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As a class of transcription regulators, numerous miRNAs have been verified to participate in regulating ovary follicular development in chickens (Gallus gallus). Previously we showed that gga-miR-135a-5p has significant differential expression between high and low-yield chicken ovaries, and the abundance of gga-miR-135a-5p is significantly higher in follicular theca cells than in granulosa cells. However, the exact role of gga-miR-135a-5p in chicken follicular theca cells is unclear. In this study, primary chicken follicular theca cells were isolated and then transfected with gga-miR-135a-5p inhibitor. Transcriptome sequencing was performed in chicken follicular theca cells with or without transfection. Differentially expressed genes (DEGs) were analyzed using bioinformatics. A dual-luciferase reporter assay was used to verify the target relationship between gga-miR-135a-5p and predicted targets within the DEGs. Compared with the normal chicken follicle theca cells, 953 up-regulated and 1060 down-regulated genes were detected in cells with gga-miR-135a-5p inhibited. The up-regulated genes were significantly enriched in Gene Ontology terms and pathways involved in cell proliferation and differentiation. In chicken follicular theca cells, Krüppel-like factor 4 (KLF4), ATPase phospholipid transporting 8A1 (ATP8A1), and Complexin-1 (CPLX1) were significantly up-regulated when the expression of gga-miR-135a-5p was inhibited. In addition, KLF4, ATP8A1, and CPLX1 confirmed as targets of gga-miR-135a-5p by using a dual-luciferase assay in vitro. The results suggest that gga-mir-135a-5p may involve in proliferation and differentiation in chicken ovarian follicular theca cells by targeting KLF4, ATP8A1, and CPLX1.

Background miRNAs play critical roles in growth and development. Various studies of chicken muscle development have focused on identifying miRNAs that are important for embryo or adult muscle development. However, little is known about the role of miRNAs in the whole muscle development process from embryonic to post-hatching periods. Here, we present a comprehensive investigation of miRNA transcriptomes at 12-day embryo (E12), E17, and day 1 (D1), D14, D56 and D98 post-hatching stages. Results We identified 337 differentially expressed miRNAs (DE-miRNAs) during muscle development. A Short Time-Series Expression Miner analysis identified two significantly different expression profiles. Profile 4 with downregulated pattern contained 106 DE-miRNAs, while profile 21 with upregulated pattern contained 44 DE-miRNAs. The DE-miRNAs with the upregulated pattern mainly played regulatory roles in cellular turnover, such as pyrimidine metabolism, DNA replication, and cell cycle, whereas DE-miRNAs with the downregulated pattern directly or indirectly contributed to protein turnover metabolism such as glycolysis/gluconeogenesis, pyruvate metabolism and biosynthesis of amino acids. Conclusions The main functional miRNAs during chicken muscle development differ between embryonic and post-hatching stages. miRNAs with an upregulated pattern were mainly involved in cellular turnover, while miRNAs with a downregulated pattern mainly played a regulatory role in protein turnover metabolism. These findings enrich information about the regulatory mechanisms involved in muscle development at the miRNA expression level, and provide several candidates for future studies concerning miRNA-target function in regulation of chicken muscle development.

Chicken body size (BS) is an economically important trait, which has been assessed in many studies for genetic selection. However, previous reports detected functional chromosome mutations or regions using gene chips. The present study used the specific-locus amplified fragment sequencing (SLAF-seq) technology to perform a genome-wide association study (GWAS) of purebred Wenshang Barred chickens. A total of 250 one-day-old male chickens were assessed in this study. Body size in individual birds was measured at 56 days. SLAF-seq was used to genotype and GWAS analysis was carried out using the general linear model (GLM) of the TASSEL program. A total of 1,286,715 single-nucleotide polymorphisms (SNPs) were detected, of which 175,211 were tested as candidate SNPs for genome-wide association analysis using the TASSEL general linear model. Three SNPs markers reached genome-wide significance. Of these, chrZ:81729634, chrZ:81841715, and chrZ:81954149 at 81,729,634, 81,841,715, and 81,954,149 bp of GGA Z were significantly associated with body diagonal length at 56 days (BDL56); and tibia length at 56 days (TL56). These SNPs were close to three genes, including ZCCHC7, PAX5, and MELK. These results open new horizons for studies on BS and should promote the use of Chinese chickens, especially Wenshang Barred chickens.

Avian leukosis virus (ALV) is a tumor-inducing virus that spreads among most chicken species, causing serious financial losses for the poultry industry. Subgroup J avian leukosis virus (ALV-J) is a recombinant exogenous ALV, which shows more extensive host range in comparison with other subgroups, especially in Chinese local chickens. To identify the relationship between ALV-J host range and the polymorphism of its cellular receptors, we performed a wide range epidemiological investigation of current ALV-J infection in Chinese local chickens, and discovered that all the 18 local chicken breeds being investigated from main local chicken breeding provinces were ALV-J positive. Furthermore, we cloned ALV-J cellular receptor genes of chNHE1 and chANXA2 of these 18 chicken breeds. Sequence alignment demonstrated that despite several regular mutations at the nucleotide level, there were no corresponding amino acid mutations for either chNHE1 gene or chANXA2 gene. Additionally, virus entry assay indicated that the level of viral enter into cells is stable among different chicken breeds. Results of this study indicated that the wide host range of ALV-J in Chinese local chickens was partially due to the high conservatism of its cellular receptors, and also provide target sites for drug design of resistance to ALV-J infection.

Chicken body weight (BW) is an economically important trait, and many studies have been conducted on genetic selection for BW. However, previous studies have detected functional chromosome mutations or regions using gene chips. The present study used the specific-locus amplified fragment sequencing (SLAF-seq) technology to perform a genome-wide association study (GWAS) on purebred Wengshang Barred chicken. A total of 1,286,715 single-nucleotide polymorphisms (SNPs) were detected, and 175,211 SNPs were selected as candidate SNPs for genome-wide association analysis using TASSEL general linear models. Six SNP markers reached genome-wide significance. Of these, rs732048524, rs735522839, rs738991545, and rs15837818 were significantly associated with body weight at 28 days (BW28), while rs314086457 and rs315694878 were significantly associated with BW120. These SNPs are close to seven genes (PRSS23, ME3, FAM181B, NABP1, SDPR, TSSK6L2, and RBBP8). Moreover, 24 BW-associated SNPs reached “suggestive” genome-wide significance. Of these, 6, 13, 1, and 4 SNPs were associated with BW28, BW56, BW80, and BW120, respectively. These results would enrich the studies on BW and promote the use of Chinese chicken, especially the Wenshang Barred chicken.

The SNPs of MC3R and MC4R genes in Jining Bairi chicken,Wenshang Luhua chicken and Laiwu Black chicken were detected by PCR-RFLP method.And the correlation between the polymorphism loci and the carcass traits were analyzed.As a result,an A/G mutation at nt 1 424 in MC3R gene and a G/T mutation at nt 923 in MC4R gene were identified,and the corresponding detection systems of Dde Ⅰ and FbrⅠwere established.The statistical analysis showed that the MC3R polymorphism locus had a significant association with live weight (P=0.041 8) and breast muscle weight (P=0.012 6).The MC4R polymorphism locus had a significant association with intermuscular fat width (P=0.036 6).The multiple-range test indicated that in MC3R polymorphism locus,the LSM of live body weight,carcass weight,semi-eviscerated weight,eviscerated weight and breast muscle weight of MN chickens were significantly higher than MM chickens or NN chickens (P 0.05).In MC4R polymorphism locus,the percentage of LSM of carcass and intermuscular fat width of AB chickens were significantly higher than BB chickens (P 0.05).

BMP15 (Bone morphogenetic protein 15) is an oocyte-secreted growth factor required for ovarian follicle development and ovulation in mammals, but its effects on reproduction in chickens are unclear. In this study, the association between BMP15 polymorphisms and reproduction traits were analyzed, and its expression characteristics in different tissues were explored in LaiWu Black chickens. Three single nucleotide polymorphisms (SNPs) were identified in four hundred LaiWu Black chickens. One SNP (NC_006091.3:g.1773T>C) located in exon 2 which was significantly associated with egg weight at first egg (EWFE) (P = 0.0389), was novel. Diplotypes based on the three SNPs were found to be significantly associated with egg weight at age of 43W (EW43) (P = 0.0058). The chickens with H3H3 diplotype had their first egg 0.57 days later than chickens with H5H5 diplotype and 1.21 days-3.96 days earlier than the other five diplotype chickens. The egg production at age of 43W (E43), egg production at age of 46W (E46) and egg production at age of 48W (E48) for chickens with H3H3 diplotype were the highest among all the chickens, and the E48 of chickens with H3H3 diplotype had 11.83 eggs higher than chickens with H1H5 diplotype. RT-qPCR results showed that the expression level of BMP15 gene in ovarian follicle was in the order of 4 mm>6 mm -8 mm> 15 mm -19 mm> 23 mm -29 mm > 33 mm -34 mm in diameter. The mRNA level in follicles of 4 mm and 6–8 mm in diameter were significantly higher than that in the other follicles (P

CTSD (Cathepsin D) is a key enzyme in yolk formation, and it primarily affects egg yolk weight and egg weight. However, recent research has mostly focused on the genomic structure of the CTSD gene and the enzyme’s role in pathology, and less is known about the enzyme’s functions in chickens. In this paper, the correlations between CTSD polymorphisms and egg quality traits were analyzed in local Shandong chicken breeds. CTSD polymorphisms were investigated by PCR-SSCP (polymerase chain reaction single strand conformation polymorphism) and sequencing analysis. Two variants were found to be associated with egg quality traits. One variant (2614T>C), located in exon 3, was novel. Another variant (5274G>T), located in intron 4, was previously referred to as rs16469410. Overall, our results indicated that CTSD would be a useful candidate gene in selection programs for improving yolk traits.