The combination of SMRT sequencing and Illumina sequencing highlights organ-specific and age-specific expression patterns of miRNAs in Sika Deer.

Due to the lack of high-quality Sika Deer (Cervus nippon) transcriptome and sRNAome across multiple organs or development stages, it is impossible to comprehensively analyze the mRNA and miRNA regulatory networks related to growth, development and immunity response. In this study, we used single molecule-real time sequencing (SMRT-seq) and Illumina sequencing methods to generate transcriptome and sRNAome from ten tissues and four age groups of Sika Deer to help us understand molecular characteristics and global miRNA expression profiles. The results showed that a total of 240,846 consensus transcripts were generated with an average length of 2,784 bp. 4,329 Transcription factors (TFs), 109,000 Simple Sequence Repeats (SSRs) and 18,987 Long non-coding RNAs (LncRNAs) were identified. Meanwhile, 306 known miRNAs and 143 novel miRNAs were obtained. A large number of miRNAs showed organ-specific and age-specific differential expression patterns. In particular, we found that the organ-specific miRNAs were enriched in the brain, some of which shared only between the brain and adrenal. These miRNAs were involved in maintaining specific functions within the brain and adrenal. By constructing miRNA96mRNA interaction networks associated with Sika Deer immunity, we found that miRNAs (miR-148a, miR-26a, miR-214, let-7b, etc.) and mRNAs (CD6, TRIM38, C3, CD163, etc.) might play an important role in the immune response of Sika Deer spleen. Together, our study generated an improved transcript annotation for Sika Deer by SMRT-seq and revealed the role of miRNA in regulating the growth, development and immunity response of Sika Deer.

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.

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.

Effect on Serum Parameters and Immune Responses of Carassius auratus gibelio Exposed to Dietary Lead and Bacillus subtilis.

Lead (Pb), a heavy metal and an environmental stressor, may affect many physiological processes, including the serum index and the immune response. The aim of this study was to explore the toxic effects of Pb on the serum index and the immune response of Carassius auratus gibelio (C. gibelio) fed 0, 120, or 240 mg/kg Pb, and 109 cfu/g Bacillus subtilis (B. subtilis). After 15 and 30 days of dietary exposure, the serum indices and the immune responses of the fish were assessed. Dietary Pb exposure significantly affected various components of the serum index, including calcium, magnesium, glucose, cholesterol, total protein, glutamic-pyruvic transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH). However, sIgA activity in the gut increased significantly following B. subtilis supplementation. Notable changes were also observed in the expression levels of immune-related genes, including HSP70, IgM, HSP90, IL-1ß, IL-6, and TNF-α. B. subtilis supplementation effectively attenuated the effects of dietary Pb exposure.

The survey of porcine teschoviruses, porcine circovirus and porcine transmissible gastroenteritis virus infecting piglets in clinical specimens in China.

A multiplex PCR assay was developed and evaluated for its ability to simultaneously detect three viral infections of swine. Specific primers were carefully selected from articles published for each of the following three viruses: porcine circovirus type II (PCV2), porcine teschovirus (PTV) and porcine transmissible gastroenteritis virus (TGEV). Each target produced a specific amplicon with a size of 353 bp (PCV2), 168 bp (PTV) and 499 bp (TGEV). The sensitivity of the multiplex PCR using purified plasmid constructs containing the specific viral target fragments was 6.60 × 10(2), 8.43 × 10(2) and 7.30 × 10(2) copies for PCV2, PTV and TGEV, respectively. Among 127 samples which were collected from Heilongjiang, Jilin, Henan and Guangxi provinces, the single infection of PCV2, PTV and TGEV was 99.21, 46.88 and 65.35 %, respectively, and co-infection of the three viruses was 26.77 %. In conclusion, the multiplex PCR has the potential to be useful for routine molecular diagnosis and epidemiology.