nonbreeding season. Importantly, the levels of circulating FSH and LH were remarkably higher during the breeding season. Taken together, these results suggested that gonadotropins may affect the function of muskrat scented gland via the locally expressed receptors in a season-dependent manner.

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Seasonal expressions of follicle-stimulating hormone receptor and luteinizing hormone receptor in the scented gland of the male muskrat (Ondatra zibethicus).

The Chinese forest musk deer (Moschus berezovskii Flerov) is an endangered artiodactyl mammal. The musk secreted by sexually mature males is highly valued for alleged pharmaceutical properties and perfume manufacturing. However, the genomic and transcriptomic resources of musk deer remain deficiently represented and poorly understood. Next-generation sequencing technique is an efficient method for generating an enormous amount of sequence data that can represent a large number of genes and their expression levels. In the present study, we used Illumina HiSeq technology to perform de novo assembly of heart and musk gland transcriptomes from the Chinese forest musk deer. A total of 239,383 transcripts and 176,450 unigenes were obtained, of which 37,329 unigenes were matched to known sequences in the NCBI nonredundant protein (Nr) database; 31,039 unigenes were assigned to 61 GO terms, and 11,782 to 332 KEGG pathways. Additionally, 592 and 2282 differentially expressed genes were found to be specifically expressed in the heart and musk gland, respectively. The abundant transcriptomic data generated in the present report will provide a comprehensive sequence resource for Chinese forest musk deer as well as lay down a foundation which will help in accelerating genetic and functional genomics research in this species.

Illumina-based de novo transcriptome sequencing and analysis of Chinese forest musk deer

Forest musk deer (Moschus berezovskii), a rare wild medicinal animal, is listed under the category of the state key protected wildlife list of China. Musk, secreted by the musk glands, is with high economic and medicinal value and used as precious traditional medicine in China. In order to meet the needs of musk in Chinese traditional medicine, forest musk deer farming was conducted in 1950s, but the research progress on musk secretion mechanism was slow. Therefore, by reviewing the histological and anatomical structure of forest musk deer musk gland, the relationship between sex hormones and the musk secretion process, and the molecular mechanism of the musk secretion, the existing problems in investigating the musk secretion mechanism were analyzed and the development trends in this field were also discussed, in order to provide a reference for further studies on the musk secretion mechanism and improve musk production of forest musk deer.

Research progress on musk secretion mechanism of forest musk deer

Epicauta aptera Kaszab (E. aptera), an endangered species used to extract cantharidin in Chinese traditional medicine, has a limited geographical distribution in the southwest China. The complete mitochondrial genome of E. aptera is 15 645bp which contains an A + T rich region and 37 genes, including 13 protein-coding genes, 22 tRNA genes and two rRNA genes (GenBank accession No. KX023302). All protein-coding genes are initiated by ATN start codon. Moreover, the largest non-coding A + T-rich region with a length of 1039 bp is at the end of 12S rRNA. It is the first report involved in the complete mitochondrial genome of E. aptera.

The complete nucleotide sequence of the mitochondrial genome of Epicauta aptera Kaszab.

Research of epithelial cells in musk gland is lacking. There are no good characterized epithelial cell lines that can provide complementary in vitro models for in vivo research. We successfully cultivated epithelial cells of musk gland for the first time. The protocol described here produces epithelial cell lines from the mature secreting musk gland. Based on morphological observation, epithelial cells of musk gland were isolated and cultured in vitro. After the third passage, the musk gland-derived cells were filled with many lipid droplets and proliferated well. We used gas chromatography and mass spectrometry to explore the chemical composition of lipid droplets in the musk gland-derived cells. The main components of secreted lipid droplet were alkanes, esters, amines, alcohols, ketones, organic acids, and aldehydes. Muscone, which is the main active compound of musk, was not found. This is a new attempt in the field of animal musk to obtain naturally secreted animal musk in vitro by cloning specialized cells. In conclusion, this study provides a reference at the cellular level to further analyze the biology and physiology of the musk gland epithelium and secretion mechanism of musk deer.

Isolation, primary culture, and morphological characterization of gland epithelium from forest musk deer (Moschus berezovskii).

3 Zhongxian Xu, Hang Jie, Binlong Chen, Uma Gaur 1 , Mingyao Yang 1 , Nan Wu 1 , 4 Jian Gao, Pinming Li, Guijun Zhao, Dejun Zeng and Diyan Li 5 1 Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan 6 Province, Sichuan Agricultural University, Chengdu, China 7 2 Laboratory of Medicinal Animal, Chongqing Institute of Medicinal Plant Cultivation, Nanchuan, 8 Chongqing, China 9 10 Corresponding Author: 11 Diyan Li 12 NO. 211 of Huimin Road, Wenjiang District, Chengdu, Sichuan Province, 611130, China 13 Email address: diyanli@sicau.edu.cn 14

Zeng Dejun

Papers

Illumina-based de novo transcriptome sequencing and analysis of Chinese forest musk deer

Research progress on musk secretion mechanism of forest musk deer

The complete nucleotide sequence of the mitochondrial genome of Epicauta aptera Kaszab.

Isolation, primary culture, and morphological characterization of gland epithelium from forest musk deer (Moschus berezovskii).

De novo assembly of Chinese forest musk deer (Moschus berezovskii) transcriptome from Next-Generation mRNA Sequencing