Discovery of exercise-related genes and pathway analysis based on comparative genomes of Mongolian originated Abaga and Wushen horse.

The Mongolian horses have excellent endurance and stress resistance to adapt to the cold and harsh plateau conditions. Intraspecific genetic diversity is mainly embodied in various genetic advantages of different branches of the Mongolian horse. Since people pay progressive attention to the athletic performance of horse, we expect to guide the exercise-oriented breeding of horses through genomics research. We obtained the clean data of 630,535,376,400 bp through the entire genome second-generation sequencing for the whole blood of four Abaga horses and ten Wushen horses. Based on the data analysis of single nucleotide polymorphism, we severally detected that 479 and 943 positively selected genes, particularly exercise related, were mainly enriched on equine chromosome 4 in Abaga horses and Wushen horses, which implied that chromosome 4 may be associated with the evolution of the Mongolian horse and athletic performance. Four hundred and forty genes of positive selection were enriched in 12 exercise-related pathways and narrowed in 21 exercise-related genes in Abaga horse, which were distinguished from Wushen horse. So, we speculated that the Abaga horse may have oriented genes for the motorial mechanism and 21 exercise-related genes also provided a molecular genetic basis for exercise-directed breeding of the Mongolian horse.

The complete mitochondrial genome of Melanoides tuberculata (Müller, 1774) in Guangdong, China.

The Melanoides tuberculata is an invasive species, which is natively distributed in Africa and Southeast Asia. This study describes the first mitochondrial genome of the M. tuberculata based on the whole genome sequencing data. The complete sequence length of the mitogenome is 15,821 bp, including 37 genes (2 rRNA genes, 22 tRNA genes and 13 protein-coding genes). Phylogenetic analysis using the 13 species of Cerithioidea species showed that the M. tuberculata is closely related to P. dartevellei, forming the sister group to C. sinensis and C. obtuse.

Multiplexed Visualization Method to Explore Complete Targeting Regulatory Relationships Among Circadian Genes for Insomnia Treatment.

Understanding the complete map of melatonin synthesis, the information transfer network among circadian genes in pineal gland, promises to resolve outstanding issues in endocrine systems and improve the clinical diagnosis and treatment level of insomnia, immune disease and hysterical depression. Currently, some landmark studies have revealed some genes that regulate circadian rhythm associated with melatonin synthesis. However, these studies don't give a complete map of melatonin synthesis, as transfer information among circadian genes in pineal gland is lost. New biotechnology, integrates dynamic sequential omics and multiplexed imaging method, has been used to visualize the complete process of melatonin synthesis. It is found that there are two extremely significant information transfer processes involved in melatonin synthesis. In the first stage, as the light intensity decreased, melatonin synthesis mechanism has started, which is embodied in circadian genes, Rel, Polr2A, Mafk, and Srbf1 become active. In the second stage, circadian genes Hif1a, Bach1, Clock, E2f6, and Per2 are regulated simultaneously by four genes, Rel, Polr2A, Mafk, and Srbf1 and contribute genetic information to Aanat. The expeditious growth in this technique offer reference for an overall understanding of gene-to-gene regulatory relationship among circadian genes in pineal gland. In the study, dynamic sequential omics and the analysis process well provide the current state and future perspectives to better diagnose and cure diseases associated with melatonin synthesis disorder.

Identification of sodium homeostasis genes in Camelus bactrianus by whole transcriptome sequencing.

Salt dietary intake is tightly coupled to human health, and excessive sodium can cause strokes and cardiovascular diseases. Research into the renal medulla of camels exhibiting high salt resistance may aid identification of the mechanisms governing resistance to high salinity. In this study, we used RNA sequencing (RNA-seq) to show that in the renal medulla of camels under salt stress, 22 mRNAs, 2 long noncoding RNAs (lncRNAs), and 31 microRNAs (miRNAs) exhibited differential expression compared with the free salt-intake diet group. Using fluorescence in situ hybridization and dual-luciferase reporter assays, we demonstrated that the lncRNA LNC003834 can bind miRNA-34a and thereby relieve suppression of the salt-absorption-inhibiting SLC14A1 mRNA from miRNA-34a, suggesting that the above lncRNA-miRNA-mRNA act as competing endogenous RNAs (ceRNAs). We subsequently performed short hairpin RNA and small RNA interference and reactive oxygen species (ROS) detection assays to show that SLC6A1, PCBP2, and PEX5L can improve the antioxidation capacity of renal medulla cells of camel by decreasing ROS levels. Our data suggest that camels achieve sodium homeostasis through regulating the expression of salt-reabsorption-related genes in the renal medulla, and this involves ceRNAs (SLC14A1 mRNA, LNC003834, and miRNA-34a) and antioxidant genes (SLC6A1, PCBP2, and PEX5L). These data may assist in the development of treatments for diseases induced by high salt diets.

KRAB family is involved in network shifts in response to osmotic stress in camels.

A feature of the camel is its tolerance to osmotic stress. However, few studies of osmotic stress in vivo or comparative analyses between different tissues of the camel have been performed. Here, we report the roles of Krüppel-associated box domain containing zinc-finger repressor proteins (KRAB-ZFPs) in transcriptional networks under osmotic stress in camels by analyzing transcriptomes of four different tissues under various osmotic conditions. We found that 273 of 278 KRAB-ZFPs were expressed in our data set, being involved in all of the 65 identified networks and exhibiting their extensive functional diversity. We also found that 110 KRAB-ZFPs were hub genes involved in more than half of the networks. We demonstrated that the osmotic stress response is involved in network shifts and that KRAB-ZFPs mediate this process. Finally, we presented the diverse mechanisms of osmotic stress responses in different tissues. These results revealed the genetic architecture of systematic physiological response in vivo to osmotic stress in camels. Our work will lead to new directions for studying the mechanism of osmotic stress response in anti-arid mammals.

Integrated Analysis of Mutations, miRNA and mRNA Expression in Glioblastoma.

BACKGROUND: Glioblastoma multiforme (GBM) is a common, malignant brain tumor in adults, with a median survival of only 15-23 months. Organisms respond to disease stress through sophisticated mechanisms at the physiological, transcriptional and metabolic levels. However, the molecular regulatory networks responsible for occurrence, progression and recurrence of glioma have yet to be elucidated. METHODS: In this study, we sought to determine the cause of gliomas by developing an RNA-seq technique that analyzes mRNA and small RNA (sRNA) with the aim of discovering potential methods for precisely blocking key signaling pathways in occurrence, progression, and recurrence. The explication of mechanisms leading to GBM formation has become a feasible and promising new therapeutic method. RESULTS: GBM-associated genes were identified based on their expression during the disease stress response. Analysis of the inverse correlations between microRNAs (miRNAs) and target mRNAs revealed 43 mRNA-miRNA interactions during disease progression. BOC-SMO and BOC-RAS were found to promote the malignant progression of glioma. A total of 3088 differentially expressed genes were identified as involved in several biological processes, such as amino acid metabolism, protein transport associated with immune response, cell proliferation, and cell apoptosis. Fifteen miRNAs were also identified as being differentially expressed in GBM and control groups. CONCLUSION: The results of this study provide an important foundation for understanding the pathogenesis of glioma and discovering new therapeutic targets.

Evidence from ileum and liver transcriptomes of resistance to high-salt and water-deprivation conditions in camel.

Camels have evolved various resistance characteristics adaptive to their desert habitats. In the present study, we used high-throughput sequencing to investigate stress-induced alternative splicing events as well as different genes involved in resistance to water deprivation and salt absorption in the ileum and liver in Camelus bactrianus. Through association analyses of mRNA, miRNA and lncRNA, we sought to explicate how camels respond to high salt and water scarcity conditions. There were two modes by which genes driven by alternative splicing were enriched to molecular functions, invoking of which was potentially fixed by organ and stress types. With qRT-PCR detection, the differentially expressed MUC6, AQP5, LOC105076960, PKP4, CDH11, TENM1, SDS, LOC105061856, PLIN2 and UPP2 were screened as functionally important genes, along with miR-29b, miR-484, miR-362-5p, miR-96, miR-195, miR-128 and miR-148a. These genes contributed to cellular stress resistance, for instance by reducing water loss, inhibiting excessive import of sodium, improving protective barriers and sodium ion homeostasis, and maintaining uridine content. The underlying competing endogenous RNAs referred to LNC001664, let-7e and LOC105076960 mRNA in ileum, and LNC001438, LNC003417, LNC001770, miR-199c and TENM1 mRNA in liver. Besides competent interpretation to resistance, there may be inspirations for curing human diseases triggered by high-salt intake.

Screening of drought-resistance related genes and analysis of promising regulatory pathway in camel renal medulla.

Camels as a sort of animal long living in desert have evolved stress-resistance characteristics to adapt to environment with high temperature and water shortage environment. However, the research of non-coding RNA (ncRNA)-mediated molecular regulation about how camel responds to arid condition in post-transcriptional regulation level is deficient. Under water-deprivation stress, by RNA-sequencing of camel renal medulla associated with regulating water metabolism, we detected significantly differential 575 alternative splicing events (ASEs) and 17 mRNAs, 26 miRNAs and 0 lncRNA. The down-regulated ACLY and LOC105061856, up-regulated PCBP2 and miR-195 potentially targeting LOC105061856 and PCBP2 mRNA were selected as candidate resistance-related genes. In quantitative experiment, the expression level of above four genes was consistent with RNA-seq data by qRT-PCR. The suppressive cell dehydration with down-regulated ACLY, inhibitive aerobic respiration with down-regulated LOC105061856 targeted by miR-195 and strong anti-oxidative capability with PCBP2 aimed by miR-195 may be regulatory modes of camel renal medulla adapting to water-deprivation condition.

Key genes differential expressions and pathway involved in salt and water-deprivation stresses for renal cortex in camel.

BACKGROUND: Camels possess the characteristics of salt- and drought-resistances, due to the long-time adaption to the living environment in desert. The camel resistance research on transcriptome is rare and deficient, especially reabsorption in renal cortex. Non-coding RNAs are normally considered as the RNA molecules that are not translated into proteins, their current roles remain mostly in regulation of information flux from DNA to protein, further on normal life activities and diseases. In order to reveal the mysterious veil of the post-transcriptional regulation of ncRNAs in renal cortex for the first time as far as we know, we designed and carried out the experiment of salt stress and water-deprivation stress in camel. RESULTS: By means of RNA-seq in renal cortex of Alxa Bactrian Camel (Camelus bactrianus), we identified certain significantly differential RNAs, including 4 novel lncRNAs, 11 miRNAs and 13 mRNAs under salt stress, 0 lncRNAs, 18 miRNAs and 14 mRNAs under water-deprivation stress. By data analysis, the response pathway of post-transcriptional regulation concerning salt and water-deprivation stresses was put forward, involving preventing sodium from entering the cell, purifying of water and compensating neutral amino acids by miR-193b, miR-542-5p interaction with SLC6A19 mRNA. CONCLUSION: Based on the resistance-related lncRNAs, miRNAs, and mRNAs, we proposed the post-transcriptional regulation pathway to explain how camels respond to salt and water-deprivation stresses in the ncRNAs regulation level of renal cortex for the first time, thus hoping to provide a theoretical basis for therapy of disease that is similar to high blood pressure in humans.

Identification of key genes and pathways involved in response to pain in goat and sheep by transcriptome sequencing.

PURPOSE: This aim of this study was to investigate the key genes and pathways involved in the response to pain in goat and sheep by transcriptome sequencing. METHODS: Chronic pain was induced with the injection of the complete Freund's adjuvant (CFA) in sheep and goats. The animals were divided into four groups: CFA-treated sheep, control sheep, CFA-treated goat, and control goat groups (n = 3 in each group). The dorsal root ganglions of these animals were isolated and used for the construction of a cDNA library and transcriptome sequencing. Differentially expressed genes (DEGs) were identified in CFA-induced sheep and goats and gene ontology (GO) enrichment analysis was performed. RESULTS: In total, 1748 and 2441 DEGs were identified in CFA-treated goat and sheep, respectively. The DEGs identified in CFA-treated goats, such as C-C motif chemokine ligand 27 (CCL27), glutamate receptor 2 (GRIA2), and sodium voltage-gated channel alpha subunit 3 (SCN3A), were mainly enriched in GO functions associated with N-methyl-D-aspartate (NMDA) receptor, inflammatory response, and immune response. The DEGs identified in CFA-treated sheep, such as gamma-aminobutyric acid (GABA)-related DEGs (gamma-aminobutyric acid type A receptor gamma 3 subunit [GABRG3], GABRB2, and GABRB1), SCN9A, and transient receptor potential cation channel subfamily V member 1 (TRPV1), were mainly enriched in GO functions related to neuroactive ligand-receptor interaction, NMDA receptor, and defense response. CONCLUSIONS: Our data indicate that NMDA receptor, inflammatory response, and immune response as well as key DEGs such as CCL27, GRIA2, and SCN3A may regulate the process of pain response during chronic pain in goats. Neuroactive ligand-receptor interaction and NMDA receptor as well as GABA-related DEGs, SCN9A, and TRPV1 may modulate the process of response to pain in sheep. These DEGs may serve as drug targets for preventing chronic pain.

Production of microhomologous-mediated site-specific integrated LacS gene cow using TALENs.

Gene editing tools (Zinc-Finger Nucleases, ZFN; Transcription Activator-Like Effector Nucleases, TALEN; and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas)9, CRISPR-Cas9) provide us with a powerful means of performing genetic engineering procedures. A combinational approach that utilizes both somatic cell nuclear transfer (SCNT) and somatic cell gene editing facilitates the generation of genetically engineered animals. However, the associated research has utilized markers and/or selected genes, which constitute a potential threat to biosafety. Microhomologous-mediated end-joining (MMEJ) has showed the utilization of micro-homologous arms (5-25 bp) can mediate exogenous gene insertion. Dairy milk is a major source of nutrition worldwide. However, most people are not capable of optimally utilizing the nutrition in milk because of lactose intolerance. Sulfolobus solfataricus ß-glycosidase (LacS) is a lactase derived from the extreme thermophilic archaeon Sulfolobus solfataricus. Our finally aim was to site-specific integrated LacS gene into cow's genome through TALEN-mediated MMEJ and produce low-lactose cow. Firstly, we constructed TALENs vectors which target to the cow's ß-casein locus and LacS gene expression vector which contain TALEN reorganization sequence and micro-homologous arms. Then we co-transfected these vectors into fetal derived skin fibroblasts and cultured as monoclone. Positive cell clones were screened using 3' junction PCR amplification and sequencing analysis. The positive cells were used as donors for SCNT and embryo transfer (ET). Lastly, we detected the genotype through PCR of blood genomic DNA. This resulted in a LacS knock-in rate of 0.8% in TALEN-treated cattle fetal fibroblasts. The blastocyst rate of SCNT embryo was 27%. The 3 months pregnancy rate was 20%. Finally, we obtained 1 newborn cow (5%) and verified its genotype. We obtained 1 site-specific marker-free LacS transgenic cow. It provides a basis to solve lactose intolerance by gene engineering breeding. This study also provides us with a new strategy to facilitate gene knock-ins in livestock using techniques that exhibit improved biosafety and intuitive methodologies.

Isolation and identification of bovine nasopharyngeal mucosal epithelial cells and establishment of cell models of acute infection by foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) commonly occurs via the respiratory tract, and bovine nasopharyngeal mucosal epithelial cells are the primary infection cells in cattle. The aim of the present study was to isolate and culture epithelial cells from the bovine nasopharyngeal mucosa in vitro using a mechanical separation method. The cells were expanded, established in continuous cell culture, and used for immunofluorescence cytochemistry and establishment of infection models. We detected pan-cytokeratin markers of bovine nasopharyngeal mucosal epithelial cells by immunofluorescence. Bovine nasopharyngeal mucosal epithelial cells were then infected with foot-and-mouth disease virus (FMDV) serum type O. RT-PCR demonstrated the successful establishment of acute FMDV infection in the cell models. This infection model provides the basis for clarification of the interaction between FMDV and host bovine nasopharyngeal mucosal epithelial cells in vitro.

Identification of key genes and pathways involved in response to pain in goat and sheep by transcriptome sequencing

PURPOSE: This aim of this study was to investigate the key genes and pathways involved in the response to pain in goat and sheep by transcriptome sequencing. METHODS: Chronic pain was induced with the injection of the complete Freund's adjuvant (CFA) in sheep and goats. The animals were divided into four groups: CFA-treated sheep, control sheep, CFA-treated goat, and control goat groups (n = 3 in each group). The dorsal root ganglions of these animals were isolated and used for the construction of a cDNA library and transcriptome sequencing. Differentially expressed genes (DEGs) were identified in CFA-induced sheep and goats and gene ontology (GO) enrichment analysis was performed. RESULTS: In total, 1748 and 2441 DEGs were identified in CFA-treated goat and sheep, respectively. The DEGs identified in CFA-treated goats, such as C-C motif chemokine ligand 27 (CCL27), glutamate receptor 2 (GRIA2), and sodium voltage-gated channel alpha subunit 3 (SCN3A), were mainly enriched in GO functions associated with N-methyl-D-aspartate (NMDA) receptor, inflammatory response, and immune response. The DEGs identified in CFA-treated sheep, such as gamma-aminobutyric acid (GABA)-related DEGs (gamma-aminobutyric acid type A receptor gamma 3 subunit [GABRG3], GABRB2, and GABRB1), SCN9A, and transient receptor potential cation channel subfamily V member 1 (TRPV1), were mainly enriched in GO functions related to neuroactive ligand-receptor interaction, NMDA receptor, and defense response. CONCLUSIONS: Our data indicate that NMDA receptor, inflammatory response, and immune response as well as key DEGs such as CCL27, GRIA2, and SCN3A may regulate the process of pain response during chronic pain in goats. Neuroactive ligand-receptor interaction and NMDA receptor as well as GABA-related DEGs, SCN9A, and TRPV1 may modulate the process of response to pain in sheep. These DEGs may serve as drug targets for preventing chronic pain.

[Construction of Fat-1 eukaryotic expression vector of excision markers and the establishment of transgenic sheep cell lines].

In order to establish marker-free transgenic cell lines, we cloned Fat-1 gene, attB and Loxp sequences by PCR. Then we inserted these sequences to pN1-EGFP vector and got pEGFP-N1-Fat-1 expression vector. PhiC31 integrase mRNA which was generated by in vitro transcription and a pEGFP-N1-Fat-1 expression vector co-electroporated into sheep fetal fibroblasts, and then we got transgenic cell lines expressing green fluorescence. Prokaryotic expression and purification of Cre recombinant protein was performed. Cre recombinant protein was transducted into stably-transfected cell colonies. We identified cell colonies by sequencing and established marker-free transgenic cell lines and eventually- established marker-free transgenic cell lines which were building more safely basic for producing Fat-1 transgenic animals.

Isolation, Culture, Differentiation, and Nuclear Reprogramming of Mongolian Sheep Fetal Bone Marrow-Derived Mesenchymal Stem Cells.

We have characterized the differentiation potentiality and the developmental potential of cloned embryos of fetal bone marrow mesenchymal stem cells (BMSCs) isolated from Mongolian sheep. BMSCs were harvested by centrifuging after the explants method and the mononuclear cells obtained were cultured. The isolated BMSCs were uniform, with a fibroblast-like spindle or stellate appearance, and we confirmed expression of OCT4, SOX2, and NANOG genes at passage 3 (P3) by RT-PCR. We measured the growth of the passage 1, 5, and 10 cultures and found exponential growth with a population doubling time of 29.7±0.05 h. We cultured the P3 BMSCs in vitro under inductive environments and were able to induce them to undergo neurogenesis and form cardiomyocytes and adipocytes. Donor cells at passages 3-4 were used for nuclear transfer (NT). We found the BMSCs could be expanded in vitro and used as nuclear donors for somatic cell nuclear transfer (SCNT). Thus, BMSCs are an attractive cell type for large-animal autologous studies and will be valuable material for somatic cell cloning and future transgenic research.

The genome of a Mongolian individual reveals the genetic imprints of Mongolians on modern human populations.

Mongolians have played a significant role in modern human evolution, especially after the rise of Genghis Khan (1162[?]-1227). Although the social cultural impacts of Genghis Khan and the Mongolian population have been well documented, explorations of their genome structure and genetic imprints on other human populations have been lacking. We here present the genome of a Mongolian male individual. The genome was de novo assembled using a total of 130.8-fold genomic data produced from massively parallel whole-genome sequencing. We identified high-confidence variation sets, including 3.7 million single nucleotide polymorphisms (SNPs) and 756,234 short insertions and deletions. Functional SNP analysis predicted that the individual has a pathogenic risk for carnitine deficiency. We located the patrilineal inheritance of the Mongolian genome to the lineage D3a through Y haplogroup analysis and inferred that the individual has a common patrilineal ancestor with Tibeto-Burman populations and is likely to be the progeny of the earliest settlers in East Asia. We finally investigated the genetic imprints of Mongolians on other human populations using different approaches. We found varying degrees of gene flows between Mongolians and populations living in Europe, South/Central Asia, and the Indian subcontinent. The analyses demonstrate that the genetic impacts of Mongolians likely resulted from the expansion of the Mongolian Empire in the 13th century. The genome will be of great help in further explorations of modern human evolution and genetic causes of diseases/traits specific to Mongolians.

Camelid genomes reveal evolution and adaptation to desert environments.

Bactrian camel (Camelus bactrianus), dromedary (Camelus dromedarius) and alpaca (Vicugna pacos) are economically important livestock. Although the Bactrian camel and dromedary are large, typically arid-desert-adapted mammals, alpacas are adapted to plateaus. Here we present high-quality genome sequences of these three species. Our analysis reveals the demographic history of these species since the Tortonian Stage of the Miocene and uncovers a striking correlation between large fluctuations in population size and geological time boundaries. Comparative genomic analysis reveals complex features related to desert adaptations, including fat and water metabolism, stress responses to heat, aridity, intense ultraviolet radiation and choking dust. Transcriptomic analysis of Bactrian camels further reveals unique osmoregulation, osmoprotection and compensatory mechanisms for water reservation underpinned by high blood glucose levels. We hypothesize that these physiological mechanisms represent kidney evolutionary adaptations to the desert environment. This study advances our understanding of camelid evolution and the adaptation of camels to arid-desert environments.

Genome-wide detection of copy number variations among diverse horse breeds by array CGH.

Recent studies have found that copy number variations (CNVs) are widespread in human and animal genomes. CNVs are a significant source of genetic variation, and have been shown to be associated with phenotypic diversity. However, the effect of CNVs on genetic variation in horses is not well understood. In the present study, CNVs in 6 different breeds of mare horses, Mongolia horse, Abaga horse, Hequ horse and Kazakh horse (all plateau breeds) and Debao pony and Thoroughbred, were determined using aCGH. In total, seven hundred CNVs were identified ranging in size from 6.1 Kb to 0.57 Mb across all autosomes, with an average size of 43.08 Kb and a median size of 15.11 Kb. By merging overlapping CNVs, we found a total of three hundred and fifty-three CNV regions (CNVRs). The length of the CNVRs ranged from 6.1 Kb to 1.45 Mb with average and median sizes of 38.49 Kb and 13.1 Kb. Collectively, 13.59 Mb of copy number variation was identified among the horses investigated and accounted for approximately 0.61% of the horse genome sequence. Five hundred and eighteen annotated genes were affected by CNVs, which corresponded to about 2.26% of all horse genes. Through the gene ontology (GO), genetic pathway analysis and comparison of CNV genes among different breeds, we found evidence that CNVs involving 7 genes may be related to the adaptation to severe environment of these plateau horses. This study is the first report of copy number variations in Chinese horses, which indicates that CNVs are ubiquitous in the horse genome and influence many biological processes of the horse. These results will be helpful not only in mapping the horse whole-genome CNVs, but also to further research for the adaption to the high altitude severe environment for plateau horses.