miR-460b-5p promotes proliferation and differentiation of chicken myoblasts and targets RBM19 gene.

The meat production of broilers is crucial to economic benefits of broiler industries, while the slaughter performance of broilers is directly determined by skeletal muscle development. Hence, the broiler breeding for growth traits shows a great importance. As a kind of small noncoding RNA, microRNA (miRNA) can regulate the expression of multiple genes and perform a wide range of regulation in organisms. Currently, more and more studies have confirmed that miRNAs are closely associated with skeletal muscle development of chickens. Based on our previous miR-seq analysis (accession number: PRJNA668199), miR-460b-5p was screened as one of the key miRNAs probably involved in the growth regulation of chickens. However, the regulatory effect of miR-460b-5p on the development of chicken skeletal muscles is still unclear. Therefore, miR-460b-5p was further used for functional validation at the cellular level in this study. The expression pattern of miR-460b-5p was investigated in proliferation and differentiation stages of chicken primary myoblasts. It was showed that the expression level of miR-460b-5p gradually decreased from the proliferation stage (GM 50%) to the lowest at 24 h of differentiation. As differentiation proceeded, miR-460b-5p expression increased significantly, reaching the highest and stabilizing at 72 h and 96 h of differentiation. Through mRNA quantitative analysis of proliferation marker genes, CCK-8 and Edu assays, miR-460b-5p was found to significantly facilitate the transition of myoblasts from G1 to S phase and promote chicken myoblast proliferation. mRNA and protein quantitative analysis of differentiation marker genes, as well as the indirect immunofluorescence results of myotubes, revealed that miR-460b-5p significantly stimulated myotube development and promote chicken myoblast differentiation. In addition, the target relationship was validated for miR-460b-5p according to the dual-luciferase reporter assay and mRNA quantitative analysis, which indicates that miR-460b-5p was able to regulate RBM19 expression by specifically binding to the 3' UTR of RBM19. In summary, miR-460b-5p has positive regulatory effects on the proliferation and differentiation of chicken myoblasts, and RBM19 is a target gene of miR-460b-5p.

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

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.

Comparative Analysis of miRNA Expression Profiles in Skeletal Muscle of Bian Chickens at Different Embryonic Ages.

MicroRNAs (miRNAs) are widely involved in the growth and development of skeletal muscle through the negative regulation of target genes. In order to screen out the differentially expressed miRNAs (DEMs) associated with skeletal muscle development of Bian chickens at different embryonic ages, we used the leg muscles of fast-growing and slow-growing Bian chickens at the 14th and 20th embryonic ages (F14, F20, S14 and S20) for RNA-seq. A total of 836 known miRNAs were identified, and 121 novel miRNAs were predicted. In the F14 vs. F20 comparison group, 127 DEMs were screened, targeting a total of 2871 genes, with 61 miRNAs significantly upregulated and 66 miRNAs significantly downregulated. In the S14 vs. S20 comparison group, 131 DEMs were screened, targeting a total of 3236 genes, with 60 miRNAs significantly upregulated and 71 miRNAs significantly downregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the predicted target genes were significantly enriched in 706 GO terms and 6 KEGG pathways in the F14 vs. F20 group and 677 GO terms and 5 KEGG pathways in the S14 vs. S20 group. According to the interaction network analysis, we screened five coexpressed DEMs (gga-miR-146a-3p, gga-miR-2954, gga-miR-34a-5p, gga-miR-1625-5p and gga-miR-18b-3p) with the highest connectivity degree with predicted target genes between the two comparison groups, and five hub genes (HSPA5, PKM2, Notch1, Notch2 and RBPJ) related to muscle development were obtained as well. Subsequently, we further identified nine DEMs (gga-let-7g-3p, gga-miR-490-3p, gga-miR-6660-3p, gga-miR-12223-5p, novel-miR-327, gga-miR-18a-5p, gga-miR-18b-5p, gga-miR-34a-5p and gga-miR-1677-3p) with a targeting relationship to the hub genes, suggesting that they may play important roles in the muscle development of Bian chickens. This study reveals the miRNA differences in skeletal muscle development between 14- and 20-day embryos of Bian chickens from fast- and slow-growing groups and provides a miRNA database for further studies on the molecular mechanisms of the skeletal muscle development in Bian chickens.

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-1ß (IL-1ß), 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.

[A genome-wide association study on body weight traits of Jinghai yellow chicken].

Body weight traits are important economic characters of broilers. This study was carried out to screen for molecular markers and candidate genes that can be used to improve the body weight traits. A herd of 400 female Jinghai yellow chickens were measured for body weights from 0 to 14 weeks of age. Genome-wide association study (GWAS) was carried out using specific-locus amplified fragment sequencing (SLAF-seq) technology to detect SNPs associated with body weight traits of Jinghai yellow chicken. Finally, 100 SNPs that associated with body weight traits were detected. The results showed that effects of 15 SNPs reached 5% Bonferroni genome-wide significance and 85 SNPs reached potential genome-wide significance. Genes in the candidate regions with 1 Mb windows (SNP position±0.5 Mb) surrounding each significant SNP were screened. Finally, nine candidate genes were obtained, among which four genes of FAM124A (Family with sequence similarity 124A), QDPR (Quinoid dihydropteridine reductase), WDR1 (WD repeat domain 1) and SLC2A9 (Solute carrier family 2 (facilitated glucose transporter), member 9) might be important candidate genes influencing body weight traits of Jinghai yellow chicken. Furthermore, it was also found that most SNPs associated with mid and late growth and body weights were intensively located in the region of 75.6-80.7 Mb on chromosome 4. Our study thus provides a basis for genetic understanding of the Jinghai yellow chicken body weight traits.

[Polymorphism of exon 10 of prolactin receptor gene and its relationship with prolificacy of Jining Grey goats].

Prolactin receptor (PRLR) gene was studied as a candidate gene for the prolificacy of Jining Grey goats. Five pairs of primers were designed to detect single nucleotide polymorphisms of exon 10 and part of 3'untranslated region (UTR) of PRLR gene in both high fecundity breed (Jining Grey goat) and low fecundity breeds (Liaoning Cashmere goat, Boer goat and Angora goat) by PCR-SSCP. The nucleotide sequence of exon 10 and part of 3'UTR of caprine PRLR gene was spliced in this study for the first time. The length of this sequence was 1,118 bp. This sequence shared 98.33%, 93.92%, 74.52% homology with the published mRNA of PRLR gene of sheep, cow and human separately, and shared 78.29% homology with the published partial genomic sequence of PRLR gene of the alpaca. Only the products amplified by primers P1, P2, P4 displayed polymorphisms. For primer P1, two genotypes (AA and AB) were detected in both Jining Grey goats and Liaoning Cashmere goats, two genotypes (AA and AC) were detected in Angora goats, and only genotype AA was detected in Boer goats. Sequencing revealed two mutations (186G-->A and 220T-->C) of PRLR gene in the genotype AB in comparison to the genotype AA. The former mutation resulted in an amino acid change of Asp-->Asn, and the latter mutation resulted in an amino acid change of Leu-->Pro. Only one mutation (140A-->G) was found in the genotype AC in comparison to the genotype AA, and this mutation did not cause any amino acid change. The difference of the least squares means (LSM) for litter size between AA and AB was non-significant (P>0.05) in Jining Grey goats. For primer P2, two genotypes (DD and DE) were detected in Jining Grey goats, Liaoning Cashmere goats and Boer goats, and only genotype DD was detected in Angora goats. Sequencing revealed two mutations (52G-->A and 122G-->A) of PRLR gene in the genotype DE in comparison to the genotype DD. The former mutation did not cause any amino acid change, and the latter mutation resulted in an amino acid change of Arg-->Gly. The difference of LSM for litter size between DD and DE was non-significant (P>0.05) in Jining Grey goats. For primer P4, two genotypes (FF and FG) were detected in Jining Grey goats, two genotypes (FF and GG) were detected in Liaoning Cashmere goats, only genotype FF was detected in Boer goats, and three genotypes (FF, FG and GG) were detected in Angora goats. Sequencing revealed one mutation (143A-->G) of PRLR gene in the genotype GG in comparison to the genotype FF, and this mutation resulted in an amino acid change of Met-->Val. The Jining Grey does with genotype FG had 0.76 (P< 0.05) kids more than those with genotype FF. These results preliminarily showed that the PRLR gene is either a major gene that influences the prolificacy of Jining Grey goats or a molecular marker in close linkage with such a gene.