Reciprocal negative feedback between Prrx1 and miR-140-3p regulates rapid chondrogenesis in the regenerating antler.

During growth phase, antlers exhibit a very rapid rate of chondrogenesis. The antler is formed from its growth center reserve mesenchyme (RM) cells, which have been found to be the derivatives of paired related homeobox 1 (Prrx1)-positive periosteal cells. However, the underlying mechanism that drives rapid chondrogenesis is not known. Herein, the miRNA expression profiles and chromatin states of three tissue layers (RM, precartilage, and cartilage) at different stages of differentiation within the antler growth center were analyzed by RNA-sequencing and ATAC-sequencing. We found that miR-140-3p was the miRNA that exhibited the greatest degree of upregulation in the rapidly growing antler, increasing from the RM to the cartilage layer. We also showed that Prrx1 was a key upstream regulator of miR-140-3p, which firmly confirmed by Prrx1 CUT&Tag sequencing of RM cells. Through multiple approaches (three-dimensional chondrogenic culture and xenogeneic antler model), we demonstrated that Prrx1 and miR-140-3p functioned as reciprocal negative feedback in the antler growth center, and downregulating PRRX1/upregulating miR-140-3p promoted rapid chondrogenesis of RM cells and xenogeneic antler. Thus, we conclude that the reciprocal negative feedback between Prrx1 and miR-140-3p is essential for balancing mesenchymal proliferation and chondrogenic differentiation in the regenerating antler. We further propose that the mechanism underlying chondrogenesis in the regenerating antler would provide a reference for helping understand the regulation of human cartilage regeneration and repair.

The Genetic Diversity of Mink (Neovison vison) Populations in China.

The American mink (Neovison vison) is a semiaquatic species of Mustelid native to North America that is now widespread in China. However, the knowledge of genetic diversity of mink in China is still limited. In this study, we investigated the genetic diversity and identified significant single nucleotide polymorphisms (SNPs) in mink populations of five different color types in three different mink farms in China. Using double-digest restriction site-associated DNA sequencing, we identified a total of 1.3 million SNPs. After filtering the SNPs, phylogenetic tree, Fst, principal component, and population structure analyses were performed. The results demonstrated that red mink and black mink grouped, with separate clustering of all other color types. The population divergence index (Fst) study confirmed that different mink populations were distinct (K = 4). Two populations with different coat colors were subjected to the selection signature analysis, and 2300 genes were found to have a clear selection signature. The genes with a selection signature were subjected to Gene Ontology (GO) categorization and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the results revealed that the genes with a selection signature were enriched in the melanogenesis pathway. These study's findings have set the stage for improved breeding and conservation of genetic resources in real-world practical mink farming.

A population of stem cells with strong regenerative potential discovered in deer antlers.

The annual regrowth of deer antlers provides a valuable model for studying organ regeneration in mammals. We describe a single-cell atlas of antler regrowth. The earliest-stage antler initiators were mesenchymal cells that express the paired related homeobox 1 gene (PRRX1+ mesenchymal cells). We also identified a population of "antler blastema progenitor cells" (ABPCs) that developed from the PRRX1+ mesenchymal cells and directed the antler regeneration process. Cross-species comparisons identified ABPCs in several mammalian blastema. In vivo and in vitro ABPCs displayed strong self-renewal ability and could generate osteochondral lineage cells. Last, we observed a spatially well-structured pattern of cellular and gene expression in antler growth center during the peak growth stage, revealing the cellular mechanisms involved in rapid antler elongation.

Single-cell transcriptome reveals core cell populations and androgen-RXFP2 axis involved in deer antler full regeneration.

Deer antlers constitute a unique mammalian model for the study of both organ formation in postnatal life and annual full regeneration. Previous studies revealed that these events are achieved through the proliferation and differentiation of antlerogenic periosteum (AP) cells and pedicle periosteum (PP) cells, respectively. As the cells resident in the AP and the PP possess stem cell attributes, both antler generation and regeneration are stem cell-based processes. However, the cell composition of each tissue type and molecular events underlying antler development remain poorly characterized. Here, we took the approach of single-cell RNA sequencing (scRNA-Seq) and identified eight cell types (mainly THY1+ cells, progenitor cells, and osteochondroblasts) and three core subclusters of the THY1+ cells (SC2, SC3, and SC4). Endothelial and mural cells each are heterogeneous at transcriptional level. It was the proliferation of progenitor, mural, and endothelial cells in the activated antler-lineage-specific tissues that drove the rapid formation of the antler. We detected the differences in the initial differentiation process between antler generation and regeneration using pseudotime trajectory analysis. These may be due to the difference in the degree of stemness of the AP-THY1+ and PP-THY1+ cells. We further found that androgen-RXFP2 axis may be involved in triggering initial antler full regeneration. Fully deciphering the cell composition for these antler tissue types will open up new avenues for elucidating the mechanism underlying antler full renewal in specific and regenerative medicine in general.

Calreticulin Identified as One of the Androgen Response Genes That Trigger Full Regeneration of the Only Capable Mammalian Organ, the Deer Antler.

Deer antlers are male secondary sexual characters that develop to become bone; they are unique appendages that, once lost, can fully regenerate from the permanent bony protuberances or pedicles. Pedicle periosteum (PP) is the tissue that gives rise to the regenerating antlers with three differentiation stages, namely, dormant (DoPP), potentiated (PoPP), and activated (AcPP). Thus far, the transition from the PoPP to the AcPP has not been studied. Our results showed that the AcPP cells maintained their original stem cell features by expressing mesenchymal stem cell (MSC) markers CD73, CD90, and CD105, although they had entered the proliferation mode. The differentially expressed genes (DEGs) in the AcPP compared with those of the PoPP were mainly involved in protein processing, cell cycle, and calcium signaling pathways. Calreticulin (CALR), an androgen response gene, was significantly differentially upregulated in the AcPP cells, and its expression level was negatively regulated by androgens, in contrast to the currently known model systems where all regulation is positive. The downregulation of CALR expression in the AcPP cells in vitro inhibited cell proliferation, induced apoptosis, and inhibited cell cycle progression at G1-S transition. Therefore, CALR is likely a downstream mediator of androgen hormones for triggering initiation of antler regeneration. We believe that the identification of CALR has not only discovered "one critical piece" of the "jigsaw puzzle" in the initiation of antler regeneration but also helps in revealing the mechanism underlying this unique mammalian epimorphic regeneration and has also opened a new avenue for the study of the nature of CALR regulation by androgen (putative binding partners), thus facilitating the identification of potential molecule(s) for investigation as targets for clinical evaluation.

Cross-Species Analysis Reveals Co-Expressed Genes Regulating Antler Development in Cervidae.

Antlers constitute an interesting model for basic research in regenerative biology. Despite decades of being studied, much is still unknown about the genes related to antler development. Here, we utilized both the genome and antlerogenic periosteum (AP) transcriptome data of four deer species to reveal antler-related genes through cross-species comparative analysis. The results showed that the global gene expression pattern matches the status of antler phenotypes, supporting the fact that the genes expressed in the AP may be related to antler phenotypes. The upregulated genes of the AP in three-antlered deer showed evidence of co-expression, and their protein sequences were highly conserved. These genes were growth related and likely participated in antler development. In contrast, the upregulated genes in antler-less deer (Chinese water deer) were involved mainly in organismal death and growth failure, possibly related to the loss of antlers during evolution. Overall, this study demonstrates that the co-expressed genes in antlered deer may regulate antler development.

Integrated analysis of miRNA and mRNA transcriptomic reveals antler growth regulatory network.

The growth of antler is driven by endochondral ossification in the growth center of the apical region. Antler grows faster than cancer tissues, but it can be stably regulated and regenerated periodically. To elucidate the molecular mechanisms of how antler grows rapidly without carcinogenesis, in this study, we used RNA-seq technology to evaluate the changes of miRNA and mRNA profiles in antler at four different developmental stages, including 15, 60, 90, and 110 days. We identified a total of 55004 unigenes and 246 miRNAs of which, 10182, 13258, 10740 differentially expressed (DE) unigenes and 35, 53, 27 DE miRNAs were identified in 60-day vs. 15-day, 90-day vs. 60-day, and 110-day vs. 90-day. GO and KEGG pathway analysis indicated that DE unigenes and DE miRNA were mainly associated with chondrogenesis, osteogenesis and inhibition of oncogenesis, that were closely related to antler growth. The interaction networks of mRNA-mRNA and miRNA-mRNA related to chondrogenesis, osteogenesis and inhibition of oncogenesis of antler were constructed. The results indicated that mRNAs (COL2A1, SOX9, WWP2, FGFR1, SPARC, LOX, etc.) and miRNAs (miR-145, miR-199a-3p, miR-140, miR-199a-5p, etc.) might have key roles in chondrogenesis and osteogenesis of antler. As well as mRNA (TP53, Tpm3 and ATP1A1, etc.) and miRNA (miR-106a, miR-145, miR-1260b and miR-2898, etc.) might play important roles in inhibiting the carcinogenesis of antler. In summary, we constructed the mRNA-mRNA and miRNA-mRNA regulatory networks related to chondrogenesis, osteogenesis and inhibition of oncogenesis of antler, and identified key candidate mRNAs and miRNAs among them. Further developments and validations may provide a reference for in-depth analysis of the molecular mechanism of antler growth without carcinogenesis.

Chromosome-level genome assembly of Tarim red deer, Cervus elaphus yarkandensis.

Tarim red deer (Cervus elaphus yarkandensis) is the only subspecies of red deer (of 22 subspecies) from Central Asia. This species is a desert dweller of the Tarim Basin of southern Xinjiang, China, and exhibits some unique adaptations to the dry and extreme hot climate. We report here the assembly of a Tarim red deer genome employing a 10X Genomics library, termed CEY_v1. Our genome consisted of 2.6 Gb with contig N50 and scaffold N50 of 275.5 Kb and 31.7 Mb, respectively. Around 96% of the assembled sequences were anchored onto 34 chromosomes based on the published high-quality red deer genetic linkage map. More than 94% BUSCOs complete genes (including 90.5% single and 3.6% duplicated ones) were detected in the CEY_v1 and 20,653 genes were annotated. The CEY_v1 is expected to contribute to comparative analysis of genome biology, to evolutionary studies within Cervidae, and to facilitating investigation of mechanisms underlying adaptation of this species to the extreme dry and hot climate.

Molecular evidence for adaptive evolution of olfactory-related genes in cervids.

BACKGROUND: Cervids have evolved very successful means for survival and thriving to adapt to various climates and environments. One of these successful means might be the effective and efficient way of communication. To support this notion, cervids are well equipped with a variety of skin glands that distribute in different body regions. However, studies relevant to adaptive evolution in cervids, particularly on olfactory reception at the molecular level, have thus far not been reported. OBJECTIVE: To provide valuable insights into molecular evidence for the adaptive evolution of olfactory-related gene in cervids. METHODS: Based on recently sequenced genomes of cervids and closely-related-species, we performed comparative genomic analysis at genome level using bioinformatics tools. RESULTS: Tree topology strongly supported that Bovidae was the sister group of Moschidae and both formed a branch that was then clustered with Cervidae. Expansion of heavy chain genes of the dynein family and 51 rapidly evolving genes could be associated with adaptation of cilia that serve as sensory organelles and act as cellular antennae. Based on the branch-site model test along the deer branch spanning 7-21 mammalian species, 14 deer olfactory receptor genes were found to be undergoing positive selection pressure and 89 positive selection sites (probability > 60%) had amino acid substitutions unique to deer. CONCLUSION: This study, for the first time, provides significant molecular evidence for adaption of olfactory-related genes of cervids according to their olfactory behavior.

Phylodynamic analysis of two amino acid substitutions in the hemagglutinin protein of canine distemper virus strains detected in fur-bearing animals in China.

Canine distemper virus (CDV) causes a highly contagious disease in a wide range of carnivores. The hemagglutinin (H) protein of viruses shows the highest variability and plays an important role in modulation of viral antigenicity, virulence, and receptor recognition. Since 2012, canine distemper (CD) outbreaks in fur-bearing animals (minks, foxes, raccoon dogs) caused by CDV variants with I542N and Y549H substitutions in the H protein have been frequently reported in China. To characterize the molecular evolutionary dynamics and epidemiological dynamics of CDV, 235 H gene sequences of CDV wild-type strains collected from 22 countries between 1975 and 2015, including 44 strains predominant in fur-bearing animals in China, were analyzed. The phylogenetic relationships and evolutionary rates of the CDV strains were determined by Bayesian phylogenetics. The CDV strains clustered into distinct geographic genotypes, irrespective of the species of isolation. All the variant strains formed a distinct monophyletic cluster and belonged to the F sub-genotype within the Asia-1 genotype-currently the predominant sub-genotype in fur-bearing animals in China. Evolutionary analysis suggested that the variant strains originated in 2006. Furthermore, the selection pressure analysis revealed that the Y549H substitution was under positive selection pressure for adaptation toward the fur-bearing animals. The residue at position 549 also showed structural interaction with the V domain of the mink signaling lymphocyte-activation molecule (SLAM) receptor based on the homology modeling of the H-SLAM complex. Our results suggested that the Y549H substitution contributed to the molecular adaptation of CDV variants in the fur-bearing animals during the viral evolutionary phase in China.

Identification of interactive molecules between antler stem cells and dermal papilla cells using an in vitro co-culture system.

Deer antlers are the only mammalian organs capable of complete renewal. Antler renewal is a stem cell-based [antler stem cells (ASCs)] process. Maintenance and activation of the ASCs require them to be located in a specialized microenvironment (niche), and to interact with the cells resident in the niche. Based on previous experiments we found that niche of the ASCs is provided by the closely associated enveloping skin, which currently was known includes dermal papilla cells (DPCs) and epidermal cells. Antler generation/regeneration are triggered by the interactions between ASCs and the niche. In the present study, we established an in vitro co-culture system in which ASCs and DPCs, were cultured together to mimic the in vivo state. A MLEFF strategy was adopted to identify the interactive molecules from the co-culture system. In total, 128 molecules were identified and over 60% belonged to exosomes. Important biological processes that were activated by these molecules included osteoblast differentiation, angiogenesis, and the PI3K-AKT signaling pathway. In so doing, we have significantly simplified the process for identifying interactive molecules, which may be the key signals for triggering antler formation/renewal. Further study of these molecules will help us to gain insights into the mechanism of mammalian organ regeneration.

S100A4: a novel partner for heat shock protein 47 in antler stem cells and insight into the calcium ion-induced conformational changes.

S100A4 is a multiple-function protein highly expressed in tumor or stem cells. We found S100A4 was a novel protein partner for heat shock protein 47 (HSP47) in deer antlerogenic periosteum cells (AP cells), indicating that S100A4 could bind with HSP47. S100A4 had both calcium-dependent and calcium-independent patterns (labeled as SCd and SCi, respectively) to execute different biological activities. Homology models of HSP47, SCd and SCi were constructed. HSP47:collagen model, HSP47:collagen I-V, HSP47:SCd and HSP47:SCi complexes were built using ZDOCK software. Together with free SCd and SCi, 200 ns molecular dynamic (MD) simulations were performed to analyze binding free energies and SCi/SCd conformational changes. The energetic results showed that SCi had the strongest affinity to HSP47, and followed by collagens. SCd had little interaction with HSP47. Decomposition energy results showed that collagen model interacted with HSP47 mainly though neutral amino acids. When SCi bound with HSP47, the majority of mediated amino acids were charged. These results indicated that SCi could compete with collagen on the binding site of HSP47. Root mean square fluctuation (RMSF) values and cross-correlation matrices of principal component analysis (PCA) were calculated to evaluate the SCi/SCd structural variation during MD simulation. Both HSP47 and Ca2+ could stabilize the conformation of SCi/SCd. The loops interacting with Ca2+s and linking the two EF-hand motifs were impacted particularly. The relative moving directions of α-helices in EF-hands were distinct by the binding effect of HSP47 and Ca2+. We found that SCi may regulate the differentiation of AP cells by disturbing the interaction between HSP47 and collagen. Communicated by Ramaswamy H. Sarma.

Identification of proteins that mediate the role of androgens in antler regeneration using label free proteomics in sika deer (Cervus nippon).

Deer antlers offer a unique model to study organ regeneration in mammals. Antler regeneration relies on the pedicle periosteum (PP) cells and is triggered by a decrease in circulating testosterone (T). The molecular mechanism for antler regeneration is however, unclear. Label-free liquid chromatography-mass spectrometry (LC-MS/MS) was used to identify differentially-expressed proteins (DEPs) in the regeneration-potentiated PP (under low T environment) over the non-regeneration-potentiated PP (under high T environment). Out of total 273 DEPs, 189 were significantly up-regulated and 84 were down-regulated from these comparisons: after castration vs before castration, natural T vs before castration, and exogenous T vs before castration. We focused on the analysis only of those DEPs that were present in fully permissive environment to antler regeneration (low T). Nine transduction pathways were identified through the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, including the estrogen signaling pathway. A total of 639 gene ontology terms were found to be significantly enriched in regeneration-potentiated PP (low T) from the DEPs. Reliability of the label free LC-MS/MS was determined by qRT-PCR to estimate the expression level of selected genes. The results suggest that up-regulated heat shock proteins (HSP90AB1, HSP90B1), peptidyl-prolyl cis-trans isomerase 4 (FKBP4), mitogen-activated protein kinase 3 (MAPK3) and calreticulin (CALR) and down-regulated SHC-transforming protein 1 (SHC1), heat shock protein family A member 1A (HSPA1A) and proto-oncogene tyrosine-protein kinase (SRC) may be associated directly or indirectly with antler regeneration. Further studies are required to investigate the roles of these proteins in regeneration using appropriate in vivo models.

Deer antler stem cells are a novel type of cells that sustain full regeneration of a mammalian organ-deer antler.

Deer antlers are extraordinary mammalian organs that can fully regenerate annually. Antler renewal is a stem cell-based epimorphic process and antler stem (AS) cells can initiate de novo generation of antlers in postnatal mammals. However, although being called stem cells, the AS cells have not been characterized at molecular level based on the stem cell criteria. Comprehensive characterization of the AS cells would undoubtedly help to decipher the mechanism underlying the full regeneration of deer antlers, the only case of stem cell-based epimorphic regeneration in mammals. In the present study, three types of AS cells (antlerogenic periosteal cells APCs, for initial pedicle and first antler formation; pedicle periosteal cells PPC, for annual antler regeneration; and reserve mesenchyme cells RMCs, for rapid antler growth), were isolated for comprehensive molecular characterization. A horn-growth-related gene, RXFP2, was found to be expressed only in AS cells lineages but not in the facial periosteal cells (FPCs, locates geographically in the vicinity of the APCs or PPCs), suggesting the RXFP2 might be a specific marker for the AS cell lineage in deer. Our results demonstrated that AS cells expressed classic MSC markers including surface markers CD73, CD90, CD105 and Stro-1. They also expressed some of the markers including Tert, Nestin, S100A4, nucleostemin and C-Myc, suggesting that they have some attributes of the ESCs. Microinjection of male APC into deer blastocysts resulted in one female foetus (110 days gestation) recovered with obvious pedicle primordia with both male and female genotype detected in the ovary. In conclusion, the AS cells should be defined as MSCs but with partial attributes of ESCs.

Transcriptome Changes in the Mink Uterus during Blastocyst Dormancy and Reactivation.

Embryo implantation in the mink follows the pattern of many carnivores, in that preimplantation embryo diapause occurs in every gestation. Details of the gene expression and regulatory networks that terminate embryo diapause remain poorly understood. Illumina RNA-Seq was used to analyze global gene expression changes in the mink uterus during embryo diapause and activation leading to implantation. More than 50 million high quality reads were generated, and assembled into 170,984 unigenes. A total of 1684 differential expressed genes (DEGs) in uteri with blastocysts in diapause were compared to the activated embryo group (p < 0.05). Among these transcripts, 1527 were annotated as known genes, including 963 up-regulated and 564 down-regulated genes. The gene ontology terms for the observed DEGs, included cellular communication, phosphatase activity, extracellular matrix and G-protein couple receptor activity. The KEGG pathways, including PI3K-Akt signaling pathway, focal adhesion and extracellular matrix (ECM)-receptor interactions were the most enriched. A protein-protein interaction (PPI) network was constructed, and hub nodes such as VEGFA, EGF, AKT, IGF1, PIK3C and CCND1 with high degrees of connectivity represent gene clusters expected to play an important role in embryo activation. These results provide novel information for understanding the molecular mechanisms of maternal regulation of embryo activation in mink.

Transcriptomic analysis of different tissue layers in antler growth Center in Sika Deer (Cervus nippon).

BACKGROUND: With the unprecedented rapid growth rate (up to 2.75 cm/day), velvet antler is an invaluable model for the identification of potent growth factors and signaling networks for extremely fast growing tissues, mainly cartilage. Antler growth center (AGC) locates in its tip and consists of five tissue layers: reserve mesenchyme (RM), precartilage (PC), transition zone (TZ), cartilage (CA) and mineralized cartilage (MC). The aim of this study was to investigate the transcription dynamics in the AGC using RNA-seq technology. RESULTS: Five tissue layers in the AGC were collected from three 3-year-old male sika deer using our previously reported sampling method (morphologically distinguishable). After sequencing (15 samples; triplicates/tissue layer), we assembled a reference transcriptome de novo and used RNA-seq to measure gene expression profiles across these five layers. Nine differentially expressed genes (DEGs) were selected from our data and subsequently verified using qRT-PCR. The results showed a high consistency with the RNA-seq results (R2 = 0.80). Nine modules were constructed based on co-expression network analysis, and these modules contained 370 hub genes. These genes were found to be mainly involved in mesenchymal progenitor cell proliferation, chondrogenesis, osteogenesis and angiogenesis. Combination of our own results with the previously published reports, we found that Wnt signaling likely plays a key role not only in stimulating the antler stem cells or their immediate progeny, but also in promoting chondrogenesis and osteogenesis during antler development. CONCLUSION: We have successfully assembled a reference transcriptome, generated gene expression profiling across the five tissue layers in the AGC, and identified nine co-expressed modules that contain 370 hub genes and genes predorminantly expressed in and highly relevant to each tissue layer. We believe our findings have laid the foundation for the identification of novel genes for rapid proliferation and chondrogenic differentiation of antler cells.

Single-cell transcriptome provides novel insights into antler stem cells, a cell type capable of mammalian organ regeneration.

Antler regeneration, a stem cell-based epimorphic process, has a potential as a valuable model for regenerative medicine. A pool of antler stem cells (ASCs) for antler development is located in the antlerogenic periosteum (AP). However, whether this ASC pool is homogenous or heterogeneous has not been fully evaluated. In this study, we produced a comprehensive transcriptome dataset at the single-cell level for the ASCs based on the 10× Genomics platform (scRNA-seq). A total of 4565 ASCs were sequenced and classified into a large cell cluster, indicating that the ASC resident in the AP are likely to be a homogeneous population. The scRNA-seq data revealed that tumor-related genes were highly expressed in these homogeneous ASCs, i.e., TIMP1, TMSB10, LGALS1, FTH1, VIM, LOC110126017, and S100A4. Results of screening for stem cell markers suggest that the ASCs may be considered as a special type of stem cell between embryonic (CD9) and adult (CD29, CD90, NPM1, and VIM) stem cells. Our results provide the first comprehensive transcriptome analysis at the single-cell level for the ASCs and identified only one major cell type resident in the AP and some key stem cell genes, which may hold the key to why antlers, the unique mammalian organ, can fully regenerate once lost.

Proteomic Analysis of Plasma Membrane Proteins of Antler Stem Cells Using Label-Free LC⁻MS/MS.

Deer antlers are unusual mammalian organs that can fully regenerate after annual shedding. Stem cells resident in the pedicle periosteum (PPCs) provide the main cell source for antler regeneration. Central to various cellular processes are plasma membrane proteins, but the expression of these proteins has not been well documented in antler regeneration. In the present study, plasma membrane proteins of PPCs and facial periosteal cells (FPCs) were analyzed using label-free liquid chromatography⁻mass spetrometry (LC⁻MS/MS). A total of 1739 proteins were identified. Of these proteins, 53 were found solely in the PPCs, 100 solely in the FPCs, and 1576 co-existed in both PPCs and FPCs; and 39 were significantly up-regulated in PPCs and 49 up-regulated in FPCs. In total, 226 gene ontology (GO) terms were significantly enriched from the differentially expressed proteins (DEPs). Five clusters of biological processes from these GO terms comprised responses to external stimuli, signal transduction, membrane transport, regulation of tissue regeneration, and protein modification processes. Further studies are required to demonstrate the relevancy of these DEPs in antler stem cell biology and antler regeneration.

Development of Diagnostic SNP Markers To Monitor Hybridization Between Sika Deer (Cervus nippon) and Wapiti (Cervus elaphus).

Sika deer (Cervus Nippon) and wapiti (Cervus elaphus) are closely related species and their hybridization can result in significant allele-shift of their gene pool. Additive genetic effects and putative heterotic effects of their hybridization on growth performance could confer considerable economic advantage in deer farming. Here, we used double-digest restriction site-associated DNA sequencing technology (ddRAD-seq) and detected ∼320,000 genome-wide SNPs from 30 captive individuals: 7 sika deer, 6 wapiti and 17 F1 hybrids (reciprocal cross). By screening observed heterozygosity of each SNP across four taxonomic groups, we report for the first time a resource of 2,015 putative diagnostic SNP markers (species-specific SNPs for sika deer and wapiti), which can be used to design tools for assessing or monitoring the degree of hybridization between sika deer and wapiti. These ddRAD-seq data and SNP datasets are also valuable resources for genome-wide studies, including trait discovery for breeders of domestic deer.