Chromosome-level genome assembly of the Arctic fox (Vulpes lagopus) using PacBio sequencing and Hi-C technology.

The Arctic fox (Vulpes lagopus) is the only fox species occurring in the Arctic and has adapted to its extreme climatic conditions. Currently, the molecular basis of its adaptation to the extreme climate has not been characterized. Here, we applied PacBio sequencing and chromosome structure capture technique to assemble the first V. lagopus genome assembly, which is assembled into chromosome fragments. The genome assembly has a total length of 2.345 Gb with a contig N50 of 31.848 Mb and a scaffold N50 of 131.537 Mb, consisting of 25 pseudochromosomal scaffolds. The V. lagopus genome had approximately 32.33% repeat sequences. In total, 21,278 protein-coding genes were predicted, of which 99.14% were functionally annotated. Compared with 12 other mammals, V. lagopus was most closely related to V. Vulpes with an estimated divergence time of ~7.1 Ma. The expanded gene families and positively selected genes potentially play roles in the adaptation of V. lagopus to Arctic extreme environment. This high-quality assembled genome will not only promote future studies of genetic diversity and evolution in foxes and other canids but also provide important resources for conservation of Arctic species.

[Activity and transcriptional regulatory elements of the promoter in Arctic fox (Vulpes lagopus) ß-defensin103 gene].

The aim of this study was to screen the active regions and transcription factor binding sites in the promoter of the CBD103 gene related to Arctic fox coat color, and to provide a basis for revealing the molecular genetic mechanism of CBD103 gene regulating the coat color formation. The 5'-flanking region fragment 2 123 bp of Arctic fox CBD103 gene was cloned, and 4 truncated promoter reporter vectors of different lengths were constructed. The promoter activity was detected by the dual-luciferase reporter assay system. Point mutations were performed on the 3 predicted specificity protein 1 (Sp1) transcription factor binding sites in the highest promoter active region, and 3 mutant vectors were constructed. The activity was then detected by the dual-luciferase reporter assay system. The results showed that the region 1 656 (-1 604/+51) had the highest activity in the 4 truncated promoters of different lengths, and the promoter activity of the three mutant vectors constructed in this region were significantly lower than that of the wild type (fragment 1 656). The region of -1 604 /+51 was the core promoter region of CBD103 gene in Arctic fox and -1 552/-1 564, -1 439/-1 454 and -329/-339 regions were positive regulatory regions. This study successfully obtained the core promoter region and positive regulation regions of the Arctic fox CBD103 gene, which laid a foundation for further study on the molecular genetic mechanism of this gene regulating Arctic fox coat color.

Genome-wide differential expression of long noncoding RNAs and mRNAs in ovarian follicles of two different chicken breeds.

Bashang long-tail chickens are an indigenous breed with dual purpose in China (meat and eggs) but have low egg laying performance. To improve the low egg laying performance, a genome-wide analysis of mRNAs and long noncoding RNAs (lncRNAs) from Bashang long-tail chickens and Hy-Line brown layers was performed. A total of 16,354 mRNAs and 8691 lncRNAs were obtained from ovarian follicles. Between the breeds, 160 mRNAs and 550 lncRNAs were found to be significantly differentially expressed. Integrated network analysis suggested some differentially expressed genes were involved in ovarian follicular development through oocyte meiosis, progesterone-mediated oocyte maturation, and cell cycle. The impact of lncRNAs on cis and trans target genes, indicating some lncRNAs may play important roles in ovarian follicular development. The current results provided a catalog of chicken ovarian follicular lncRNAs and genes for further study to understand their roles in regulation of egg laying performance.

Improved Cytotoxic T Lymphocyte Responses to Vaccination with Porcine Reproductive and Respiratory Syndrome Virus in 4-1BB Transgenic Pigs.

Vaccination is the most reliable measure to prevent infectious diseases in domestic animals. Development of novel vaccines demands extensive studies with new technologies, such as using novel adjuvants and immunomodulatory molecules. The co-stimulatory molecule 4-1BB provides a key signal that directs the fate of T cells during activation, and thus is important to their function in immune protection. To determine whether host immune responses to viral infection could be promoted by enhancing 4-1BB co-stimulation, in this study, we produced transgenic pig clones expressing an extra copy of the 4-1BB gene by clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9-mediated homologous recombination at the Rosa26 locus. The immune responses of transgenic pigs to porcine reproductive and respiratory syndrome virus (PRRSV) vaccine were determined on day 14. We show that peripheral blood lymphocytes of transgenic pigs expressed around twice the level of 4-1BB mRNA than those of control pigs. We also found IL-2, TNF-α, and granzyme B mRNA levels as well as PRRSV-specific IFN-γ response were significantly upregulated in 4-1BB transgenic pigs, leading to more efficient cytotoxic T lymphocyte (CTL) killing, whereas the expressions of IL-4, IL-17, and Foxp3 were not affected. These results indicate that higher levels of 4-1BB expression involve in promoting Th1 differentiation and enhancing specific CTL responses to PRRSV, and provide a novel approach to increase the efficacy of current vaccines to control the infectious diseases.

Genetic variations of the coding region of the melanocortin receptor 1 (MC1R) gene in the fox.

BACKGROUND: The melanocortin 1 receptor (MC1R) gene plays an important role in the control of coat colour in mammals. Genetic variation of the MC1R gene and the relationship between genotype and coat colour are not well understood. Studies in the fox may improve our understanding of gene influence on coat colour in dogs and cats. HYPOTHESIS/OBJECTIVES: To investigate coat colour associated mutations in the coding region of MC1R gene in foxes. ANIMALS: A total of 118 foxes, comprising 70 red foxes (Vulpes vulpes) (19 red, 10 white silver, 29 silver and 12 chocolate foxes) and 48 arctic foxes (Vulpes lagopus) (9 dominant white blue foxes and 39 normal blue foxes) were included in the study. METHODS: Evaluation of the DNA sequence of the coding region of MC1R gene and its polymorphisms. RESULTS: Eight polymorphic sites (single nucleotide polymorphisms, SNPs) distributed throughout the 954-bp coding region of the fox MC1R gene were detected. Among them, c.13G>T, c.124A>G, c.289G>A, c.373T>C and c.839 T>G were mis-sense mutations, which resulted in codon change of p.G5C, p.N42D, p.V97I, p.C125R and p.F280C, respectively. Mutation and haplotype analysis indicated that c.373T>C was associated with black and brown pigmented phenotypes in foxes, and c.13G>T and c.839T>G were important in distinguishing V. lagopus and V. vulpes. CONCLUSIONS AND CLINICAL IMPORTANCE: SNP c.373T>C in the coding region of the MC1R gene is probably associated with the brown phenotype of chocolate foxes.

Gene silencing of porcine MUC13 and ITGB5: candidate genes towards Escherichia coli F4ac adhesion.

BACKGROUND: Integrin beta-5 (ITGB5) and mucin 13 (MUC13) genes are highly expressed on the apical surface of intestinal epithelia and are thought to be candidate genes for controlling the expression of the receptor for enterotoxigenic Escherichia coli (ETEC) F4ac. Human MUC13 protein has an expected role in protecting intestinal mucosal surfaces and porcine ITGB5 is a newly identified potential receptor for ETEC F4ac. METHODOLOGY/PRINCIPAL FINDINGS: To test the hypothesis that ITGB5 and MUC13 both play key roles in protection of the intestinal mucosa against pathogenic bacterium, porcine intestinal epithelial cells (IPEC-J2) were transfected with ITGB5-targeting, MUC13-targeting or negative control small interfering RNA (siRNA), respectively. Firstly, we measured mRNA expression levels of mucin genes (MUC4, MUC20), pro-inflammatory genes (IL8, IL1A, IL6, CXCL2), anti-inflammatory mediator SLPI, and PLAU after RNAi treatments with and without ETEC infection. Secondly, we compared the adhesions of ETEC to the pre- and post-knockdown IPEC-J2 cells of ITGB5 and MUC13, respectively. We found that ITGB5 and MUC13 knockdown both had small but significant effects in attenuating the inflammation induced by ETEC infection, and both increased bacterial adhesion in response to F4ac ETEC exposure. CONCLUSIONS/SIGNIFICANCE: Our current study first reported that ITGB5 and MUC13 are important adhesion molecules of mucosal epithelial signaling in response to Escherichia coli in pigs. These data suggest that both ITGB5 and MUC13 play key roles in defending the attachment and adhesion of ETEC to porcine jejunal cells and in maintaining epithelial barrier and immunity function.

Molecular cloning and protein characterization of swine 4-1BB.

4-1BB is expressed on activated T cells and other immune and non-immune cells. It plays important roles in human and mouse T cell function. However, the swine 4-1BB sequence remains unknown and its role in swine T cell response has not been studied. In the present study, we for the first time described the cloning of the swine 4-1BB gene and the property of the protein. Two 4-1BB variants were detected in swine. The coding sequences of variant 1 and variant 2 were 768 and 726 nucleotides in length, respectively, and both variants were coded by 7 exons in the swine genome. Comparison of nucleotide and amino acid sequences showed that both swine 4-1BB variants were more closely related to bovine and human sequences than to either the mouse or rat sequence. Prediction analysis showed that swine 4-1BB belonged to the tumor necrosis factor receptor (TNFR) superfamily like human and mouse 4-1BB and the tertiary structures of the swine 4-1BB variants were much more similar to mouse 4-1BB than to human 4-1BB. The 1556bp 5' regulatory sequence cloned by nested PCR efficiently induced green fluorescent protein expression in porcine peripheral blood mononuclear cells (PBMC) post nucleofection. Moreover, 4-1BB protein was widely expressed in pig tissues and both variants of swine 4-1BB protein were transmembrane proteins and expressed on the membrane of porcine PBMCs.

Differential gene expression profiling of porcine epithelial cells infected with three enterotoxigenic Escherichia coli strains.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is one of the most important pathogenic bacteria causing severe diarrhoea in human and pigs. In ETEC strains, the fimbrial types F4 and F18 are commonly found differently colonized within the small intestine and cause huge economic losses in the swine industry annually worldwide. To address the underlying mechanism, we performed a transcriptome study of porcine intestinal epithelial cells (IPEC-J2) with and without infection of three representative ETEC strains. RESULTS: A total 2443, 3493 and 867 differentially expressed genes were found in IPEC-J2 cells infected with F4ab ETEC (C(F4ab)), with F4ac ETEC (C(F4ac)) and with F18ac ETEC (C(F18ac)) compared to the cells without infection (control), respectively. The number of differentially expressed genes between C(F4ab) and C(F4ac), C(F4ab) and C(F18ac), and C(F4ac) and C(F18ac) were 77, 1446 and 1629, respectively. The gene ontology and pathway analysis showed that the differentially expressed genes in C(F4ab)vs control are significantly involved in cell-cycle progress and amino acid metabolism, while the clustered terms of the differentially expressed genes in C(F4ac)vs control comprise immune, inflammation and wounding response and apoptosis as well as cell cycle progress and proteolysis. Differentially expressed genes between C(F18ac)vs control are mainly involved in cell-cycle progression and immune response. Furthermore, fundamental differences were observed in expression levels of immune-related genes among the three ETEC treatments, especially for the important pro-inflammatory molecules, including IL-6, IL-8, TNF-α, CCL20, CXCL2 etc. CONCLUSIONS: The discovery in this study provides insights into the interaction of porcine intestinal epithelial cells with F4 ETECs and F18 ETEC, respectively. The genes induced by ETECs with F4 versus F18 fimbriae suggest why ETEC with F4 may be more virulent compared to F18 which seems to elicit milder effects.

High transfection efficiency of porcine peripheral blood T cells via nucleofection.

Transgenic technology is an effective approach to assess the roles of specific genes in the activation and differentiation of T cells and modify T cell qualities. However, porcine T cell transfection is poorly documented. Here, we developed a non-virus-based method for the transfection of resting and ConA-stimulated porcine peripheral blood T cells using "Nucleofection" gene transfer technology; both plasmid DNA- and mRNA-mediated nucleofection systems were developed. The results demonstrated for the first time that plasmid DNA encoding green fluorescent protein (GFP) and in vitro transcribed GFP mRNA could be delivered efficiently into resting and activated porcine T cells. For both methods, the onset of gene expression was rapid and occurred within 2h post-nucleofection. Optimised plasmid DNA-mediated nucleofection induced approximately 40% transgene expression with 51% cell viability in resting T cells and approximately 20% transgene expression with 53% cell viability in activated T cells at 24h post-gene delivery. However, optimised mRNA-based nucleofection resulted in higher transfection efficiencies and cell viability, with more than 50% transgene expression and 62% viability for resting T cells and approximately 40% transgene expression and 59% viability for activated T cells. Finally, we measured the impact of the developed nucleofection systems on T cell function by detecting the mRNA levels of the activation markers CD25, CD69 and the cytokine IFN-γ; cell proliferation of the nucleofected resting peripheral blood mononuclear cells (PBMC) after ConA stimulation was also examined. The nucleofected resting PBMCs proliferated normally and up-regulated CD25, CD69 and IFN-γ mRNA expression levels in a manner comparable to non-nucleofected cells. These results indicate that the developed nucleofection systems have no adverse effects on T cell function and can be utilised in swine immunological research.

Study on tandem repeat sequence variation in sheep mtDNA D-loop region.

The 75-nt-long tandem repeat sequence in the control region of mtDNA of 77 individuals, of which 69 were from different indigenous sheep breeds in China and 8 were from imported breeds, was sequenced and analyzed to investigate the origin and differentiation of Chinese indigenous sheep breeds and also the genetic diversities and relationships among them. A total of 28 variable sites were detected within 309 repeated sequences, among which 7 sites were singleton variable sites with two variants, 1 site was a singleton variable site with three variants, and 20 sites were parsimony informative sites with two variants. A total of 63 haplotypes were sorted from 28 polymorphic sites, among which two main and basic haplotypes, namely, Hap 1 and Hap 3 were present at a much higher proportion, at 12.94% and 30.42%, respectively. It could be inferred that Chinese indigenous sheep breeds originated from two maternal ancestors because of the maternal inheritance characteristics of the mtDNA. Altay sheep and Kazakstan sheep are closely related and do not differentiate significantly. Mongolian sheep and Ujumuqin sheep also share a close relationship. Tibetan sheep, Mongolian sheep, and Ujumuqin sheep have lower genetic diversity than Altay sheep and Kazakstan sheep.

SNP identification and analysis in part of intron 2 of goat MSTN gene and variation within and among species.

Part of intron 2 of the myostatin (MSTN) gene of 140 goats from 24 populations and 38 sheep from 8 breeds were sequenced, and similar sequences of different species from Gene bank were also obtained to study MSTN diversity within and among species. The results indicated that there were seven polymorphic sites in the sequenced region of goat, which have not been separated by recombination (or recurrent mutation), presented complete linkage disequilibrium, and could be sorted into three haplotypes. There was no polymorphic site in the sequenced region of sheep. The haplotype diversity, nucleotide diversity, and average number of single nucleotide polymorphism (SNP) differences of goats from the South group are higher than those of North group, and the corresponding value of the Foreign group is also higher than that of Chinese. The genetic differentiation (0.7558) between the Foreign and Chinese group is significant. There are two main haplotypes of the MSTN intron 2 in the goat, which may represent two ancestral types, in support of the theory that domestic goats in the world mainly originated from two ancestors based on morphology, history, archaeology, and molecular markers. The sequence differences of the MSTN intron 2 among species are greater than those within species.

[Study on mtDNA D-loop of Chinese main indigenous sheep breeds using PCR-RFLP].

The polymorphism of mtDNA D-loop of 83 individuals from 9 Chinese indigenous sheep breeds and 2 imported sheep breeds were studied with 5 endonucleases, Hinf I, Msp I, Sau3A I, Xsp I and Taq I, using PCR-RFLP. The results indicated that there existed two basic haplotypes in the region of mtDNA D-loop. It could be inferred that Chinese indigenous sheep breeds originated from two maternal ancestors. The averaged polymorphic degree (Pi value=0.0421%) of mtDNA D-loop showed that the genetic diversity of mtDNA of Chinese indigenous sheep breeds was very poor.

Study on polymorphisms of microsatellites DNA of six Chinese indigenous sheep breeds.

The polymorphisms of 17 microsatellites loci of six indigenous sheep breeds, including Mongolian sheep, Ujumuqin sheep, Kazakstan sheep, Aletai sheep and Tibetan sheep, were studied using polypropylene gel electrophoresis in order to investigate their genetic diversity,origin, differentiation and relationships. The results indicated that there was a significant difference in genetic diversity between different loci (P < 0.01). There was no significant difference in PIC, Fis and observed heterozygosity (Obs. Het) among populations (P > 0.05), but a significant difference in gene diversity and expected heterozygosity (Exp. Het) (P < 0.05). Chinese indigenous sheep breeds had similar genetic diversity as those from Europe,but with higher inbreeding coefficient. It could be inferred from the cluster of individuals and populations that Chinese indigenous sheep breeds might originate from two ancestors. The cluster of populations showed that Mongolian sheep and Ujumuqin sheep had close relationship and did not differentiate obviously. Mongolian sheep and Tibetan sheep had far relationship and differentiated significantly. Aletai sheep differentiated from Kazakstan sheep but not significantly. Tan sheep,Aletai sheep and Tibetan sheep also had close relationships. The Fst of Chinese indigenous sheep breeds was close to some Spanish sheep breeds,but much smaller than that of other European sheep breeds. More loci and samples should be studied in the future in order to obtain more accurate results.

Study on SNP of melanocyte stimulating hormone receptor gene in several Chinese indigenous sheep breeds using DHPLC.

Single nucleotide polymorphism (SNP) of melanocyte stimulating hormone receptor (MSHR) gene was studied by sequencing and denaturing high performance liquid chromatography (DHPLC) in Mongolian sheep, Kazakstan sheep, Tan sheep and Tibetan sheep. The results showed that there is a mutation at position 317 (T317C) within the length of 415bp and the DHPLC is a high-throughput and simple method for screening this mutation. The heterozygote (TC) with shoulder peak could be detected quickly at the first time of DHPLC, and two homozygotes (TT or CC) could be discriminated easily through two times of DHPLC when each homozygous DNA was mixed with a known homozygous reference sample at the second time of DHPLC. All of the populations for this site are in Hardy-Weinberg equilibrium. The results also indicated that Mongolian sheep and Kazakstan sheep had close relationship, Tibetan sheep and Tan sheep had close relationship. The relationship among breeds was consistent with that of microsatellite DNA.

[Inheritance of head and neck color and black hoof of Boer goat].

The inheritance of head and neck color and black hoof of Boer goats and their offspring were analyzed by observing,breeding data and statistic test in the Crossbreeding and Improving Research Center of Boer goat in Qinhuangdao city. The results indicated that the color of head and neck and black hoof of Boer goat were all controlled respectively by two linked recessive genes on one autosome. The rate of crossing-over between the genes of head and neck color and black hoof was 7.5 genetic units.

[Studies of random amplified polymorphic DNA(RAPD) of main indigenous sheep breeds in China].

The genetic polymorphism and relationship of 7 indigenous sheep breeds of China and 3 imported sheep breeds were studied using random amplified polymorphic DNA (RAPD). The results indicated that the RAPD was an effective marker for the analysis of genetic relationship among sheep breeds. Among 43 arbitrary primers,35 were polymorphic. The percentage of polymorphic markers was 66.24%,which indicated that the RAPD had higher efficiency of polymorphism detection and sensitivity in studying the genetic variation among sheep breeds. The average index of genetic polymorphism for whole population (Hsp) was 0.9139,which showed that the genetic polymorphism was abundant between sheep populations. The genetic relationship between different indigenous sheep breeds in China was in accord with their localities,the results from archeology and cytogenetics and the genetic relationship between imported sheep breeds was in accord with their breeding history.