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Xue Q.,Yangzhou University | Xue Q.,Key Laboratory for Animal Genetics | Zhang G.,Yangzhou University | Zhang G.,Key Laboratory for Animal Genetics | And 8 more authors.
PLoS ONE | Year: 2017

The early growth pattern, especially the age of peak growth, of broilers affects the time to market and slaughter weight, which in turn affect the profitability of the poultry industry. However, the underlying mechanisms regulating chicken growth and development have rarely been studied. This study aimed to identify candidate genes involved in chicken growth and investigated the potential regulatory mechanisms of early growth in chicken. RNA sequencing was applied to compare the transcriptomes of chicken muscle tissues at three developmental stages during early growth. In total, 978 differentially expressed genes (DEGs) (fold change ≥ 2; false discovery rate < 0.05) were detected by pairwise comparison. Functional analysis showed that the DEGs are mainly involved in the processes of cell growth, muscle development, and cellular activities (such as junction, migration, assembly, differentiation, and proliferation). Many of the DEGs are well known to be related to chicken growth, such as MYOD1, GH, IGF2BP2, IGFBP3, SMYD1, CEBPB, FGF2, and IGFBP5. KEGG pathway analysis identified that the DEGs were significantly enriched in five pathways (P < 0.1) related to growth and development: extracellular matrix-receptor interaction, focal adhesion, tight junction, insulin signaling pathway, and regulation of the actin cytoskeleton. A total of 42 DEGs assigned to these pathways are potential candidate genes inducing the difference in growth among the three developmental stages, such as MYH10, FGF2, FGF16, FN1, CFL2, MAPK9, IRS1, PHKA1, PHKB, and PHKG1. Thus, our study identified a series of genes and several pathways that may participate in the regulation of early growth in chicken. These results should serve as an important resource revealing the molecular basis of chicken growth and development. © 2017 Xue et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Zhang Y.,Yangzhou University | Zhang Y.,Key Laboratory for Animal Genetics | Wang Y.,Yangzhou University | Wang Y.,Key Laboratory for Animal Genetics | And 20 more authors.
PLoS ONE | Year: 2017

An efficient genome editing approach had been established to construct the stable transgenic cell lines in the domestic chicken (Gallus gallus domesticus) at present. Our objectives were to investigate gene function in the differentiation process of chicken embryonic stem cells (ESCs) into spermatogonial stem cells(SSCs). Three guides RNA (gRNAs) were designed to knockout the Stra8 gene, and knockout efficiency was evaluated in domestic chicken cells using cleavage activity of in vitro transcription of gRNA, Luciferase-SSA assay, T7 endonuclease I assay(T7E1) and TA clone sequence. In addition, the Cas9/ gRNA plasmid was transfected into ESCs to confirm the function of Stra8. SSA assay results showed that luciferase activity of the vector expressing gRNA-1 and gRNA- 2 was higher than that of gRNA-3. TA clone sequencing showed that the knockdown efficiency was 25% (10/40) in DF-1 cells, the knockdown efficiency was 23% (9/40) in chicken ESCs. T7E1 assay indicated that there were cleavage activity for three individuals, and the knockdown efficiency was 12% (3/25). Cell morphology, qRT-PCR, immunostaining and FCS indicated that Cas9/gRNA not only resulted in the knockout of Stra8 gene, but also suggested that the generation of SSCs was blocked by the Stra8 gene knockdown in vitro. Taken together, our results indicate that the CRISPR/Cas9 system could mediate stable Stra8 gene knockdown in domestic chicken's cells and inhibit ECSs differentiation into SSCs. Copyright: © 2017 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Wei Y.,Yangzhou University | Wei Y.,Key Laboratory for Animal Genetics | Wei Y.,Jiangxi Academy of Agricultural science | Zhang G.X.,Yangzhou University | And 11 more authors.
Genetics and Molecular Research | Year: 2016

The growth trait is important in poultry production. We analyzed the association between single nucleotide polymorphisms (SNPs) in the Myf5 and MyoG gene and Bian chicken growth traits. SNPs in candidate genes of the Bian chickens were detected by the polymerase chain reaction-single strand conformation polymorphism method. Two mutation loci and six genotypes were identified in each candidate gene. In terms of growth traits, least square analysis showed that the FF genotype of the MyoG was the advantage genotype and the IJ genotype of the Myf5 was the disadvantage genotype for growth trait in Bian chicken. Correlation analysis suggested that the different combination genotypes between Myf5 and MyoG genes had a significant effect on growth traits in Bian chickens. The result suggested that MyoG and Myf5 genes can be used in marker-assisted selection for improving the growth trait in Bian chicken. © 2016 The Authors.


Zhang T.,Yangzhou University | Zhang T.,Key Laboratory for Animal Genetics | Zhang G.X.,Yangzhou University | Zhang G.X.,Key Laboratory for Animal Genetics | And 13 more authors.
Genetics and Molecular Research | Year: 2015

The gonadotropin-releasing hormone (GnRH) plays an important role in the control of reproductive functions. Recent studies have reported the occurrence of GnRH molecular variants in numerous species. In this study, the GnRH1 gene from Jinghai yellow chicken was cloned by reverse transcriptase-polymerase chain reaction and transformed into BL21 (DE3) competent cells. The GnRH1 gene and amino acid sequences were subjected to bioinformatic analyses. The GnRH1 gene nucleotide sequence was discovered to be 352 bp long, containing a coding, promoter, and section of the 3'-regions. The GnRH1 gene shared 93, 81, 54, 58, 61, 76, 76, 59, 76, and 66% sequence identity with Meleagris gallopavo, Columba livia, Homo sapiens, Bos taurus, swines, Capra hircus, Ovis aries, Pantholops hodgsonii, Equus caballus, and Rattus norvegicus, respectively. The GnRH1 gene showed conserved domains. The GnRH1 protein was a secreted protein comprising 92 amino acids, with a molecular weight of 10205.6 Da and a theoretical pI of 5.67. Most of the amino acid residues were observed to be hydrophilic, indicating water solubility. The predicted secondary structures of proteins included α-helices (h; 23.08%), β-extensions (e; 10.92%), and random coils (c; 66.0%). The successful construction of prokaryotic expression vector pET32a-GnRH1 was confirmed by restriction and sequence analysis. SDS-PAGE analysis showed the successful expression of recombinant plasmid in Escherichia coli BL21 (molecular weight = 25-28 kDa). Larger quantities of protein were expressed in supernatant, indicating greater expression in soluble form. Western blot analysis confirmed the expression of the target protein. © FUNPEC-RP.


Zhang Y.,Yangzhou University | Zhang Y.,Key Laboratory for Animal Genetics | Wang Y.,Yangzhou University | Wang Y.,Key Laboratory for Animal Genetics | And 10 more authors.
PLoS ONE | Year: 2016

Several inducers have been used to differentiate embryonic stem cells (ESCs) into male germ cells but the induction process has been inefficient. To solve the problem of low efficiency of inducer for ESCs differentiation into male germ cells, all-trans retinoic acid (ATRA), Am80(the retinoic acid receptor agonist), and estradiol (E2) was used to induce ESCs to differentiate into male germ cells in vitro. ESCs were cultured in media containing ATRA, Am80, or E2 respectively which can differentiate ESCs into a germ cell lineage. In process of ATRA and Am80 induction Group, germ cell-like cells can be observed in 10 days; but have no in E2 induction Group. The marker genes of germ cell: Dazl, Stra8, C-kit, Cvh, integrinα6, and integrinβ1 all showed a significant up-regulation in the expression level. The ATRA-induction group showed high expression of C-kit and Cvh around 4 days, and integrinα6 and integrinβ1 were activated on day 10, respectively, while the E2-,Am80-induction group showed a high expression of C-kit as early as 4 days immunocytochemistry results shown that, integrinα6 and integrinβ1 could be detected in the ATRA-, Am80-, and E2-induction group, Positive clones in the ATRA group were greater in number than those in the other two groups. we conclued that ATRA, Am80, and E2 can promote the expression of the corresponding genes of germ cells, and had different effect on the differentiation of ESCs into male germ cells. ATRA was the most effective inducer of germ cell differentiation. © 2016 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Abdalhag M.A.,Yangzhou University | Abdalhag M.A.,Key Laboratory for Animal Genetics | Zhang T.,Yangzhou University | Zhang T.,Key Laboratory for Animal Genetics | And 11 more authors.
Genetics and Molecular Research | Year: 2015

Body weight is one of the most important economic traits in the poultry industry. In the present study, a custom SNP Beadchip was used to analyze the association between those 15 SNPs and 12 growth traits of Jinghai yellow chickens, and other important genetic parameters were also calculated and analyzed. The results indicated that nine of the 15 SNPs were associated with growth traits in Jinghai yellow chickens (P < 0.05), and the identified SNPs were also in linkage disequilibrium. Five of the nine identified SNPs were mainly associated with all of the growth traits, which indicated that those five SNPs might have significant influence on Jinghai yellow chicken growth traits. Polymorphism information content (PIC) analyses indicated that five of the nine SNPs exhibited moderate polymorphism (0.25 < PIC < 0.5), which reflected intermediate genetic diversity. Six candidate genes surrounding the significant SNPs were obtained and subjected to Gene Ontology annotation analyses and pathway analyses. The functions of six important candidate genes (SETDB2, ATP7B, INTS6, KPNA3, DLEU7, and FOXO1A) were discussed. The present study provided basic data for marker-assisted selection in Jinghai yellow chickens. © FUNPEC-RP.


Tang Y.,Yangzhou University | Tang Y.,Key Laboratory for Animal Genetics | Zhang T.,Yangzhou University | Zhang T.,Key Laboratory for Animal Genetics | And 13 more authors.
Molecular Biology Reports | Year: 2014

The objective of this study was to analyze possible associations between single nucleotide polymorphisms (SNPs) in the Myf5 gene with chicken growth and reproductive traits. SNPs in Myf5 of the Jinghai yellow chicken were detected by the polymerase chain reaction single-strand conformation polymorphism method and the haplotypes were analyzed. Eight SNPs were identified in the exons of Myf5. Nine haplotypes were established in a group of 379 Jinghai yellow chickens. In terms of growth traits, least square analysis showed that haplotype H1H5 had significant effects on weight at weeks 8 and 12 (P < 0.05). Haplotype H2H6 had significant effects on weight at weeks 12 and 14 (P < 0.05). For reproductive traits, H1H5 had higher body weight for the first egg than H1H4 and H2H4 (P < 0.05), and H1H3 (P < 0.01). H1H3 had a poor performance in average egg weight at 300 days. On the other hand, H1H3 had an advantage in egg number at 300 days. The results showed that SNPs of Myf5 have certain effects on growth and reproductive traits in Jinghai yellow chickens, which can be used in marker-assisted selection to accelerate chicken genetic progress. © 2014, Springer Science+Business Media Dordrecht.


Genxi Z.,Yangzhou University | Genxi Z.,Key Laboratory for Animal Genetics | Ying T.,Yangzhou University | Ying T.,Key Laboratory for Animal Genetics | And 5 more authors.
Molecular Biology Reports | Year: 2014

In order to analyze the association of single nucleotide polymorphisms (SNPs) in the MyoG and Myf5 genes with chicken growth traits, PCR-SSCP approach was used to detect the (SNPs). The general linear model was used to analyze gene interaction and genetic effects between different genotypes and growth traits of the Jinghai yellow chicken. For the MyoG gene, three genotypes (AA, AB and BB) were detected in the Jinghai yellow chicken population. Gene sequencing revealed one mutation (T36C) in the genotype BB in comparison to the genotype AA. For the Myf5 gene, three genotypes (CC, CD and DD) were detected in the Jinghai yellow chicken population. Gene sequencing revealed one mutation (A1313G) in the genotype DD in comparison to the genotype CC. Gene interaction effect has significant influence on 6, 8-week-weight and 300-day-weight. The least square analysis showed that individuals with BB genotype of the MyoG gene had higher bodyweight at 2, 4, 10, 12, 14 and 16 weeks compared to individuals with AA and AB genotypes. Individuals with CD genotype of the Myf5 gene had higher birth weight than individuals with CC genotype (P < 0.05). The interactive genotype AB*DD performs well at 6, 8 weeks and 300 days bodyweight. The results suggested that SNPs of the MyoG and Myf5 genes had certain effects on growth traits of the Jinghai yellow chicken. © 2014, Springer Science+Business Media Dordrecht.


Wang W.,Yangzhou University | Wang W.,Key Laboratory for Animal Genetics | Zhang T.,Yangzhou University | Zhang T.,Key Laboratory for Animal Genetics | And 8 more authors.
Journal of Applied Genetics | Year: 2015

Newcastle disease (ND) and avian infectious bronchitis (IB) are contagious diseases of chickens. To identify genes associated with antibody levels against ND and IB, a genome-wide association study was performed using specific-locus amplified fragment sequencing (SLAF-seq) technology in Jinghai yellow chickens. This determined six single-nucleotide polymorphisms (SNPs) that were associated with antibody levels against Newcastle disease virus (NDV): rsZ2494661, rsZ2494710, rs1211307701, rs1211307711, rs1218289310 and rs420701988. Of these, rsZ2494661 and rsZ2494710 reached the 5 % Bonferroni genome-wide significance level (5.5E-07) and they were both 134.7 kb downstream of the SETBP1 gene. The remaining four SNPs had ‘suggestive’ genome-wide significance levels (1.1E-05) and they were within or near the Plexin B1, LRRN1 and PDGFC genes. IB had two SNPs associated with antibody levels: rs149988433 and rs16170823; both reached chromosome-wide significance levels and they were near the USP7 and TRIM27 genes, respectively. Bioinformatics, GO annotation and pathway analysis indicated that five of these genes (Plexin B1, TRIM27, PDGFC, SETBP1 and USP7) may be important for the generation of protective antibodies against NDV and infectious bronchitis virus (IBV). This paves the way for further research on host immune responses against NDV. © 2015, Institute of Plant Genetics, Polish Academy of Sciences, Poznan.


Zhang T.,Yangzhou University | Zhang T.,Key Laboratory for Animal Genetics | Fan Q.C.,Yangzhou University | Fan Q.C.,Key Laboratory for Animal Genetics | And 7 more authors.
Genetics and Molecular Research | Year: 2015

Meat quality traits are very important in the poultry industry. To identify single nucleotide polymorphisms (SNPs) and candidate genes affecting meat quality traits, a genome-wide association study was performed using the Illumina chicken 60K SNP beadchip in Jinghai yellow chicken. Four meat quality traits were measured. Two SNPs reached 5% Bonferroni genome-wide significance (P < 1.8E-6) and 7 SNPs reached "suggestive" genome-wide significance (P < 3.59E-6) with meat quality. These SNPs were located nearby or in 7 candidate genes, including CBLN2, HPGDS, SETD2, and ANKRD46, among others. A total of 5650 haplotpyes were established and only 1 was found to be associated with fat content in leg muscle. These results indicate that the 9 SNPs and 7 genes are important candidate markers and may influence meat quality traits in chicken. © FUNPEC-RP.

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