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.

Development of novel EST microsatellite markers for genetic diversity analysis and correlation analysis of velvet antler growth characteristics in Sika deer.

BACKGROUND: Sika deer is one of the most popular and valued animals in China. However, few studies have been conducted on the microsatellite of Sika deer, which has hampered the progress of genetic selection breeding. To develop and characterize a set of microsatellites for Sika deer which provide helpful information for protection of Sika deer natural resources and effectively increase the yield and quantity of velvet antler. RESULTS: We conducted a transcriptome survey of Sika deer using next-generation sequencing technology. One hundred eighty-two thousand two hundred ninety-five microsatellite markers were identified in the transcriptome, 170 of 200 loci were successfully amplified across panels of 140 individuals from Shuangyang Sika deer population. And 29 loci were found to be obvious polymorphic. Number of alleles is from 3 to 14. The expected heterozygosity ranged from 0.3087 to 0.7644. The observed heterozygosity ranged from 0 to 0.7698. The polymorphism information content values of those microsatellites varied ranged from 0.2602 to 0.7507. The marker-trait association was tested for 6 important and kernel characteristics of two-branched velvet antler in Shuangyang Sika deer through one-way analysis of variance. The results showed that marker-trait associations were identified with 8 different markers, especially M009 and M027. CONCLUSIONS: This study not only provided a large scale of microsatellites which were valuable for future genetic mapping and trait association in Sika deer, but also offers available information for molecular breeding in Sika deer.

Altered miRNA and mRNA Expression in Sika Deer Skeletal Muscle with Age.

Studies of the gene and miRNA expression profiles associated with the postnatal late growth, development, and aging of skeletal muscle are lacking in sika deer. To understand the molecular mechanisms of the growth and development of sika deer skeletal muscle, we used de novo RNA sequencing (RNA-seq) and microRNA sequencing (miRNA-seq) analyses to determine the differentially expressed (DE) unigenes and miRNAs from skeletal muscle tissues at 1, 3, 5, and 10 years in sika deer. A total of 51,716 unigenes, 171 known miRNAs, and 60 novel miRNAs were identified based on four mRNA and small RNA libraries. A total of 2,044 unigenes and 11 miRNAs were differentially expressed between adolescence and juvenile sika deer, 1,946 unigenes and 4 miRNAs were differentially expressed between adult and adolescent sika deer, and 2,209 unigenes and 1 miRNAs were differentially expressed between aged and adult sika deer. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that DE unigenes and miRNA were mainly related to energy and substance metabolism, processes that are closely associate with the growth, development, and aging of skeletal muscle. We also constructed mRNA-mRNA and miRNA-mRNA interaction networks related to the growth, development, and aging of skeletal muscle. The results show that mRNA (Myh1, Myh2, Myh7, ACTN3, etc.) and miRNAs (miR-133a, miR-133c, miR-192, miR-151-3p, etc.) may play important roles in muscle growth and development, and mRNA (WWP1, DEK, UCP3, FUS, etc.) and miRNAs (miR-17-5p, miR-378b, miR-199a-5p, miR-7, etc.) may have key roles in muscle aging. In this study, we determined the dynamic miRNA and unigenes transcriptome in muscle tissue for the first time in sika deer. The age-dependent miRNAs and unigenes identified will offer insights into the molecular mechanism underlying muscle development, growth, and maintenance and will also provide valuable information for sika deer genetic breeding.

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.

Isolation of novel microsatellite markers and their application for genetic diversity and parentage analyses in sika deer.

Every part of the sika deer (Cervus nippon) body is valuable traditional Chinese medicine. And sika deer is the most important semi-domestic medicinal animal that is widely bred in Jilin province northeast of China. But few studies had been conducted to characterize the microsatellite markers derived from sika deer. We firstly used IlluminaHiSeq™2500 sequencing technology obtained 125Mbp genomic data of sika deer. Using microsatellite identification tool (MISA), 22,479 microsatellites were identified. From these data, 100 potential primers were selected for further polymorphic validation, finally, 76 primer pairs were successfully amplified and 29 primer pairs were found to be obvious polymorphic in 8 different individuals. Using those polymorphic microsatellite markers, we analyzed the genetic diversity of Jilin sika deer population. The mean number of alleles of the 29 loci is 9.31 based on genotyping blood DNA from 96 Jilin sika deer; The mean expected heterozygosity and polymorphic information content (PIC) value of the 29 loci is 0.72 and 0.68 respectively, and among which 26 loci are highly polymorphic (PIC>0.50). According to the electrophoretic results and PIC value of these 29 loci, 10 loci with combined paternity exclusion probabilities>99.99% were selected to use in parentage verification for 16 sika deer. All the offspring of a family could be successfully assigned to their biological father. These microsatellite markers generated in this study could greatly facilitate future studies of molecular breeding in sika deer.

Genome-Wide SNP Discovery and Analysis of Genetic Diversity in Farmed Sika Deer (Cervus nippon) in Northeast China Using Double-Digest Restriction Site-Associated DNA Sequencing.

Sika deer are an economically valuable species owing to their use in traditional Chinese medicine, particularly their velvet antlers. Sika deer in northeast China are mostly farmed in enclosure. Therefore, genetic management of farmed sika deer would benefit from detailed knowledge of their genetic diversity. In this study, we generated over 1.45 billion high-quality paired-end reads (288 Gbp) across 42 unrelated individuals using double-digest restriction site-associated DNA sequencing (ddRAD-seq). A total of 96,188 (29.63%) putative biallelic SNP loci were identified with an average sequencing depth of 23×. Based on the analysis, we found that the majority of the loci had a deficit of heterozygotes (FIS >0) and low values of Hobs, which could be due to inbreeding and Wahlund effects. We also developed a collection of high-quality SNP probes that will likely be useful in a variety of applications in genotyping for cervid species in the future.

Transcriptome analysis of sika deer in China.

Sika deer is of great commercial value because their antlers are used in tonics and alternative medicine and their meat is healthy and delicious. The goal of this study was to generate transcript sequences from sika deer for functional genomic analyses and to identify the transcripts that demonstrate tissue-specific, age-dependent differential expression patterns. These sequences could enhance our understanding of the molecular mechanisms underlying sika deer growth and development. In the present study, we performed de novo transcriptome assembly and profiling analysis across ten tissue types and four developmental stages (juvenile, adolescent, adult, and aged) of sika deer, using Illumina paired-end tag (PET) sequencing technology. A total of 1,752,253 contigs with an average length of 799 bp were generated, from which 1,348,618 unigenes with an average length of 590 bp were defined. Approximately 33.2 % of these (447,931 unigenes) were then annotated in public protein databases. Many sika deer tissue-specific, age-dependent unigenes were identified. The testes have the largest number of tissue-enriched unigenes, and some of them were prone to develop new functions for other tissues. Additionally, our transcriptome revealed that the juvenile-adolescent transition was the most complex and important stage of the sika deer life cycle. The present work represents the first multiple tissue transcriptome analysis of sika deer across four developmental stages. The generated data not only provide a functional genomics resource for future biological research on sika deer but also guide the selection and manipulation of genes controlling growth and development.