BackgroundThe widely observed RNA-DNA differences (RDDs) have been found to be due to nucleotide alteration by RNA editing. Canonical RNA editing (i.e., A-to-I and C-to-U editing) mediated by the adenosine deaminases acting on RNA (ADAR) family and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) family during the transcriptional process is considered common and essential for the development of an individual. To date, an increasing number of RNA editing sites have been reported in human, rodents, and some farm animals; however, genome-wide detection of RNA editing events in sheep has not been reported. The aim of this study was to identify RNA editing events in sheep by comparing the RNA-seq and DNA-seq data from three biological replicates of the kidney and spleen tissues.ResultsA total of 607 and 994 common edited sites within the three biological replicates were identified in the ovine kidney and spleen, respectively. Many of the RDDs were specific to an individual. The RNA editing-related genes identified in the present study might be evolved for specific biological functions in sheep, such as structural constituent of the cytoskeleton and microtubule-based processes. Furthermore, the edited sites found in the ovine BLCAP and NEIL1 genes are in line with those in previous reports on the porcine and human homologs, suggesting the existence of evolutionarily conserved RNA editing sites and they may play an important role in the structure and function of genes.ConclusionsOur study is the first to investigate RNA editing events in sheep. We screened out 607 and 994 RNA editing sites in three biological replicates of the ovine kidney and spleen and annotated 164 and 247 genes in the kidney and spleen, respectively. The gene function and conservation analysis of these RNA editing-related genes suggest that RNA editing is associated with important gene function in sheep. The putative functionally important RNA editing sites reported in the present study will help future studies on the relationship between these edited sites and the genetic traits in sheep.Electronic supplementary materialThe online version of this article (10.1186/s40104-019-0331-z) contains supplementary material, which is available to authorized users.

Summary Chicken plumage color, the genetic basis of which is often affected by epistasis, has long interested scientists. In the current study, a population of complex epistasis was constructed by crossing dominant White Leghorn chickens with recessive white feather chickens. Through a genome‐wide association study, we identified single nucleotide polymorphisms and genes significantly associated with white and colored plumage in hens at different developmental stages. Interestingly, white plumage in adulthood was associated with the recessive white feather gene (TYR), whereas white feathers at birth stage were associated with the dominant white feather gene (PMEL), indicating age‐related roles for these genes. TYR was shown to exert an epistatic effect on PMEL in adult hens. Additionally, TYR had an epistatic effect on barred plumage, while barred plumage had an epistatic effect on black plumage. TYR had no epistatic effect on the yellow plumage. We confirmed that the barred plumage gene is CDKN2A, as reported in previous studies. Golgb1 and REEP3, which play important roles in the Golgi network and affect the formation of feather pigments, are important candidate genes for yellow plumage. The candidate genes for black plumage are CAMKK1 and IFT22. Further research is warranted to elucidate the molecular mechanisms underlying these traits.

Hyperpigmentation in Silky Fowl (SF) results in aberrant immune cell development. However, how melanocytes regulate B-cell proliferation in the bursa of Fabricius (BF) is unclear. To resolve this conundrum, we collected BFs from three-week-old SF and White Leghorn (WL) female chickens for RNA sequencing. The BF development was relatively weaker in SF than in WL. The transcriptome analyses identified 4848 differentially expressed genes, 326 long noncoding RNAs (lncRNAs), and 67 microRNAs in the BF of SF. The genes associated with melanogenesis was significantly higher, but that of the genes associated with the cytokine-cytokine receptor interactions and JAK-STAT signalling pathway was significantly lower in SF than in WL. Crucial biological processes, such as the receptor activity, cell communication, and cellular responses to stimuli, were clustered in SF. The predicted target lncRNAs genes were mainly associated with cell proliferation pathways such as JAK-STAT, WNT, MAPK, and Notch signalling pathways. Except for the above pathways, the target microRNA genes were related to the metabolism, melanogenesis, autophagy, and NOD-like and Toll-like receptor signalling pathways. The lncRNAs and microRNAs were predicted to regulate the JAK2, STAT3, and IL-15 genes. Thus, B-cell development in the BF of SF might be regulated and affected by noncoding RNAs.

Ghrelin O-acyltransferase (GOAT), ghrelin, and GHSR have been reported to play important roles that influence feed intake in mammals. LEAP2, an endogenous antagonist of GHSR, plays an important role in the regulation of feed intake. However, chicken ghrelin has also been reported to have an inhibitory effect on feed intake. The role of the GOAT–Ghrelin–GHSR–LEAP2 axis in chicken-feed intake remains unclear. Therefore, it is necessary to systematically evaluate the changes in the tissue expression levels of these genes under different energy states. In this study, broiler chicks in different energy states were subjected to starvation and feeding, and relevant gene expression levels were measured using quantitative real-time PCR. Different energy states significantly modulated the expression levels of LEAP2 and GHSR but did not significantly affect the expression levels of GOAT and ghrelin. A high expression level of LEAP2 was detected in the liver and the whole small intestine. Compared to the fed group, the fasted chicks showed significantly reduced LEAP2 expression levels in the liver and the small intestine; 2 h after being refed, the LEAP2 expression of the fasted chicks returned to the level of the fed group. Transcription factor prediction and results of a dual luciferase assay indicated that the transcription factor CDX4 binds to the LEAP2 promoter region and positively regulates its expression. High expression levels of GHSR were detected in the hypothalamus and pituitary. Moreover, we detected GHSR highly expressed in the jejunum—this finding has not been previously reported. Thus, GHSR may regulate intestinal motility, and this aspect needs further investigation. In conclusion, this study revealed the function of chicken LEAP2 as a potential feed-intake regulator and identified the potential mechanism governing its intestine-specific expression. Our study lays the foundations for future studies on avian feed-intake regulation.

We previously reported that blue eggshell color in chickens is associated with a partial endogenous retroviral ( EAV-HP ) insertion in the promoter region of the solute carrier organic anion transporter family member 1B3 ( SLCO1B3 ) gene. The EAV-HP sequence includes numerous regulatory elements, which may modulate the expression of adjacent genes. To determine whether this insertion influences the expression of neighboring genes, we screened the expression of solute carrier organic anion transporter family members 1C1, 1B1 ( SLCO1C1 , SLCO1B1 ), and SLCO1B3 in 13 and 10 tissues from female and male Yimeng chickens, respectively. We observed that the insertion only significantly modulated the expression of SLCO1B3 and did not majorly affect that of SLCO1C1 and SLCO1B1 . High expression of SLCO1B3 was detected in the shell gland, magnum, isthmus, and vagina of the oviduct in female blue-eggshell chickens. We also observed ectopic expression of SLCO1B3 in the testes of male chickens. SLCO1B3 is typically highly expressed in the liver; however, the EAV-HP insertion significantly reduces SLCO1B3 expression. As a liver-specific transporter, a reduction in the expression of SLCO1B3 may affect liver metabolism, particularly that of bile acids. We also detected higher ectopic expression of SLCO1B3 in the lungs of birds heterozygous for the EAV-HP insertion than in homozygous genotypes. In conclusion, we confirmed that the EAV-HP insertion modifies SLCO1B3 expression, and showed, for the first time, similar expression profile of this gene in all parts of the oviduct in females and testis in males. We also observed different levels of SLCO1B3 expression in the liver, which were associated with the EAV-HP insertion, and significantly higher expression in the lungs of birds with heterozygous genotype. The effects of these changes in the SLCO1B3 expression pattern on the function of the tissues warrant further investigation.

The eggshell blueness is an interesting object for chicken genetic studies and blue-shelled chicken industry, especially after the discovery of the causative mutation of chicken blue eggshell. In the present study, genome wide association study (GWAS) was conducted in Chinese Dongxiang blue-shelled chicken underlying four traits of blue eggshell pigments: quantity of biliverdin (QB), quantity of protoporphyrin (QP), quantity of total pigment (QT), and color density trait (CD). A total of 139 individuals were randomly collected for GWAS. We detected two SNPs in genome-wise significance and 35 in suggestive significance, 24 out of the 37 SNP were located either within intron/exon or near 15 genes in a range of ~1.17 Mb on GGA21. For further confirmation of the identified SNP loci by GWAS, the follow-up replication studies were performed in two populations. A total of 146 individuals of the second generation derived from the former GWAS population, as well as 280 individuals from an alternative independent population were employed for genotyping by MALDI-TOF MS in a genotype-phenotype association study. Eighteen SNPs evenly distributed on the GGA21 significant region were successfully genotyped in the two populations, of which 4 and 6 SNP loci were shown significantly associated with QB, QT and QP in the two repeat populations, respectively. Further, the SNPs were narrowed down to a region of ~ 653.819 Kb on GGA21 that harbors five candidate genes: AJAP1, TNFRSF9, C1ORF174, CAMTA1, and CEP104. Shell gland of chickens laying dark and light blue eggshell was chosen for detection of mRNA expression of the five candidate genes. The results showed differential expression levels of these genes in the two groups. The specific function of these genes has not yet been defined clearly in chickens and further in-depth studies are needed to explore the new functional role in chicken eggshell blueness.

β-Catenin is an evolutionarily conserved molecule in the canonical Wnt signaling pathway, which controls decisive steps in embryogenesis and functions as a crucial effector in the development of hair follicles. However, the molecular mechanisms underlying wool production have not been fully elucidated. In this study, we investigated the effects of ovine β-catenin on wool follicles of transgenic sheep produced by pronuclear microinjection with a skin-specific promoter of human keratin14 (k14). Both polymerase chain reaction and Southern blot analysis showed that the sheep carried the ovine β-catenin gene and that the β-catenin gene could be stably inherited. To study the molecular responses to high expression of β-catenin, high-throughput RNA-seq technology was employed using three transgenic sheep and their wild-type siblings. These findings suggest that β-catenin normally plays an important role in wool follicle development by activating the downstream genes of the Wnt pathway and enhancing the expression of keratin protein genes and keratin-associated protein genes.

Solute carrier organic anion transporter 1B3 (SLCO1B3) is an important liver primarily highly expressed gene, its encoded protein (OATP1B3) involved in the transport of multi-specific endogenous and exogenous substances. We previously reported that an EAV-HP inserted mutation (IM+) in the 5ʹ flanking region of SLCO1B3 was the causative mutation of chicken blue eggs, and a further research showed that IM+ significantly reduced the expression of SLCO1B3 in liver. Herein, we confirmed a cholate response element (IR-1) played an important role in activating SLCO1B3 and in vitro experiments showed that the activation of IR-1 can be significantly reduced by the EAV-HP IM+ . We performed transcriptome and proteomic analysis using the same set of IM+ and IM− liver tissues from Yimeng hens (a Chinese indigenous breed) to study the effect of SLCO1B3 and OATP1B3 expression reduction on chicken liver function. The results showed that common differential expression pathways were screened out from both transcriptome and proteome, in which fatty acid metabolism and drug metabolism—cytochrome P450 were significantly enriched in the KEGG analysis. The lipid-related metabolism was weakened in IM+ group, which was validated by serum biochemical assay. We unexpectedly found that EAV-HP fragment was highly expressed in the liver of the IM+ chickens. We cloned the EAV-HP full-length transcript and obtained the complete open reading frame. It is worth noting that there was some immune related differential expressed genes, such as NFKBIZ, NFKBIA, and IL1RL1, which were higher expressed in the IM+ group, which may due to the high expression of EAV-HP. Our study showed that EAV-HP IM+ reduced the expression of SLCO1B3 in liver, resulting in the decrease of fatty metabolism and exogenous substance transport capacity. The mutation itself also expressed in the liver and may be involved in the immune process. The mechanism needs further study.