Rapid and visual detection of Mycoplasma synoviae by recombinase-aided amplification assay combined with a lateral flow dipstick.

Mycoplasma synoviae (MS) is an important avian pathogen that has brought substantial economic losses to the global poultry industry. Fast and accurate diagnosis is one of the critical factors for the control of MS infection. This study established a simple, rapid and visual detection method for MS using a recombinase-aided amplification (RAA) combined with a lateral flow dipstick (LFD). The reaction temperature and time of the RAA-LFD assay were optimized after selecting the primers and probe, and the specificity and sensitivity rates were analyzed. The results showed that RAA could amplify the target gene in 20 min at a constant temperature of 38°C, and the amplification products could be visualized by LFD within 5 min. There was no cross-reaction with Mycoplasma gallisepticum (MG), Pasteurella multocida (P. multocida), Escherichia coli (E. coli), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), and avian reovirus (ARV). Furthermore, the RAA-LFD assay exhibited high sensitivity with a detection limit of 10 copies/µL. A total of 128 clinical samples with suspected infection of MS were tested by RAA-LFD, PCR, and real-time fluorescence quantitative PCR (RFQ-PCR). The coincidence rate of the detection results was 95.3% between RAA-LFD and PCR, and 98.4% between RAA-LFD and RFQ-PCR. These results suggested that the RAA-LFD method established in the present study was easy to use and was associated with strong specificity and high sensitivity. This method was very suitable for the rapid detection of MS in clinical practice.

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.

Analysis of long noncoding RNA and mRNA using RNA sequencing during the differentiation of intramuscular preadipocytes in chicken.

Long noncoding RNAs (lncRNAs) regulate metabolic tissue development and function, including adipogenesis. However, little is known about the function and profile of lncRNAs in intramuscular preadipocyte differentiation in chicken. Here, we identified lncRNAs in chicken intramuscular preadipocytes at different differentiation stages using RNA sequencing. A total of 1,311,382,604 clean reads and 25,435 lncRNAs were obtained from 12 samples. In total, 7,433 differentially expressed genes (4,698 lncRNAs and 2,735 mRNAs) were identified by pairwise comparison. These 7,433 differentially expressed genes were grouped into 11 clusters based on their expression patterns by K-means clustering. Using Weighted Gene Coexpression Network Analysis, we identified four stage-specific modules positively related to I0, I2, I4, and I6 stages and two stage-specific modules negatively related to I0 and I2 stages, respectively. Many well-known and novel pathways associated with intramuscular preadipocyte differentiation were identified. We also identified hub genes in each stage-specific module and visualized them in Cytoscape. Our analysis revealed many highly-connected genes, including XLOC_058593, BMP3, MYOD1, and LAMP3. This study provides a valuable resource for chicken lncRNA study and improves our understanding of the biology of preadipocyte differentiation in chicken.

Genome-Wide Analysis of lncRNA and mRNA Expression During Differentiation of Abdominal Preadipocytes in the Chicken.

Long noncoding RNAs (lncRNAs) regulate adipogenesis and other processes associated with metabolic tissue development and function. However, little is known about the function and profile of lncRNAs during preadipocyte differentiation in the chicken (Gallus gallus). Herein, lncRNA and mRNA expression in preadipocytes at different stages of differentiation were analyzed using RNA sequencing. A total of 1,300,074,528 clean reads and 27,023 novel lncRNAs were obtained from 12 samples. The number of genes (1336 lncRNAs and 1759 mRNAs; 3095 in total) differentially expressed across various stages declined as differentiation progressed. Differentially expressed genes were found to be involved in several pathways related to preadipocyte differentiation that have been extensively studied, including glycerolipid metabolism, and the mammalian target of rapamycin, peroxisome proliferator-activated receptor, and mitogen-activated protein kinase signaling pathways. To our knowledge, some pathways are being reported for the first time, including the propanoate metabolism, fatty acid metabolism, and oxidative phosphorylation pathways. Furthermore, 3095 differentially expressed genes were clustered into eight clusters, and their expression patterns were determined through K-means clustering. Genes involved in the K2 cluster likely play important roles in preadipocyte differentiation. Six stage-specific modules related to A0 (day 0), A2 (day 2), and A6 (day 6) stages were identified, using weighted coexpression network analysis. Nine central, highly connected .genes in stage-specific modules were subsequently identified, including XLOC_068731, XLOC_022661, XLOC_045161, XLOC_070302, CHD6, LLGL1, NEURL1B, KLHL38, and ACTR6 This study provides a valuable resource for further study of chicken lncRNA and facilitates a better understanding of preadipocyte differentiation in the chicken.

Genome-wide association study of 8 carcass traits in Jinghai Yellow chickens using specific-locus amplified fragment sequencing technology.

Carcass traits are important to the commercial chicken industry, and understanding the genetics of these traits will be useful in the development of commercially viable varieties of chickens. We conducted a genome-wide association study based on 8 carcass trait phenotypes in a population of 400 43-week-old Jinghai Yellow chickens. Specific-locus amplified fragment sequencing technology was used to identify 90,961 single nucleotide polymorphisms (SNP) distributed among 29 chromosomes and the mitochondrial genome. SNP that were significantly associated with phenotypic traits were identified by a simple general linear model. Fifteen SNP attained genome-wide significance (P < 1.87E−6) and were associated with 5 of the 8 carcass traits; only one SNP was significantly associated with 2 traits (foot weight and wing weight). Twelve genes were associated with these 15 SNP. A region of chromosome 4 between 75.5 and 76.1 Mb was associated with carcass weight, foot weight, and wing weight. An 84-kb region on chromosome 3 (51.2 Mb) was associated with eviscerated weight and semi-eviscerated weight.

Genome-wide association study of antibody level response to NDV and IBV in Jinghai yellow chicken based on SLAF-seq technology.

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.

Eight SNPs of the Myf5 gene and diplotypes associated with growth and reproductive traits in Jinghai yellow chicken.

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.