The First High-quality Reference Genome of Sika Deer Provides Insights into High-tannin Adaptation.

Sika deer are known to prefer oak leaves, which are rich in tannins and toxic to most mammals; however, the genetic mechanisms underlying their unique ability to adapt to living in the jungle are still unclear. In identifying the mechanism responsible for the tolerance of a highly toxic diet, we have made a major advancement by explaining the genome of sika deer. We generated the first high-quality, chromosome-level genome assembly of sika deer and measured the correlation between tannin intake and RNA expression in 15 tissues through 180 experiments. Comparative genome analyses showed that the UGT and CYP gene families are functionally involved in the adaptation of sika deer to high-tannin food, especially the expansion of the UGT family 2 subfamily B of UGT genes. The first chromosome-level assembly and genetic characterization of the tolerance to a highly toxic diet suggest that the sika deer genome may serve as an essential resource for understanding evolutionary events and tannin adaptation. Our study provides a paradigm of comparative expressive genomics that can be applied to the study of unique biological features in non-model animals.

Complete mitochondrial genome and phylogenetic analysis of eight sika deer subspecies in northeast Asia.

Sika deer (Cervus nippon) are large ruminants distributed throughout northeastern Asia. The phylogenetic relationship of the sika deer subspecies remains unclear. The complete mitochondrial genomes of 287 sika deer from eight subspecies (C. n. hortulorum, C. n. sichuanicus, C. n. kopschi, C. n. taiouanus, C. n. yesoensis, C. n. centralis, C. n. nippon and C. n. yakushimae) were obtained. Haplotype network and development of a phylogenetic tree revealed China clusters and Japan clusters that were well separated based on the mitochondrial whole-genome level. Our studies indicate that China sika deer are genetically distinguishable from Japanese samples. Our findings increase the understanding of the phylogenetic relationships of sika deer and could provide useful information for sika deer conservation projects as well as for sika deer genomics, emergence and geographical distribution.

Phylogeography of sika deer (Cervus nippon) inferred from mitochondrial cytochrome-b gene and microsatellite DNA.

The genetic diversity and phylogenetic relationships of sika deer of different subspecies are uncertain. In order to explore the phylogenetic relationship of different sika deer subspecies, this study used a wider sample collection to analyze mitochondrial sequences and nuclear microsatellites of sika deer. The full lengths of cytochrome-b gene of 134 sika deer were sequenced, and 16 haplotypes were obtained. Based on phylogenetic and haplotype networks analysis, the sika deer was not clustered according to subspecies but was divided into four lineages. Lineage I includes individuals from C.n.kopschi, C.n.sichuanicus, and C.n.hortulorum subspecies; Lineage II includes individuals from C.n.hortulorum subspecies; Lineage III includes individuals from C.n.centralis, C.n.yakushime, C.n.mageshimae, and C.n.keramae subspecies, namely southern Japanese population; Lineage IV includes individuals from C.n.centralis and C.n.yesoensis subspecies, namely northern Japanese population. The microsatellite analysis showed that the sika deer in China and Japan originated independently. The three subspecies of China have significant genetic differentiation, while the three subspecies of Japan have no significant differentiation. This study provides reference for the research of genetic diversity and phylogenetic relationship of sika deer, and also provides scientific data for the evaluation, protection, and utilization of sika deer resources.

Identification of key genes associated with spermatogenesis arrest in fox hybrids using weighted gene co-expression network analysis.

The silver fox and the blue fox represent different genera, but produce viable offspring. Although these hybrids show obvious heterosis, they are completely sterile due to spermatogenic arrest at the early stages of spermatogenesis, especially mitosis and meiosis I; the hybrids produce few spermatogonia and primary spermatocytes, and no secondary spermatocytes. Although the mechanisms of spermatogenic arrest have been well investigated, transcriptomic differences between hybrid and the pure-species testes have not clarified. In the present study, we used RNA sequencing (RNA-Seq) to generate testicular transcriptomic profiles for silver foxes, blue foxes, and reciprocal hybrids during the pre-breeding period and the breeding season. In total, 1,344,022 transcripts (≥200 bp) were generated; 1,057,724 genes were obtained; and 33,423 genes were shown to have fragments per kilobase of transcript per million mapped reads (FPKM) > 0.3. To identify the hub genes associated with spermatogenesis arrest, weighted gene co-expression network analysis (WGCNA) was used. Nine modules were explored. Genes in only a single module were consistently downregulated in the hybrids as compared to the pure species; these genes were significantly associated with fox hybrid male infertility. Six of the genes in this module (CATSPERD, DMRTC2, RNF17, NME5, SPEF2, SPINK2) also play key roles in mitosis and meiosis during spermatogenesis. Therefore, these six genes might be associated with fox hybrid male infertility.

Genetic diversity of Aoluguya Reindeer based on D-loop region of mtDNA and its conservation implications.

Aoluguya Reindeer is the only reindeer population in China. In recent years, habitat loss and inbreeding have led to population decline, and population growth has been slow, maintaining a thousand or so. To better protect the Aoluguya Reindeer and improve its fecundity, we have introduced reindeer from Finland, crossbreeding help us to reach this goal. However, it is lacking in the study of genetic diversity of reindeer in China and Finland. Therefore, we used the partial sequences of the D-loop region of mitochondrial DNA to analyze the genetic diversity of Chinese reindeer (Aoluguya Reindeer) and the introduced Finnish reindeer, and identified twenty-six haplotypes, including nineteen in China, five in Finland, and two in Russia. There is no shared haplotype among them. The nucleotide diversity of Aoluguya Reindeer is 0.00752, which is significantly lower than that of reindeer in Finland and other countries. The haplotype and phylogenetic analysis show that reindeer from different geographical origins are not clustered completely according to geographical distribution. Aoluguya Reindeer populations and the introduced reindeer herds from Finland are all closely related to the reindeer from Russia. AMOVA analysis showed that there was significant differentiation between reindeer populations in China and Finland, and mismatch analysis showed that both populations had not experienced expansion. In this study, we identified the genetic diversity of Aoluguya Reindeer and the introduced reindeer, and provided a scientific basis for the conservation and breeding of Aoluguya Reindeer resources.

Genome-wide study on genetic diversity and phylogeny of five species in the genus Cervus.

BACKGROUND: Previous investigations of phylogeny in Cervus recovered many clades without whole genomic support. METHODS: In this study, the genetic diversity and phylogeny of 5 species (21 subspecies/populations from C. unicolor, C. albirostris, C. nippon, C. elaphus and C. eldii) in the genus Cervus were analyzed using reduced-representation genome sequencing. RESULTS: A total of 197,543 SNPs were identified with an average sequencing depth of 16 x. A total of 21 SNP matrices for each subspecies/population and 1 matrix for individual analysis were constructed, respectively. Nucleotide diversity and heterozygosity analysis showed that all 21 subspecies/populations had different degrees of genetic diversity. C. eldii, C. unicolor and C. albirostris showed relatively high expected and observed heterozygosity, while observed heterozygosity in C. nippon was the lowest, indicating there was a certain degree of inbreeding rate in these subspecies/populations. Phylogenetic ML tree of all Cervus based on the 21 SNP matrices showed 5 robustly supported clades that clearly separate C. eldii, C. unicolor, C. albirostris, C. elaphus and C. nippon. Within C. elaphus clade, 4 subclades were well differentiated and statistically highly supported: C. elaphus (New Zealand), C. e. yarkandensis, C. c. canadensis and the other grouping the rest of C. canadensis from China. In the C. nippon clade, 2 well-distinct subclades corresponding to C. n. aplodontus and other C. nippon populations were separated. Phylogenetic reconstruction indicated that the first evolutionary event of the genus Cervus occurred approximately 7.4 millions of years ago. The split between C. elaphus and C. nippon could be estimated at around 3.6 millions of years ago. Phylogenetic ML tree of all samples based on individual SNP matrices, together with geographic distribution, have shown that there were 3 major subclades of C. elaphus and C. canadensis in China, namely C. e. yarkandensis (distributed in Tarim Basin), C. c. macneilli/C. c. kansuensis/C. c. alashanicus (distributed in middle west of China), and C. c. songaricus/C. c. sibiricus (distributed in northwest of China). Among them, C. e. yarkandensis was molecularly the most primitive subclade, with a differentiation dating back to 0.8-2.2 Myr ago. D statistical analysis showed that there was high probability of interspecific gene exchange between C. albirostris and C. eldii, C. albirostris and C. unicolor, C. nippon and C. unicolor, and there might be 2 migration events among 5 species in the genus Cervus. CONCLUSIONS: Our results provided new insight to the genetic diversity and phylogeny of Cervus deer. In view of the current status of these populations, their conservation category will need to be reassessed.

Genetic diversity and population genetic structure of the only population of Aoluguya Reindeer (Rangifer tarandus) in China.

Aoluguya Reindeer is the only reindeer species in China and currently approximately 1000 Aoluguya Reindeer remain semi-domesticated. A relative low diversity estimate was found by investigating genetic variability and demographic history of its population. Mismatch distribution curve of its nucleotide sequences and neutral test indicate its population has not experienced expansion. Genetic diversity and population structure were also analysed by using its mtDNA and microsatellites technology. Statistical results of these analyses showed there were varying degrees of population inbreeding and suggested that gene flow existed among its populations at one time. Three mutation models were also used to detect the bottleneck effect of reindeer population. The genetic variation of eight populations is relatively small. In addition, the clustering program STRUCTURE was used to analyse Aoluguya Reindeer population structure, to determine its optimal K and first time to analyse the phylogenetic status of Aoluguya Reindeer among other reindeer subspecies. It is recommended that the government establish a natural conservation area in Aoluguya Reindeer growing geography, forbade the trade and hunting of Aoluguya Reindeer, and strengthen the protection of this endangered species.

Full-length transcriptome and microRNA sequencing reveal the specific gene-regulation network of velvet antler in sika deer with extremely different velvet antler weight.

Velvet antler displays the fastest and most robust tissue proliferation in the animal world, it is a model for a complete organ development/regeneration, and alternative medicine, tonic made from velvet antler, was beneficial for human. The weight of velvet antler had high biomedical and economic value, but the related regulation mechanisms controlling velvet antler weight remain unclear. In this study, extremely heavy and light velvet antler groups were selected from a sika deer population of 100 individuals with extreme velvet antler weight. A combination of full-length transcriptome sequencing and microRNA sequencing to the proliferation zone in the tip of velvet antler was applied. A total of 55306 transcripts and 1082 microRNAs were identified. Some highly expressed genes (COL1A1, COL1A2, COL3A1, FN1, and ATP6) and microRNAs (miR-21, let-7i, and miR-27b) were highly correlated with the physiological and growth characteristics of velvet antlers. Among the 334 differentially expressed genes, we found that most of the genes were located in the developmental process, especially animal organ development process. It is exciting to see that more blood vessels were found in the growing tip of heavy velvet antler through histological observation, and GO term of blood vessel development was also significant different between two groups. The combination analysis with mRNA and microRNA data in velvet antler showed a specific regulation network involved in the development of bone, mesenchyme, cartilage, and blood vessel, and helped us clearly find out the candidate 14 genes and 6 microRNAs, which could be used for selecting significant DNA markers of velvet antler weight.

Complete mitochondrial genome sequence of Aoluguya reindeer (Rangifer tarandus).

The complete mitochondria genome of the reindeer, Rangifer tarandus, was determined by accurate polymerase chain reaction. The entire genome is 16,357 bp in length and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a D-loop region, all of which are arranged in a typical vertebrate manner. The overall base composition of the reindeer's mitochondrial genome is 33.7% of A, 23.1% of C, 30.1% of T and 13.2%of G. A termination associated sequence and several conserved central sequence block domains were discovered within the control region.

Complete mitochondrial genome sequence of northeastern red deer (Cervus elaphus xanthopygus).

The complete mitochondrial genome of the northeastern red deer, Cervus elaphus xanthopygus, was determined by accurate polymerase chain reaction. The entire genome is 16,416 bp in length and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and 1 control region, all of which are arranged in a typical vertebrate manner. The overall base composition of the northeastern red deer's mitochondrial genome is 33.3% of A, 24.3% of C, 28.9% of T and 13.5% of G. A termination-associated sequence and several conserved central sequence block domains were discovered within the control region.

Complete mitochondrial genome sequence of northeastern sika deer (Cervus nippon hortulorum).

The complete mitochondrial genome of the northeastern sika deer, Cervus nippon hortulorum, was determined by accurate polymerase chain reaction. The entire genome is 16,434 bp in length and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and 1 control region, all of which are arranged in a typical vertebrate manner. The overall base composition of the northeastern sika deer's mitochondrial genome is 33.3% of A, 24.5% of C, 28.7% of T and 13.5% of G. A termination associated sequence and several conserved central sequence block domains were discovered within the control region.