Jinghai Yellow Chicken Research Articles

Exploration of Potential Target Genes of miR-24-3p in Chicken Myoblasts by Transcriptome Sequencing Analysis.

Broiler skeletal muscle growth is significantly influenced by miRNAs. Our earlier research demonstrated that miR-24-3p significantly suppressed the proliferation of chicken myoblasts while promoting their differentiation. The purpose of this study is to investigate miR-24-3p potential target genes in chickens. We collected myoblasts of Jinghai yellow chicken and transfected four samples with mimics of miR-24-3p and another four samples with mimic NC (negative control) for RNA-seq. We obtained 54.34 Gb of raw data in total and 50.79 Gb of clean data remained after filtering. Moreover, 11,635 genes were found to be co-expressed in these two groups. The mimic vs. NC comparison group contained 189 DEGs in total, 119 of which were significantly up-regulated and 70 of which were significantly down-regulated. Important biological process (BP) terminology such as nuclear chromosomal segregation, reproduction, and nuclear division were discovered by GO enrichment analysis for DEGs in the mimic vs. NC comparison group. KEGG pathway analysis showed that focal adhesion, cytokine-cytokine receptor interaction, the TGF-β signaling pathway, and the MAPK signaling pathway were enriched in the top 20. Variation site analysis illustrated the SNP (single nucleotide polymorphisms) and INDEL (insertion-deletion) in the tested samples. By comparing the target genes predicted by miRDB (MicroRNA target prediction database) and TargetScan with the 189 DEGs found by the transcriptome sequencing, we discovered two up-regulated DEGs (NEURL1 and IQSEC3) and two down-regulated DEGs (REEP1 and ST6GAL1). Finally, we carried out qPCR experiments on eight DEGs and discovered that the qPCR results matched the sequencing outcomes. These findings will aid in identifying potential miR-24-3p target genes in chicken skeletal muscle and offer some new directions for upcoming research on broiler breeding.

Prediction of the Effect of Methylation in the Promoter Region of ZP2 Gene on Egg Production in Jinghai Yellow Chickens.

Simple SummaryJinghai yellow Chickens have a small size, high quality, early maturity, and stress resistance characteristics. There are particular individuals in its population with high and low egg numbers at 300 days of age. As a quantitative trait, egg production is not only influenced by the environment but is also controlled by micro-effect polygenes. In this paper, we focus on the ZP2 gene, which may impact the egg production of Jinghai yellow chickens. We also analyze the role of essential methylation sites in regulating the mRNA expression of the gene and further explore the molecular regulation mechanism of the ZP2 gene.Egg production in chickens is a quantitative trait. The aim of this study was to investigate the effect of promoter methylation of the Zona pellucida 2 (ZP2) gene on egg production. Real-time fluorescence quantification showed that the expression of the ZP2 gene in the ovaries of 300-day-old Jinghai yellow chickens in the high-laying group was significantly higher than that in the low-laying group (p < 0.01). A series of deletion fragments of the ZP2 gene promoter in Jinghai yellow chickens had different promoter activities in DF-1 cells, and the core region of the ZP2 gene promoter was found to be between −1552 and −1348. Four CpG islands in the promoter region of the ZP2 gene were detected by software prediction. The overall degree of methylation of the ZP2-1 amplified fragment was negatively correlated with mRNA expression to some extent (R = −0.197); the overall degree of methylation of the ZP2-2 amplified fragment was also negatively correlated with mRNA expression to some extent (R = −0.264), in which the methylation of methylcytosine (mC)-9, mC-20, and mC-21 sites was significantly negatively correlated with mRNA expression (p < 0.05). In addition, the mC-20 and mC-21 sites are located on the Sp1 transcription factor binding site, and it is speculated that these two sites may be the main sites for regulating transcription. In summary, the methylation sites mC-20 and mC-21 of the ZP2 gene may inhibit the binding of Sp1 and DNA, affect the transcription of the ZP2 gene, and then affect the number of eggs produced by the Jinghai yellow chickens.

MiRNA-seq analysis in skeletal muscle of chicken and function exploration of miR-24-3p

The regulation of skeletal muscle growth and development in chicken is complex. MicroRNAs (miRNAs) have been found to play an important role in the process, and more research is needed to further understand the regulatory mechanism of miRNAs. In this study, leg muscles of Jinghai yellow chickens at 300 d with low body weight (slow-growing group) and high body weight (fast-growing group) were collected for miRNA sequencing (miRNA-seq) and Bioinformatics analysis revealed 12 differentially expressed miRNAs (DEMs) between the two groups. We predicted 150 target genes for the DEMs, and GO and KEGG pathway analysis showed the target genes of miR-24-3p and novel_miR_133 were most enriched in the terms related to growth and development. Moreover, networks of DEMs and target genes showed that miR-24-3p and novel_miR_133 were the 2 core miRNAs. Hence, miR-24-3p was selected for further functional exploration in chicken primary myoblasts (CPMs) with molecular biology technologies including qPCR, cell counting kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU) and immunofluorescence. When proliferating CPMs were transfected with miR-24-3p mimic, the expression of cyclin dependent kinase inhibitor 1A (P21) was up-regulated and both CCK-8 and EdU assays showed that the proliferation of CPMs was inhibited. However, when the inhibitor was transfected into the proliferating CPMs, the opposite results were found. In differentiated CPMs, transfection with miR-24-3p mimic resulted in up regulation of MYOD, MYOG and MYHC after 48 h. Myotube areas also increased significantly compared to the mimic negative control (NC) group. When treated with inhibitor, differentiation CPMs produced the opposite effects. Overall, we revealed 2 miRNAs (novel_miR_133 and miR-24-3p) significantly related with growth and development and further proved that miR-24-3p could suppress the proliferation and promote differentiation of CPMs. The results would facilitate understanding the effects of miRNAs on the growth and development of chickens at the post-transcriptional level and could also have an important guiding role in yellow-feathered chicken breeding.

Qualitative and quantitative determination of tilmicosin in poultry eggs by gas chromatography tandem mass spectrometry after derivatization with acetic anhydride

A novel GC-MS/MS analytical method was established for the qualitative and quantitative determination of tilmicosin in poultry (Jinghai yellow chicken, Gaoyou duck and Yangzhou goose) eggs. The method was based on LLE and SPE for sample extraction and purification. Pyridine and acetic anhydride were used for the derivatization reaction. When tilmicosin was added to blank poultry egg samples at the LOQ and 75 μg/kg, 150 μg/kg, and 300 μg/kg, the recoveries ranged from 72.80% to 88.75%, the intraday and interday RSDs ranged from 2.31% to 4.56% and 3.29%-5.61%, respectively, and the LODs and LOQs ranged from 3.8 to 5.6 μg/kg and 8.4-10.5 μg/kg, respectively. These results confirmed that the parameters of this novel method meet the requirements of the FAO & WHO (2014) for veterinary drug residue testing. Poultry egg samples purchased from the local market were analysed according to the established method and only one egg sample was found to contain 18.9 μg/kg of tilmicosin.

Genome-Wide Association Study on Two Immune-Related Traits in Jinghai Yellow Chicken

This study was designed to discover molecular marker associated with the interferon INF-γ and avian influenza (AI) antibody titer traits in Jinghai Yellow chicken (Gallus gallus). Serum samples were taken from 400 female chickens and the INF-γ concentrations and AI antibody titer levels were measured. A genome-wide association study was carried out using specific-locus amplified fragment (SLAF) sequencing. Bioinformatics analysis was applied to detect single-nucleotide polymorphisms (SNPs) associated with the two traits. After sequencing and quality control, 103,680 SLAFs and 90,961 SNPs were obtained. The 400 samples were divided into 10 subgroups to reduce the effects of group stratification. The Bonferroni adjusted P-value of genome-wide significance was set at 1.87E−06 according to the number of independent SNP markers and linkage disequilibrium blocks. A SNP that was significantly associated with INF-γ concentration was detected in the myomesin 1 (MYOM1) gene on chromosome 2, and another SNPthat was significantly associated with the AI antibody titer level was detected in an RNA methyltransferase gene (Nsun7), which was found to have an important biological function. We propose that MYOM1 and Nsun7 are valuable candidate genes that influence the disease resistance characters of chicken. However, in-depth investigations are needed to determine the essential roles of these genes in poultry disease resistance and their possible application in breeding disease resistant poultry.

Study on the transcriptome for breast muscle of chickens and the function of key gene RAC2 on fibroblasts proliferation

BackgroundGrowth performance is significant in broiler production. In the growth process of broilers, gene expression varies at different growth stages. However, limited research has been conducted on the molecular mechanisms of muscle growth and development in yellow-feathered male chickens.ResultsIn the study, we used RNA-seq to study the transcriptome of the breast muscle of male Jinghai yellow chickens at 4 (M4F), 8 (M8F) and 12 weeks (M12F) of age. The results showed that 4608 differentially expressed genes (DEGs) were obtained by comparison in pairs of the three groups with Fold Change (FC) ≥ 2 and False Discovery Rate (FDR) ≤ 0.05, and 83, 3445 and 3903 DEGs were obtained separately from M4FvsM8F, M4FvsM12F and M8FvsM12F. Six genes were found as co-differentially expressed in the three age groups, namely SNCG, MYH1A, ARHGDIB, ENSGALG00000031598, ENSGALG00000035660 and ENSGALG00000030559. The GO analysis showed that 0, 304 and 408 biological process (BP) were significantly enriched in M4FvsM8F, M4FvsM12F and M8FvsM12F groups, respectively. KEGG pathway enrichment showed that 1, 2, 4 and 4 pathways were significantly enriched in M4FvsM8F, M4FvsM12F, M8FvsM12F and all DEGs, respectively. They were steroid biosynthesis, carbon metabolism, focal adhesion, cytokine-cytokine receptor interaction, biosynthesis of amino acids and salmonella infection. We constructed short hairpin RNA (shRNA) to interfere the differentially expressed gene RAC2 in DF-1 cells and detected mRNA and protein expression of the downstream genes PAK1 and MAPK8. Results of qPCR showed that RAC2, PAK1 and MAPK8 mRNA expression significantly decreased in the shRAC2–2 group compared with the negative control (NC) group. Western Blot (WB) results showed that the proteins of RAC2, PAK1 and MAPK8 also decreased in the shRAC2–2 group. Cell Counting Kit-8 (CCK-8) and 5-Ethynyl-2′-deoxyuridine (EdU) assay both showed that the proliferation of DF-1 cells was significantly inhibited after transfection of shRAC2–2.ConclusionsThe results of RNA-seq revealed genes, BP terms and KEGG pathways related to growth and development of male Jinghai yellow chickens, and they would have important guiding significance to our production practice. Further research suggested that RAC2 might regulate cell proliferation by regulating PAKs/MAPK8 pathway and affect growth of chickens.

Transcriptome Analysis of Long Noncoding RNAs and mRNAs in Granulosa Cells of Jinghai Yellow Chickens Illuminated With Red Light.

Jinghai Yellow chickens are a new indigenous breed with a dual purpose in China, but their egg laying performance is limited. Compared with white light (WL), exposure to red light (RL) can improve the egg laying performance of hens. Herein, to elucidate the molecular mechanism by which RL affects the egg laying performance, RNA sequencing was used to analyze long noncoding RNAs (lncRNAs) and mRNAs from granulosa cells of small yellow follicles from Jinghai Yellow chickens in RL and WL groups. A total of 12,466 lncRNAs were identified among the assembled transcripts, of which 168 lncRNAs were significantly different between the RL and WL groups (101 downregulated and 67 upregulated). Additionally, 1182 differentially expressed mRNAs were identified (958 downregulated and 224 upregulated). Integrated network analysis demonstrated that numerous differential mRNAs were involved in follicular development through steroid hormone synthesis, oocyte meiosis, and the PI3K-Akt signaling pathway. The impact of lncRNAs on cis and trans target mRNAs indicates that some lncRNAs play important roles in follicular development of small yellow follicles. The results provide a starting point for studies aimed at understanding the molecular mechanisms by which monochromatic light affects follicular development and egg production in hens.

The anti-Müllerian hormone gene's second exon is associated with the reproductive performance of Jinghai Yellow chickens.

Anti-Müllerian hormone (AMH), a member of the transforming growth factor- superfamily, plays important regulatory roles in follicular development and sex differentiation. Although much has been learned about the impact of polymorphisms of AMH on reproduction in animals, the effect on chicken reproduction is not well explored. In this study, the polymorphism of five exons of AMH gene and its effect on the reproductive performance of Jinghai Yellow chickens were studied. Primers for the amplification of AMH exons were designed, and Sanger sequencing was performed. Finally, only the polymorphism in the second exon of the AMH gene was found in the present population. Polymorphisms in the second exon of the AMH gene in 246 Jinghai Yellow hens and their associations with reproductive traits were analyzed. In total, four single nucleotide polymorphism (SNP) mutations were detected in the second exon of the AMH gene: g.1868AC (AA, aa and Aa); g.1883GA (BB, bb and Bb); g.1987GA (CC, cc and Cc); and g.1996AG (DD, dd and Dd). Only the mutation of g.1996AG affected the reproductive traits: the age of laying first egg (AFE) of dd genotype was significantly () earlier than that in the DD and Dd hens. Moreover, the egg number by 300 d old (EN300) of dd individuals was significantly higher than that of DD and Dd individuals (). Thus, we inferred that the dd genotype is the beneficial genotype. Additionally, AFE and EN300 showed significantly better performance in both the H2H2 and H7H7 diplotypes compared with other diplotype individuals (). Thus, the H2H2 and H7H7 genotype had the best combination of AFE and EN300. Our study may allow for molecular marker section in poultry breeding.

Effect of IL-8, IL-2 and IL-15 Gene Expression in Different Tissues of Jinghai Yellow Chicken Infected by Eimeria Tenella

For research the role of IL-8, IL-2 and IL-15 genes in immune regulation after E. tenella infection, with Jinghai yellow chickens as material, the mRNA expression levels of IL-8, IL-2 and IL-15 genes in ten different tissues after infection with different doses of E. tenella oocysts were compared to determine the expression difference of the three genes in the same tissue with different infection doses. The results showed that IL-8, IL-2 and IL-15 genes were expressed in all tissues at the 7th day of infection, and the infection group was higher than the control group in most tissues. The mRNA expression of IL-8 gene in infection group II in the liver was significantly higher than that in the other two groups (P<0.05), and was extremely far higher the other two groups in the spleen and cecum (P<0.01). The mRNA expression of IL-8 gene in infection group II was also far higher in the bursa and thymus than that in the control group (P<0.05). The mRNA expression of IL-2 gene in infection group II in liver and lungs were significantly (P<0.05) and extremely significant (P<0.01) higher than that in the control group, respectively. The IL-15 gene mRNA expression. The infection group was obvious (P<0.05) and extremely significantly (P<0.01) higher in the lung and cecum than that in the control group, respectively. These results indicate that the three genes play a role in the infection of E. tenella in chickens, and provide a basis for the subsequent study of the relationship between these three genes and chicken coccidiosis resistance.

Study of the Relationship between Polymorphisms in the IL-8 Gene Promoter Region and Coccidiosis Resistance Index in Jinghai Yellow Chickens.

Interleukin 8 (IL-8) participates in the immune response and has the function of inducing neutrophils to release lysosomal enzymes and eliminate pathogens. This study was to investigate the effect of single nucleotide mutations in the IL-8 gene promoter region on the coccidiosis resistance index. In this study, 180 infected Eimeria tenella (E. tenella) Jinghai yellow chickens were used as experimental samples. DNA sequencing technology was used to detect single nucleotide polymorphisms (SNPs) in the IL-8 gene promoter region. The association between these SNPs and coccidiosis resistance indexes (including superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-PX), catalase (CAT), nitric oxide (NO), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), IL-8, and interferon-γ (IFN-γ)) were analyzed. Three SNPs (T-550C, G-398T, and T-360C) were detected. Significant associations were found between each genotype at the T-550C site with NO (p-value = 0.006) and IL-8 (p-value = 0.034) indexes. Significant associations were found between each genotype at the G-398T site with SOD (p-value = 0.042), CAT (p-value = 0.049), NO (p-value = 0.008), and IL-2 (p-value = 0.044) indexes. Significant associations were found between each genotype at the T-360C site with SOD (p-value = 0.007), NO (p-value = 0.046), IL-2 (p-value = 0.041), IL-8 (p-value = 0.039), and IFN-γ (p-value = 0.042) indexes. Haplotype analysis showed that multiple indexes of the H1H3 haplotype combination were significantly higher than other haplotype combinations. Therefore, mutation of the IL-8 gene promoter region has a significant regulatory effect on the coccidiosis resistance index, with a change in transcription factor binding potentially altering IL-8 gene expression, thereby further affecting the IL-8 level in plasma. However, the specific mechanism needs further study.

Transcriptome for the breast muscle of Jinghai yellow chicken at early growth stages.

BackgroundThe meat quality of yellow feathered broilers is better than the quality of its production. Growth traits are important in the broiler industry. The exploration of regulation mechanisms for the skeletal muscle would help to increase the growth performance of chickens. At present, some progress has been made by researchers, but the molecular mechanisms of the skeletal muscle still remain unclear and need to be improved.MethodsIn this study, the breast muscles of fast- and slow-growing female Jinghai yellow chickens (F4F, F8F, F4S, F8S) and slow-growing male Jinghai yellow chickens (M4S, M8S) aged four and eight weeks were selected for transcriptome sequencing (RNA-seq). All analyses of differentially expressed genes (DEGs) and functional enrichment were performed. Finally, we selected nine DEGs to verify the accuracy of the sequencing by qPCR.ResultsThe differential gene expression analysis resulted in 364, 219 and 111 DEGs (adjusted P-value ≤ 0.05) for the three comparison groups, F8FvsF4F, F8SvsF4S, and M8SvsM4S, respectively. Three common DEGs (ADAMTS20, ARHGAP19, and Novel00254) were found, and they were all highly expressed at four weeks of age. In addition, some other genes related to growth and development, such as ANXA1, COL1A1, MYH15, TGFB3 and ACTC1, were obtained. The most common DEGs (n = 58) were found between the two comparison groups F8FvsF4F and F8SvsF4S, and they might play important roles in the growth of female chickens. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway also showed some significant enrichment pathways, for instance, extracellular matrix (ECM)-receptor interaction, focal adhesion, cell cycle, and DNA replication. The two pathways that were significantly enriched in the F8FvsF4F group were all contained in that of F8SvsF4S. The same two pathways were ECM–receptor interaction and focal adhesion, and they had great influence on the growth of chickens. However, many differences existed between male and female chickens in regards to common DEGs and KEGG pathways. The results would help to reveal the regulation mechanism of the growth and development of chickens and serve as a guideline to propose an experimental design on gene function with the DEGs and pathways.

Research Note: Correlation analysis of interleukin-6, interleukin-8, and C-C motif chemokine ligand 2 gene expression in chicken spleen and cecal tissues after Eimeria tenella infection in vivo

IL-6, IL-8, and C-C motif chemokine ligand 2 (CCLi2) are important factors in inflammatory and immune responses. To investigate their relationships in the spleen and cecum and between coccidiosis-infected and uninfected states, we performed quantitative real-time PCR to compare the relative expression difference of IL-6, IL-8, and CCLi2 in the same tissues between the infection and control groups. In addition, the correlations of the relative expression levels of these 3 genes were determined in the same and different tissues within the same group. The results showed that the expression levels of IL-6, IL-8, and CCLi2 in the spleen and cecum of the infected group were all higher than those of the uninfected group (P < 0.05). The correlation coefficients among the IL-6, IL-8, and CCLi2 expression levels in the spleen or cecum were all positive in both the infection and control groups. In the spleen tissues, CCLi2 expression was strongly correlated with IL-6 and IL-8 in the uninfected group (P < 0.01), and the correlation coefficients reached 0.853 (R2 = 0.728) and 0.996 (R2 = 0.992), respectively. The expression of CCLi2 was also strongly correlated with IL-8 (R reached 0.890, R2 = 0.792) in the infected group. In the cecal tissues, the expression levels of the 3 genes were all extremely significantly correlated in the uninfected group (P < 0.01), and the correlation coefficients ranged from 0.498 to 0.765, indicating moderate correlations. The expression of IL-6 was extremely significantly positively correlated with IL-8 and CCLi2 in the infected group (P < 0.01), with moderate correlations (R ranged from 0.469–0.639). In addition, the expression levels of the 3 genes were not significantly correlated (P > 0.05) between the spleen and cecum tissues in either the infection group or the control group. These results indicate that IL-6, IL-8, and CCLi2 were correlated and play an important role in coccidiosis infection of Jinghai yellow chicken. Our data also provide a basis for further exploring the role of these 3 genes in genetic breeding for coccidiosis resistance.

Establishing a Model for Evaluating Chicken Coccidiosis Resistance Based on Principal Component Analysis

Simple SummaryAvian coccidiosis, an infectious disease caused by seven species of Eimeria that can infect a bird’s digestive tract and significantly retard its growth, is a serious economic disease for chickens. Many studies have demonstrated that host resistance to coccidiosis related to genetic variations can be improved by selective breeding. The parameters for evaluation of resistance to coccidiosis could be objective indicators, such as body weight gain and cecal lesion score, or biochemical indices, such as immune factors or cytokines in the plasma or serum. The aim of the study is to establish an optimal comprehensive evaluation model including a resistance index that can be detected in live chickens (slaughter traits cannot be selected in breeding) based on principal component analysis. The value of individual chickens calculated with the optimal evaluation model is associated with the cecum lesion score; the larger the value, the stronger the resistance to coccidiosis. This illustrated that the optimal model is effective in coccidiosis resistance selection.To establish a coccidiosis resistance evaluation model for chicken selection, the different parameters were compared between infected and control Jinghai yellow chickens. Validation parameters were selected for principal component analysis (PCA), and an optimal comprehensive evaluation model was selected based on the significance of a correlation coefficient between coccidiosis resistance parameters and principal component functions. The following six different parameters were identified: body weight gain 3–5 days post infection and catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and γ-interferon (IFN-γ) concentrations on the eight day post inoculation. Six principal components and one accumulated contribution of up to 80% of the evaluation models were established by PCA. The results showed that the first model was significantly or highly significantly related to nine resistance parameters (p < 0.01 or p < 0.05), especially to cecal lesions (p < 0.01). The remaining models were related to only 2–3 parameters (p < 0.01 or p < 0.05) and not to cecal lesions (p > 0.05). The values calculated by the optimal model (first model) were significantly negatively correlated with cecal lesion performance; the larger the value, the more resistant to coccidiosis. The model fi1 = −0.636 zxi1 + 0.311 zxi2 + 0.801 zxi3 − 0.046 zxi4 − 0.076 zxi5 + 0.588 zxi6 might be the best comprehensive selection index model for chicken coccidiosis resistance selection.

Association Analysis of Single Nucleotide Polymorphisms in the 5′ Regulatory Region of the IL-6 Gene with Eimeria tenella Resistance in Jinghai Yellow Chickens

Interleukin 6 (IL-6) is an immunoregulatory cytokine involved in various inflammatory and immune responses. To investigate the effects of single nucleotide polymorphisms (SNPs) and haplotypes of IL-6 on resistance to Eimeria tenella (E. tenella), SNPs in the 5′ regulatory region of IL-6 were detected with direct sequencing, and the effects of SNPs and haplotypes on resistance to E. tenella were analyzed by the least square model in Jinghai yellow chickens. Nineteen SNPs were identified in the 5′ regulation region of IL-6, among which three SNPs were newly discovered. The SNP association analysis results showed that nine of the SNPs were significantly associated with E. tenella resistance indexes; the A-483G locus was significantly associated with the GSH-Px, IL-2, and IL-17 indexes (p < 0.05); the C-447G locus was significantly associated with the SOD, GSH-Px, IL-17, and IL-2 indexes (p < 0.05); and the G-357A locus had significant effects on the CAT and IL-16 indexes (p < 0.05). Haplotype analysis showed that H2H3 and H2H5 were favorable haplotype combinations with good coccidium resistance. Furthermore, we used qRT-PCR and observed that the expression of IL-6 in the infection group was higher than that in the control group in the liver, proventriculus, small intestine, thymus, kidney, and bursa of Fabricius and extremely significantly different than that in the cecum especially (p < 0.01). In summary, SNPs and haplotypes in the 5′ regulatory region of IL-6 have important effects on E. tenella resistance, and the results will provide a reference for molecular marker selection of E. tenella resistance in Jinghai yellow chickens.

Transcriptome Analysis of Differentially Expressed Genes Related to the Growth and Development of the Jinghai Yellow Chicken.

The growth traits are important traits in chickens. Compared to white feather broiler breeds, Chinese local broiler breeds have a slow growth rate. The main genes affecting the growth traits of local chickens in China are still unclear and need to be further explored. This experiment used fast-growth and slow-growth groups of the Jinghai Yellow chicken as the research objects. Three males and three females with similar body weights were selected from the two groups at four weeks old and eight weeks old, respectively, with a total of 24 individuals selected. After slaughter, their chest muscles were taken for transcriptome sequencing. In the differentially expressed genes screening, all of the genes obtained were screened by fold change ≥ 2 and false discovery rate (FDR) < 0.05. For four-week-old chickens, a total of 172 differentially expressed genes were screened in males, where there were 68 upregulated genes and 104 downregulated genes in the fast-growth group when compared with the slow-growth group. A total of 31 differentially expressed genes were screened in females, where there were 11 upregulated genes and 20 downregulated genes in the fast-growth group when compared with the slow-growth group. For eight-week-old chickens, a total of 37 differentially expressed genes were screened in males. The fast-growth group had 28 upregulated genes and 9 downregulated genes when compared with the slow-growth group. A total of 44 differentially expressed genes were screened in females. The fast-growth group had 13 upregulated genes and 31 downregulated genes when compared with the slow-growth group. Through gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, many genes were found to be related to cell proliferation and differentiation, muscle growth, and cell division such as SNCG, MCL1, ARNTL, PLPPR4, VAMP1, etc. Real-time PCR results were consistent with the RNA-Seq data and validated the findings. The results of this study will help to understand the regulation mechanism of the growth and development of Jinghai Yellow chicken and provide a theoretical basis for improving the growth rate of Chinese local chicken breeds.

Transcriptome analysis of ovary in relatively greater and lesser egg producing Jinghai Yellow Chicken

Egg production is determined by the function of ovary and is regulated by the hypothalamic-pituitary-ovary axis. The mechanism by which the ovary regulates egg production, however, is still poorly understood. The purpose of this study is to compare the transcriptome difference in ovary of relatively greater and lesser egg producing chickens, and to screen candidate genes related to egg production. A RNA sequencing was performed to analyze and compare the mRNA in ovarian tissues of relatively greater and lesser egg producing chickens. A total of 4 431 new genes expressed in the chicken ovary were mined. There were 305 differentially expressed genes (DEGs) identified between the relatively greater and lesser egg producing hens. Gene ontology analysis identified five candidate genes related to egg production, including ZP2, WNT4, AMH, IGF1, and CYP17A1 genes. Tissue expression profiles indicated these five candidate genes were highly expressed in chicken ovarian tissues, indicating a potential role in regulating chicken ovarian function and egg production. The KEGG analysis indicated the neuroactive ligand-receptor interaction pathway might have an important function in regulation of egg production. In addition, four known pathways related to reproduction were detected, including the calcium signaling, wnt signaling pathway, focal adhesion, and cytokine-cytokine receptor interaction pathways. Results of the present study indicate gene expression differences in the ovarian tissues of relatively greater and lesser egg producing chickens, and identified five important candidate genes related to egg production, which provided a theoretical basis for improving egg production of Jinghai Yellow Chickens.

Polymorphisms of the Myf6 gene exon 1 and their relationship with growth and reproductive traits in Jinghai Yellow Chicken

Polymorphisms of the Myf6 gene exon 1 and their relationship with growth and reproductive traits in Jinghai Yellow Chicken

RNA Sequencing Analysis of Chicken Cecum Tissues Following Eimeria tenella Infection in Vivo.

Eimeria tenella (E. tenella) is one of the most frequent and pathogenic species of protozoan parasites of the genus Eimeria that exclusively occupies the cecum, exerting a high economic impact on the poultry industry. To investigate differentially expressed genes (DEGs) in the cecal tissue of Jinghai yellow chickens infected with E. tenella, the molecular response process, and the immune response mechanism during coccidial infection, RNA-seq was used to analyze the cecal tissues of an E. tenella infection group (JS) and an uninfected group (JC) on the seventh day post-infection. The DEGs were screened by functional and pathway enrichment analyses. The results indicated that there were 5477 DEGs (p-value < 0.05) between the JS and the JC groups, of which 2942 were upregulated, and 2535 were downregulated. GO analysis indicated that the top 30 significantly enriched GO terms mainly involved signal transduction, angiogenesis, inflammatory response, and blood vessel development. KEGG analysis revealed that the top significantly enriched signaling pathways included focal adhesion, extracellular matrix–receptor interaction, and peroxisome proliferator-activated receptor. The key DEGs in these pathways included ANGPTL4, ACSL5, VEGFC, MAPK10, and CD44. These genes play an important role in the infection of E. tenella. This study further enhances our understanding of the molecular mechanism of E. tenella infection in chickens.

Association between Fbxl5 gene polymorphisms and partial economic traits in Jinghai Yellow chickens.

The Fbxl5 gene is a member of the F-BOX family and plays an important role in maintaining iron homeostasis in cells. In order to reveal the genetic effects of Fbxl5 gene polymorphisms on body weight (BW) traits and reproductive performance in chickens, Fbxl5 gene polymorphisms were detected in 363 Jinghai Yellow chickens by PCR-single-strand conformation polymorphism (SSCP) and DNA sequencing methods using three primers. With primer 1, three genotypes (BB, bb, Bb) were detected in the Jinghai Yellow chicken population and two mutations (g. 14257 T C and g. 14262 T C) were revealed by gene sequencing. With primer 2, two genotypes (EE, Ee) were detected in the same population and one mutation (g. 19018 G A), and for primer 3, three genotypes (FF, ff, Ff) and one mutation (g. 19018 G A) were detected. Four single nucleotide polymorphisms (SNPs) were used to estimate the frequency distributions of the eight haplotypes with PHASE 2.1 software. CTCG was the major haplotype with a frequency of 37.93 %, while the least frequent was TCTA with a frequency of 2.98 %. The BW of haplotype combination H1H8 was higher than that of the other haplotypes and was a dominant combination. In terms of reproductive performance, the age at the first egg of the haplotype combination H9H1 was later than in the other haplotypes, but the mean egg weight at 300 days was relatively optimal. The H1H2 haplotype produced the highest mean egg weight in 300 days, although the total number of eggs in 300 days was smaller in the H2H4 haplotype with the highest at first egg. Therefore, we can consider using the haplotype combination H1H2 for selection. The findings of this study expand the theoretical basis of the use of the Fbxl5 gene in the molecular breeding of poultry.

Transcriptome profile analysis of leg muscle tissues between slow- and fast-growing chickens.

Chicken is widely favored by consumers because of some unique features. The leg muscles occupy an important position in the market. However, the specific mechanism for regulating muscle growth speed is not clear. In this experiment, we used Jinghai yellow chickens with different body weights at 300 days as research subjects. The chickens were divided into fast- and slow-growing groups, and we collected leg muscles after slaughtering for use in RNA-seq. After comparing the two groups, 87 differentially expressed genes (DEGs) were identified (fold change ≥ 2 and FDR < 0.05). The fast-growing group had 42 up-regulated genes and 45 down-regulated genes among these DEGs compared to the slow-growing group. Six items were significantly enriched in the biological process: embryo development ending in birth or egg hatching, chordate embryonic development, embryonic skeletal system development, and embryo development as well as responses to ketones and the sulfur compound biosynthetic process. Two significantly enriched pathways were found in the KEGG pathway analysis (P-value < 0.05): the insulin signaling pathway and the adipocytokine signaling pathway. This study provides a theoretical basis for the molecular mechanism of chicken growth and for improving the production of Jinghai yellow chicken.