Guobin Chang

Migration and accumulation of primordial germ cells of early embryo in quail.

In this research, we have identified primordial germ cells (PGCs) in quail embryo using Quail Hemangioblastic Lineage (QH1) monoclonal antibody analysis. Quail PGCs originated from the opaca of unincubated blastodisc, and then transferred to the pellucida and the germinal crescent. At 27 hours post-incubation, a few PGCs first appeared in blood vessels of the pellucida, where many PGCs accumulated at 36 hours post-incubation. The PGCs scattered or clustered from head to omphalo mesenteric and mainly settled down in the mesenchymal blood vessels of head at 45 hours post-incubation. The size of PGCs population increased significantly (P<0.05) from stage XII (12.8±4.82 µm) to primitive streak stage (106.7±8.74 µm) and from Head process stage (95.8±19.74 µm) to tenth somite stage (199.4±19.97 µm). It is concluded that the PGCs scattered in the head area before migration to the germinal crescent and distributed randomly in both gland. The number of PGCs varied at different stages with two peaks, primitive streak stage (18 hours post-incubation) and tenth somite (36 hours post-incubation).

The analysis of genetic diversity and differentiation of six Chinese cattle populations using microsatellite markers.

A total of 321 individuals from six cattle populations of four species in a bovine subfamily in China were studied using 12 pairs of microsatellite markers. The genetic diversities within and between populations were calculated. The phylogenetic trees were constructed by (delta mu)(2) and DA distances, and the divergence times between populations were estimated by (delta mu)(2). Altogether, 144 microsatellite alleles were detected including 24 private alleles and nine shared alleles. Chinese Holstein had the largest number of private alleles (10), whereas, Bohai black and Buffalo had the smallest number of private alleles (2). Chinese Holstein showed the highest genetic variability. Its observed number of alleles (Na), mean effective number of alleles (MNA), and mean heterozygosity (He) were 7.7500, 4.9722, and 0.7719, respectively, whereas, the Buffalo and Yak showed low genetic variability. In the phylogenetic trees, Luxi and Holstein grouped first, followed by Bohai and Minnan. Yak branched next and buffalo emerged as the most divergent population from other cattle populations. Luxi and Bohai were estimated to have diverged 0.039-0.105 million years ago (MYA), however, buffalo and Holstein diverged 0.501-1.337 MYA. The divergence time of Yak versus Minnan, Holstein and buffalo was 0.136-0.363, 0.273-0.729, and 0.326-0.600 MYA, respectively.

Cloning of the Quail PIWI Gene and Characterization of PIWI Binding to Small RNAs

The PIWI protein regulates gene expression at the epigenetic and post-transcriptional level with a variety of endogenous small non-coding RNAs. In poultry, the biological function of the PIWI protein and PIWI binding to small RNAs had not been determined. The present study cloned and analyzed the sequences of the PIWIL1 protein. We also characterized PIWIL1 binding to small RNAs from adult quail testis, where the PIWIL1 protein is specifically expressed. Small RNAs showed a strong peak at 24–27 nt in the testicular RNA library, mapped primarily to repeat sequences and were similar to rasiRNAs. MicroRNAs (miRNAs) were abundant in the ovarian RNA library at a peak of 22 nt.

Identification and Expression Analysis of the Interferon-Induced Protein with Tetratricopeptide Repeats 5 (IFIT5) Gene in Duck (Anas platyrhynchos domesticus)

The interferon-induced proteins with tetratricopeptide repeats (IFITs) protein family mediates antiviral effects by inhibiting translation initiation, cell proliferation, and migration in the interferon (IFN) dependent innate immune system. Several members of this family, including IFIT1, IFIT2, IFIT3 and IFIT5, have been heavily studied in mammals. Avian species contain only one family member, IFIT5, and little is known about the role of this protein in birds. In this study, duck IFIT5 (duIFIT5) full-length mRNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE). Based on the sequence obtained, we performed a series of bioinformatics analyses, and found that duIFIT5 was most similar to homologs in other avian species. Also, duIFIT5 contained eight conserved TPR motifs and two conserved multi-domains (TPR_11 and TPR_12). Finally, we used duck hepatitis virus type 1 (DHV-1) and polyriboinosinicpolyribocytidylic acid (poly (I:C)) as a pathogen or a pathogen-associated molecular pattern induction to infect three-day-old domestic ducklings. The liver and spleen were collected to detect the change in duIFIT5 transcript level upon infection by quantitative real-time PCR (qRT-PCR). DuIFIT5 expression rapidly increased after DHV-1 infection and maintained a high level, while the transcripts of duIFIT5 peaked at 8h after poly (I:C) infection and then returned to normal. Taken together, these results provide a greater understanding of avian IFIT5.

Developmental research on the origin and phylogeny of quails

Around the world, there are 20 types of wild and about 70 domestic quail breeds or strains, including laboratory and commercial quail. Although all domestic quails were derived from wild strains, many obvious differences are evident today. However, how these differences occurred and which wild population was the first to be domesticated, remains unclear. This paper systematically presents the history of the development of domestic quail in China from 770 B.C. to the end of the 20 century. Taking into account recent research on some structural loci of domestic and wild quail, and in the light of recent survey reports of the present general situation of these birds, particularly in respect of their ecological performance and differences between wild and domestic quail, this review puts forward a new thesis for resolving the current uncertainty about the origin of domestic quail. It is suggested that unlike those of Japanese origin, Chinese quail are probably the earlier and more direct ancestor of most kinds of the domestic quail found around the world. Moreover, the review analyzes the possible evolutionary path to domestic quail, which is mainly a result of the flow of people from Japan to China. On the assumption that more and more wild quail populations are endangered, it aims to provide a basis for renewing knowledge of wild quail resources and supporting the protection and use of these valuable worldwide stocks. This is especially important in China, the last country in the world to have so many wild quail populations. Furthermore, this new insight can promote and assist the world commercial quail industry to develop and flourish.

A mutation in the NLRC5 promoter limits NF-κB signaling after Salmonella Enteritidis infection in the spleen of young chickens.

To date, the functions of the NLRC5 in chickens remain undefined. In the current study, chicken NLRC5 was cloned and an A1017G mutation was detected in its promoter region. The relative expression levels of the NLRC5 and key NF-κB pathway genes, IKKα, IKKβ, NF-κB, IL-6, IL-1β and IFN-γ, in the spleens of wild and mutant type birds, AA and GG, were determined using FQ-PCR at 7 day post-infection (DPI) with Salmonella Enteritidis. Additionally, the bacterial burden in the caecum and various immune response parameters were measured to evaluate immune responses. All of the examined immune response parameters were significantly different between the AA chickens and the GG chickens. Specifically, the mRNA expression levels of IKKα, NF-κB, IL-6, IL-1β and IFN-γ were higher in AA chickens than those in GG chickens, while the mRNA expression levels of NLRC5 were lower in AA chickens than those in GG chickens (P<0.05). Moreover, the mRNA expression levels of TLR4 and MyD88 were not affected in either group. Collectively, considering former NLRC5 functional study in vitro, the wild genotype birds presented with better resistance to Salmonella Enteritidis through the actions of the NLRC5 and subsequent inhibition of the NF-κB pathway in chickens.

Duck RIG-I CARD Domain Induces the Chicken IFN-β by Activating NF-κB

Retinoic acid-inducible gene I- (RIG-I-) like receptors (RLRs) have recently been identified as cytoplasmic sensors for viral RNA. RIG-I, a member of RLRs family, plays an important role in innate immunity. Although previous investigations have proved that RIG-I is absent in chickens, it remains largely unknown whether the chicken can respond to RIG-I ligand. In this study, the eukaryotic expression vectors encoding duRIG-I full length (duck RIG-I, containing all domains), duRIG-I N-terminal (containing the two caspase activation and recruitment domain, CARDs), and duRIG-I C-terminal (containing helicase and regulatory domains) labeled with 6∗His tags were constructed successfully and detected by western blotting. Luciferase reporter assay and enzyme-linked immunosorbent assay (ELISA) detected the duRIG-I significantly activated NF-κB and induced the expression of IFN-β when polyinosinic-polycytidylic acid (poly[I:C], synthetic double-stranded RNA) challenges chicken embryonic fibroblasts cells (DF1 cells), while the duRIG-I was inactive in the absence of poly[I:C]. Further analysis revealed that the CARDs (duRIG-I-N) induced IFN-β production regardless of the presence of poly[I:C], while the CARD-lacking duRIG-I (duRIG-I-C) was not capable of activating downstream signals. These results indicate that duRIG-I CARD domain plays an important role in the induction of IFN-β and provide a basis for further studying the function of RIG-I in avian innate immunity.

Cloning, characterization and widespread expression analysis of testicular piRNA-like chicken RNAs

Piwi-interacting RNAs (piRNAs) are small RNAs abundant in the germline that have been implicated in germline development and maintenance of genomic integrity across several animal species including human, mouse, rat, zebrafish and drosophila. Tens of thousands of piRNAs have been discovered, yet abundant piRNAs have still not been detected in various eukaryotic organisms. This is a report on the characterization, cloning and expression profiling of piRNA-like chicken RNAs. Here, we identified 19 piRNAs, each 23–39 nucleotides long, from chicken testis using a small RNA cDNA library and T-A cloning methods. Three different pilRNAs were selected according to size, homology and secondary structure for temporal and spatial expression by Q-PCR technology in different tissues at five growth and four development stages of Chinese indigenous Rugao chickens (RG) and introduced recessive white feather chickens (RW). We found that, consistent to other organisms, pilRNA-encoding sequences within the chicken genome were asymmetrically distributed on the chromosomes while displaying a preference for intergenic regions across the genome. Interestingly, unlike miRNAs with unique stem-loop structures (mature miRNAs form stem section and the rest form loop section), distinct secondary structures of pilRNAs were predicted. In addition, chicken pilRNAs were not only abundant in the germline but also existed in somatic tissues, where, expression levels were influenced mainly by different pilRNAs, breed and gender. Taken together, our results suggest that two distinct secondary structures exist between pilRNAs and miRNAs, which may clarify the splicing and processing mechanisms of the two small RNAs are possible different. Moreover, our results suggest that pilRNAs may not only be confined to development and maintenance of the germline but may also play important roles in somatic tissues. Additionally, different pilRNAs may be involved in the unique regulatory machinery of complex biological processes.

Behavior differentiation between wild Japanese quail, domestic quail, and their first filial generation

The number of wild quail has dramatically reduced in China and reached a state of endangerment with the deterioration of the environment in recent years. In this study, we examined the ecological behaviors of quails in the cage to determine the differentiation level between wild Japanese quail and domestic quail, to detect the relationship between quail behavior and evolutionary differentiation and to analyze the possibility of restoring effective size of wild population. With the on-the-spot observations and measurements, the behaviors of 3 categories of quail, namely wild Japanese quail from the Weishan Lake area in China, domestic quail, and their first filial generation (F(1)) were studied. Domestic quail differed from wild Japanese quail in morphological pattern and ecological behaviors, including some indexes of figure type and egg, vocalization, aggression and fighting, and mating, but wild Japanese quail and domestic quail could succeed in mating and reproducing fertile hybrid offspring. There were significant differences between domestic quail and wild Japanese quail in reproductive traits, involved mating times, fertility rate, hatching rate, and hatching rate of fertilized eggs (P < 0.05). The first filial generation presented significant difference from the wild Japanese quail in vocalization, aggression and fighting, mating, hatching rate, hatching rate of fertilized eggs, and some egg indexes (P < 0.05) and significantly differ from the domestic quail in vocalization, hatching rate, and hatching rate of fertilized eggs (P < 0.05). Evolutionary differentiation between wild quail and domestic quail was still at a relatively low level because no reproductive isolation existed. The advantages of the F(1) hybrids in reproductive capacity, fertilization, and hatching recommend that releasing hybrids instead of domestic quails to the wild would be a more effective way to restore the effective size of wild quail population if necessary.

Discovery of novel long non-coding RNAs induced by subgroup J avian leukosis virus infection in chicken

Abstract Avian leukosis virus subgroup J (ALV‐J) is an avian oncogenic retrovirus that has led to severe economic losses in the poultry industry in China in recent decades. Here, using high throughput transcriptome sequencing of HD11 and CEF cells infected with ALV‐J, a set of 4804 novel long non‐coding transcripts and numerous differentially expressed long non‐coding RNAs (lncRNAs) were identified. We also found that they share relatively shorter transcripts and fewer exon numbers compared to mRNA. Correlation analysis suggested that many lncRNAs may activate gene expression in an enhancer‐like manner other than through transcriptional regulation. Expression level analyses in vivo showed that three lncRNAs (NONGGAT001975.2, NONGGAT005832.2 and NONGGAT009792.2) may be associated with immune response regulation and could function as novel biomarkers for ALV‐J infection. Our findings provides new insight into the pathological process of ALV‐J infection and should serve as a high‐quality resource for further research on epigenetic influences on disease‐resistance breeding as well as immune system and genomic studies. HighlightsCatalog of lncRNAs during ALV‐J infection in both somatic and immune cells were provided.Functional differences in noncoding RNA regulation between them characterized for the first time.Chicken lncRNAs are shorter, have fewer exons and shorter exons than protein‐coding genes.Many lncRNAs may activate gene expression in an enhancer‐like manner other than transcriptional regulation.We found and validated three lncRNAs that could function as novel biomarkers for ALV‐J infection.