[The cell cycle pathway regulates chicken abdominal fat deposition as revealed by transcriptome sequencing].

With the improvement of growth traits and feed conversion rate, the abdominal fat rate of Chinese local breeds of broilers has been increasing. Excessive abdominal fat deposition not only reduces the slaughter rate and disease resistance of broiler chickens, but also produces waste due to the difficulty of fat treatment. In order to study the regulatory genes and pathways involved in abdominal fat deposition of broilers, we used high-fat diets to feed the Xinghua Chicken, which is a Chinese local breed. Two weeks after feeding, we found that the abdominal fat weight and rate of broilers in the high-fat diet group increased significantly, and the diameter and area of abdominal fat cells also increased significantly. Transcriptome sequencing of abdominal fat and livers showed that the differentially expressed genes in the abdominal fat were mainly enriched in the cell cycle, peroxisome proliferator- activated receptor (PPAR) and extracellular matrix (ECM) receptor signaling pathways. The differentially expressed genes in livers were also significantly enriched in the cell cycle pathway, as well as in the steroid biosynthesis and PPAR signaling pathway. By analyzing the common differentially expressed genes in abdominal fat and liver tissues, we found that these genes were also enriched in cell cycle. Finally, we used the chicken LMH (chicken hepatoma cell) cell line and chicken ICP (immortalized chicken preadipocytes) cell line to do the in vitro validation assays. We used high-fat and common medium to culture the cells. The results showed that after 48 hours, the high-fat medium could significantly promote cell cycle and increase the number of cells in S phase. Additionally, qRT-PCR results showed that the high-fat medium could significantly promote the expression of genes related to cell cycle. In conclusion, we found that high-fat diets activate the cell cycle progression of chicken hepatocytes and preadipocytes, promote cell proliferation, and then increase abdominal fat deposition.

The Long Intron 1 of Growth Hormone Gene from Reeves' Turtle (Chinemys reevesii) Correlates with Negatively Regulated GH Expression in Four Cell Lines.

Turtles grow slowly and have a long lifespan. Ultrastructural studies of the pituitary gland in Reeves' turtle (Chinemys reevesii) have revealed that the species possesses a higher nucleoplasmic ratio and fewer secretory granules in growth hormone (GH) cells than other animal species in summer and winter. C. reevesii GH gene was cloned and species-specific similarities and differences were investigated. The full GH gene sequence in C. reevesii contains 8517 base pairs (bp), comprising five exons and four introns. Intron 1 was found to be much longer in C. reevesii than in other species. The coding sequence (CDS) of the turtle's GH gene, with and without the inclusion of intron 1, was transfected into four cell lines, including DF-1 chicken embryo fibroblasts, Chinese hamster ovary (CHO) cells, human embryonic kidney 293FT cells, and GH4C1 rat pituitary cells; the turtle growth hormone (tGH) gene mRNA and protein expression levels decreased significantly in the intron-containing CDS in these cell lines, compared with that of the corresponding intronless CDS. Thus, the long intron 1 of GH gene in Reeves' turtle might correlate with downregulated gene expression.

Negative Glucocorticoid Response-Like Element from the First Intron of the Chicken Growth Hormone Gene Represses Gene Expression in the Rat Pituitary Tumor Cell Line.

The effects of introns, especially the first intron, on the regulation of gene expression remains unclear. Therefore, the objective of the present study was to investigate the transcriptional regulatory function of intron 1 on the chicken growth hormone (cGH) gene in the rat pituitary tumor cell line (GH4-C1). Transient transfection using first-intron-inserted cGH complete coding sequences (CDSs) and non-intron-inserted cGH CDS plasmids, quantitative RT-PCR (qRT-PCR) and western blot assays were used to detect the expression of cGH. The reporter gene assay was also used to investigate the effect of a series of fragments in the first intron of cGH on gene expression in GH4-C1. All of the results revealed that a 200-bp fragment located in the +485/+684 region of intron 1 was essential for repressing the expression of cGH. Further informatics analysis showed that there was a cluster of 13 transcriptional factor binding sites (TFBSs) in the +485/+684 region of the cGH intron 1. Disruption of a glucocorticoid response-like element (the 19-nucleotide sequence 5'-AGGCTTGACAGTGACCTCC-3') containing a T-box motif (TGACCT) located within this DNA fragment increased the expression of the reporter gene in GH4-C1. In addition, an electrophoretic mobility shift assay (EMSA) revealed a glucocorticoid receptor (GR) protein of rat binding to the glucocorticoid response-like element. Together, these results indicate that there is a negative glucocorticoid response-like element (nGRE) located in the +591/+609 region within the first intron of cGH, which is essential for the down-regulation of cGH expression.

Impact of GC content on gene expression pattern in chicken.

BACKGROUND: GC content varies greatly between different genomic regions in many eukaryotes. In order to determine whether this organization named isochore organization influences gene expression patterns, the relationship between GC content and gene expression has been investigated in man and mouse. However, to date, this question is still a matter for debate. Among the avian species, chicken (Gallus gallus) is the best studied representative with a complete genome sequence. The distinctive features and organization of its sequence make it a good model to explore important issues in genome structure and evolution. METHODS: Only nuclear genes with complete information on protein-coding sequence with no evidence of multiple-splicing forms were included in this study. Chicken protein coding sequences, complete mRNA sequences (or full length cDNA sequences), and 5' untranslated region sequences (5' UTR) were downloaded from Ensembl and chicken expression data originated from a previous work. Three indices i.e. expression level, expression breadth and maximum expression level were used to measure the expression pattern of a given gene. CpG islands were identified using hgTables of the UCSC Genome Browser. Correlation analysis between variables was performed by SAS Proprietary Software Release 8.1. RESULTS: In chicken, the GC content of 5' UTR is significantly and positively correlated with expression level, expression breadth, and maximum expression level, whereas that of coding sequences and introns and at the third coding position are negatively correlated with expression level and expression breadth, and not correlated with maximum expression level. These significant trends are independent of recombination rate, chromosome size and gene density. Furthermore, multiple linear regression analysis indicated that GC content in genes could explain approximately 10% of the variation in gene expression. CONCLUSIONS: GC content is significantly associated with gene expression pattern and could be one of the important regulation factors in the chicken genome.

Indel segregating within introns in the chicken genome are positively correlated with the recombination rates.

Insertions and deletions (Indel) are important sources of genetic diversity and phenotypic divergence. Many factors such as mutation, recombination, selection and genetic drift can jointly affect the indel distribution across the genome. Studies of the relationship between recombination and indel density can, to a certain extent, reflect the selective constrain on indel. Based on the improved genetic map, genome sequence assembly and the partial (0.25X) shotgun sequencing of three breeds of domestic chicken, we calculated the recombination rates and the indel density segregating within introns and intergenic for 4 Mb windows (n = 210). Regression analyses demonstrated that recombination rates are significantly correlated with intron indel density, but not with the intergenic indel density. After adjusted regional effect, the significant trend was remained. This implies that selection is an important factor to influence the indel distribution within introns in chicken genome. By contrast, the intergenic indel seem to be neutral. Since the intron indel density on Z chromosome is less than half of that on autosomes, we preliminarily deduced that genetic hitchhiking might be more important than background selection in producing the observed correlation. As these two processes are not mutually exclusive, it is most likely that both contribute somewhat to the observed pattern. In result similar to previous study, we also found SNP density is highly correlated with indel density. Based on this characteristic, a hypothesis suggested that there are common effects of mutation and/or selection on the occurrence of indel and point mutations. This hypothesis can not explain our observations.

863 genomes reveal the origin and domestication of chicken.

Despite the substantial role that chickens have played in human societies across the world, both the geographic and temporal origins of their domestication remain controversial. To address this issue, we analyzed 863 genomes from a worldwide sampling of chickens and representatives of all four species of wild jungle fowl and each of the five subspecies of red jungle fowl (RJF). Our study suggests that domestic chickens were initially derived from the RJF subspecies Gallus gallus spadiceus whose present-day distribution is predominantly in southwestern China, northern Thailand and Myanmar. Following their domestication, chickens were translocated across Southeast and South Asia where they interbred locally with both RJF subspecies and other jungle fowl species. In addition, our results show that the White Leghorn chicken breed possesses a mosaic of divergent ancestries inherited from other subspecies of RJF. Despite the strong episodic gene flow from geographically divergent lineages of jungle fowls, our analyses show that domestic chickens undergo genetic adaptations that underlie their unique behavioral, morphological and reproductive traits. Our study provides novel insights into the evolutionary history of domestic chickens and a valuable resource to facilitate ongoing genetic and functional investigations of the world's most numerous domestic animal.

Author Correction: 863 genomes reveal the origin and domestication of chicken.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Extent of linkage disequilibrium in wild and domestic chicken populations.

Linkage disequilibrium (LD) analyses play a fundamental role in gene mapping, both as a tool for fine mapping of complex trait gene and in genome-wide association studies. The use of LD analyses in practice depends crucially on the understanding of the patterns of LD in the genome. In the present study, a total of 36 SNP were selected initially in a region (200 kb) of Contig.060226.1 on GGA1 based on the average physical distance. After verifying their level of polymorphism, 21 SNP were selected finally to genotype one wild and two domestic chicken populations, Red Jungle Fowl (RJF), Taihe Silkie chicken (TS) and White Recessive Rock chicken (WRR). Two distinct measures of linkage disequilibrium, D' and r(2), between marker pairs were used. The D'map of RJF is spurious. Many marker pairs showed complete LD. TS and WRR showed distinct characteristic of decreasing D' value over increasing physical distance. The r(2) showed much less inflation than did D' in RJF population, and also showed a characteristic decreasing value over increasing physical distance. In TS and WRR populations, although the two measures differed in scale, their decay profiles were similar. The data in the present study suggested that the extent of LD in this region is about 150 kb, corresponding to 0.4 cM. Our results imply that a very dense map of SNP markers will be required for LD mapping methods. Thus, association studies based on polymorphisms within all known genes, and candidate QTL mapping, may ultimately prove to be a more effective strategy.

[Analysis on molecular evolution of MC1R gene in dog].

Melanocortin 1 receptor (MC1R) gene regulates pigment synthesis in mammals, and therefore is regarded as an important candidate gene for dog coat color. Based on MC1R amino acids and cDNA sequences of 10 vertebrate animals released by NCBI, molecular evolution of dog MC1R gene was analyzed with bioinformatic software and internet resource. Results showed that 10 vertebrate animals were divided into two major groups, a compact group A (7 mammals) and an incompact group B (chicken, zebrafish and fugu). This phylogenetic tree was consistent with putative evolutionary relationship within these 10 species. Positive selection was detected during the evolutionary process of dog (also cat and pig) from cattle by PAML branch model (omega = 90.8177), and five amino acids of 2V, 25E, 184N, 197V and 314L of dog MC1R were predicted under positive selection by site model. Comparative linkage analysis of chromosome showed that "ZFP276-MC1R-GAS8" linkage group was conservative in human, chimpanzee, chicken and dog.

[Single nucleotide polymorphism and its use in chicken QTL mapping].

Single nucleotide polymorphism (SNP) refers to the change of single nucleotide in DNA sequence. Because of its high density in genomes and easy in detection and analysis statistically,SNP can be used in genetic linkage map construction and QTL mapping.Here,the characters and detecting technology of SNP,as well as the status and foreground of the use of candidate gene SNP in chicken QTL mapping are introduced.