A Study on the Growth and Development Characteristics of Lindian Chickens.

As an excellent chicken breed found in a high-altitude zone of northern China, Lindian chickens are characterized by good egg and meat production, strong adaptability, cold tolerance, rough feeding resistance, excellent egg quality, and delicious meat quality. To facilitate the exploitation of the unique qualities of the Lindian chicken, the varying patterns and correlations of various body size and carcass traits of 3-22-week-old Lindian chickens were analyzed in this study. The optimal growth model of these traits was determined by growth curve fitting analysis. The results showed that most traits of Lindian chickens increased steadily with increasing age, and most of them increased rapidly before 10 weeks of age. In addition, the inflection point age of each trait was predicted to be between 4 and 10 weeks. Furthermore, this study revealed that body size traits were closely related to carcass traits in Lindian chickens. In summary, Lindian chickens are in a rapid growth stage before the age of 10 weeks, and better slaughter performance can be achieved through good feeding management during this stage. The reproductive traits and muscles are the main developmental focus after the age of 19 weeks, so it is important to adequately meet their energy requirements for subsequent good breeding performance.

HBP1 promotes chicken preadipocyte proliferation via directly repressing SOCS3 transcription.

Preadipocyte proliferation is an essential process in adipose development. During proliferation of preadipocytes, transcription factors play crucial roles. HMG-box protein 1 (HBP1) is an important transcription factor of cellular proliferation. However, the function and underlying mechanisms of HBP1 in the proliferation of preadipocytes remain unclear. Here, we found that the expression level of HBP1 decreased first and then increased during the proliferation of chicken preadipocytes. Knockout of HBP1 could inhibit the proliferation of preadipocytes, while overexpression of HBP1 could promote the proliferation of preadipocytes. ChIP-seq data showed that HBP1 had the unique DNA binding motif in chicken preadipocytes. By integrating ChIP-Seq and RNA-Seq, we revealed a total of 3 candidate target genes of HBP1. Furthermore, the results of ChIP-qPCR, RT-qPCR, luciferase reporter assay and EMSA showed that HBP1 could inhibit the transcription of suppressor of cytokine signaling 3 (SOCS3) by binding to its promoter. Moreover, we confirmed that SOCS3 can mediate the regulation of HBP1 on the proliferation of preadipocytes through RNAi and rescue experiments. Altogether, these data demonstrated that HBP1 directly targets SOCS3 to regulate chicken preadipocyte proliferation. Our findings expand the transcriptional regulatory network of preadipocyte proliferation, and they will be helpful in formulating a molecular breeding scheme to control excessive abdominal fat deposition and to improve meat quality in chickens.

Comparative analyses of laying performance and follicular development characteristics between fat and lean broiler lines.

The deposition of high levels of fat in broiler breeder hens can have a profound impact on follicular development and laying performance. This study was formulated with the goal of comparing egg production and follicular development characteristics at different laying stages in the Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF). The egg production was analyzed using the birds from the 19th to 24th generations of NEAUHLF; the follicular development characteristics were analyzed by hematoxylin-eosin staining and quantitative real-time polymerase chain reaction using the birds from the 24th generation of NEAUHLF. The results showed that the age at first egg of lean hens was significantly earlier than that of fat hens in this study. While no significant differences in total egg output from the first egg to 50 wk of age were noted when comparing these 2 chicken lines, lean hens laid more eggs from the first egg to 35 wk of age relative to fat hens, whereas fat hens laid more eggs from wk 36 to 42 and 43 to 50 relative to their lean counterparts. No differences in ovarian morphology and small yellow follicle (SYF) histological characteristics were noted when comparing these 2 chicken lines at 27 wk of age. At 35 and 52 wk of age, however, lean hens exhibited significantly lower ovarian weight, ovarian proportion values, numbers of hierarchical follicles, hierarchical follicle weight, and SYF granulosa layer thickness as compared to fat hens, together with a significant increase in the number of prehierarchical follicles relative to those in fat hens. Gene expression analyses suggested that follicle selection was impaired in the fat hens in the early laying stage, whereas both follicle selection and maturation were impaired in the lean hens in the middle and late laying stages. Overall, these data highlight that fat deposition in broiler hens can have a range of effects on follicular development and egg production that are laying stage-dependent.

Associations of Transcription Factor 21 Gene Polymorphisms with the Growth and Body Composition Traits in Broilers.

This study aims to identify molecular marker loci that could be applied in broiler breeding programs. In this study, we used public databases to locate the Transcription factor 21 (TCF21) gene that affected the economically important traits in broilers. Ten single nucleotide polymorphisms were detected in the TCF21 gene by monoclonal sequencing. The polymorphisms of these 10 SNPs in the TCF21 gene were significantly associated (p < 0.05) with multiple growth and body composition traits. Furthermore, the TT genotype of g.-911T>G was identified to significantly increase the heart weight trait without affecting the negative traits, such as abdominal fat and reproduction by multiple methods. Thus, it was speculated that the g.-911T>G identified in the TCF21 gene might be used in marker-assisted selection in the broiler breeding program.

Genetic parameters estimation and genome-wide association studies for internal organ traits in an F2 chicken population.

Chicken internal organs are indispensable parts of the body, but their genetic architectures have not been commonly understood. Herein, we estimated the genetic parameters for heart weight (HW), liver weight (LW), spleen weight (SpW), testis weight (TW), glandular stomach weight (GSW), muscular stomach weight (MSW) and identified single nucleotide polymorphisms (SNPs) and potential candidate genes associated with internal organ weights in an F2 population constructed by crossing broiler cocks derived from Arbor Acres with high abdominal fat content and Baier layer dams (a Chinese native breed). The restricted maximum likelihood (REML) method was applied for genetic parameters estimation of internal organ weights using GCTA software. The results showed that heritabilities of internal organ traits ranged from 0.336 to 0.673 and most of the genetic and phenotypic correlations amongst internal organs weights were positive. A genome-wide association study (GWAS) was performed based on a mixed linear model (MLM) in GEMMA software. Genotypic data were produced from the whole genome re-sequenced (26 F0 individuals were re-sequenced at 10 × coverage; 519 F2 individuals were re-sequenced at 3 × coverage). A total of 7,890,258 SNPs remained to be analysed after quality control and genotype imputation. The GWAS results indicated that significant SNPs responsible for internal organ traits were scattered on the different chicken chromosomes 1-5, 8, 11, 14, 16, 18, 19 and 27. Amongst the annotated genes, fibronectin type III domain containing 3A (FNDC3A), LOC101748122, membrane palmitoylated protein 6 (MPP6), LOC107049584 and KAT8 regulatory NSL complex subunit 1 (KANSL1) were the most promising candidates for internal organ traits. The findings will provide instrumental information for understanding the genetic basis of internal organ development.

Genome-wide association studies for growth traits in broilers.

BACKGROUND: The identification of markers and genes for growth traits may not only benefit for marker assist selection /genomic selection but also provide important information for understanding the genetic foundation of growth traits in broilers. RESULTS: In the current study, we estimated the genetic parameters of eight growth traits in broilers and carried out the genome-wide association studies for these growth traits. A total of 113 QTNs discovered by multiple methods together, and some genes, including ACTA1, IGF2BP1, TAPT1, LDB2, PRKCA, TGFBR2, GLI3, SLC16A7, INHBA, BAMBI, APCDD1, GPR39, and GATA4, were identified as important candidate genes for rapid growth in broilers. CONCLUSIONS: The results of this study will provide important information for understanding the genetic foundation of growth traits in broilers.

Genome-wide survey and functional analysis reveal TCF21 promotes chicken preadipocyte differentiation by directly upregulating HTR2A.

BACKGROUND/AIM: Previously, we showed that transcription factor 21 (TCF21) promotes chicken preadipocyte differentiation. However, the genome-wide TCF21 binding sites and its downstream target genes in chicken adipogenesis were unknown. METHODS: ChIP-Seq and RNA-Seq were used to screen candidate targets of TCF21. qPCR and luciferase reporter assay were applied to verify the sequencing results. Western blotting, oil red-O staining and pharmacological treatments were performed to investigate the function of 5-hydroxytryptamine receptor 2A (HTR2A), one of the bonafide direct downstream binding targets of TCF21. RESULTS: A total of 94 candidate target genes of TCF21 were identified. ChIP-qPCR, RT-qPCR, and luciferase reporter assay demonstrated that HTR2A is one of the bonafide direct downstream binding targets of TCF21. HTR2A expression in adipose tissue was upregulated in fat line broilers. Also, the abundance of HTR2A gradually increased during the adipogenesis process. Interestingly, pharmacological enhancement or inhibition of HTR2A promoted or attenuated the differentiation of preadipocytes, respectively. Furthermore, HTR2A inhibition impaired the TCF21 promoted adipogenesis. CONCLUSIONS: We profiled the genome-wide TCF21 binding sites in chicken differentiated preadipocytes revealing HTR2A as the direct downstream target of TCF21 in adipogenesis.

Estimation of the genetic parameters of traits relevant to feed efficiency: result from broiler lines divergent for high or low abdominal fat content.

Feed consumption represents a major cost in poultry production and improving feed efficiency is one of the important goals in breeding strategies. The present study aimed to analyze the relationship between feed efficiency and relevant traits and find the proper selection method for improving feed efficiency by using the Northeast Agricultural University High and Low Fat broiler lines that were divergently selected for abdominal fat content. A total of 899 birds were used to measure the feed intake (FI), abdominal fat weight (AFW), and body weight traits. The abdominal fat percentage (AFP), feed conversion ratio (FCR), and the residual feed intake (RFI) were calculated for each individual broiler. The differences in the AFW, AFP, and in traits relevant to feed efficiency, such as FCR and RFI, between the fat line and the lean line were analyzed, and the genetic parameters were estimated for AFW, AFP, and feed efficiency relevant traits. The results showed that AFW, AFP, body weight gain (BWG), FI, FCR, and RFI were significantly higher in the fat line compared with the lean line. The heritability of FI, BWG, FCR, RFI, AFW, and AFP were 0.45, 0.28, 0.36, 0.38, 0.33, and 0.30, respectively. Both FCR and RFI showed high positive genetic correlations with FI, AFW, and AFP and relatively low, negative genetic correlations with BWG. The RFI showed much higher positive genetic correlation with the abdominal fat traits than FCR. In addition, the FCR showed negative genetic correlation with body weight of 4 wk (BW4) and 7 wk (BW7), whereas RFI showed positive genetic correlation with BW4 and BW7. The results showed that both RFI and FCR could be used for improving feed efficiency. When selecting against RFI, the AFP could be significantly reduced, and by selecting against FCR, the body weight could be improved simultaneously.

Multi-Omics Association Reveals the Effects of Intestinal Microbiome-Host Interactions on Fat Deposition in Broilers.

Growing evidence indicates that gut microbiota factors cannot be viewed as independent in the occurrence of obesity. Because the gut microbiome is highly dimensional and complex, studies on interactions between gut microbiome and host in obesity are still rare. To explore the relationship of gut microbiome-host interactions with obesity, we performed multi-omics associations of gut metagenome, intestinal transcriptome, and host obesity phenotypes in divergently selected obese-lean broiler lines. Metagenomic shotgun sequencing generated a total of 450 gigabases of clean data from 80 intestinal segment contents of 20 broilers (10 of each line). The microbiome comparison showed that microbial diversity and composition in the duodenum, jejunum, ileum, and ceca were altered variously between the lean- and fat-line broilers. We identified two jejunal microbes (Escherichia coli and Candidatus Acetothermia bacterium) and four cecal microbes (Alistipes sp. CHKCI003, Ruminococcaceae bacterium CPB6, Clostridiales bacterium, and Anaeromassilibacillus sp. An200), which were significantly different between the two lines (FDR < 0.05). When comparing functional metagenome, the fat-line broilers had an intensive microbial metabolism in the duodenum and jejunum but degenerative microbial activities in the ileum and ceca. mRNA-sequencing identified a total of 1,667 differentially expressed genes (DEG) in the four intestinal compartments between the two lines (| log2FC| > 1.5 and FDR < 0.05). Multi-omics associations showed that the 14 microbial species with abundances that were significantly related with abdominal fat relevant traits (AFRT) also have significant correlations with 155 AFRT-correlated DEG (p < 0.05). These DEG were mainly involved in lipid metabolism, immune system, transport and catabolism, and cell growth-related pathways. The present study constructed a gut microbial gene catalog of the obese-lean broiler lines. Intestinal transcriptome and metagenome comparison between the two lines identified candidate DEG and differential microbes for obesity, respectively. Multi-omics associations suggest that abdominal fat deposition may be influenced by the interactions of specific gut microbiota abundance and the expression of host genes in the intestinal compartments in which the microbes reside. Our study explored the interactions between gut microbiome and host intestinal gene expression in lean and obese broilers, which may expand knowledge on the relationships between obesity and gut microbiome.

Microevolutionary Dynamics of Chicken Genomes under Divergent Selection for Adiposity.

Decades of artificial selection have significantly improved performance and efficiency of animal production systems. However, little is known about the microevolution of genomes due to intensive breeding. Using whole-genome sequencing, we document dynamic changes of chicken genomes under divergent selection on adiposity over 19 generations. Directional selection reduced within-line but increased between-line genomic differences. We observed that artificial selection tended to result in recruitment of preexisting variations of genes related to adipose tissue growth. In addition, novel mutations contributed to divergence of phenotypes under selection but contributed significantly less than preexisting genomic variants. Integration of 15 generations genome sequencing, genome-wide association study, and multi-omics data further identified that genes involved in signaling pathways important to adipogenesis, such as autophagy and lysosome (URI1, MBL2), neural system (CHAT), and endocrine (PCSK1) pathways, were under strong selection. Our study provides insights into the microevolutionary dynamics of domestic animal genomes under artificial selection.

RXRα Positively Regulates Expression of the Chicken PLIN1 Gene in a PPARγ-Independent Manner and Promotes Adipogenesis.

Perilipin1 (PLIN1), the most abundant lipid droplet (LD)-associated protein, plays a vital role in regulating lipid storage and breakdown in adipocytes. Recently, we found that the overexpression of PLIN1 promotes chicken preadipocyte lipid accumulation. However, the mechanisms by which transcription of the chicken PLIN1 gene is regulated remain unknown. In this study, we investigated the role of retinoid X receptor α (RXRα) in transcription of the chicken PLIN1 gene. Notably, reporter gene and expression assays showed that RXRα activates transcription of the chicken PLIN1 gene in a PPARγ-independent manner. Furthermore, promoter deletion and electrophoretic mobility shift assay (EMSA) analysis revealed that the chicken PLIN1 gene promoter region (-774/-785) contains an RXRα-binding site. Further study demonstrated that RXRα overexpression promotes differentiation of an immortalized chicken preadipocyte cell line (ICP1), causing a concomitant increase in PLIN1 transcripts. Taken together, our results show for the first time that RXRα activates transcription of the chicken PLIN1 gene in a PPARγ-independent manner, which might be at least in part responsible for RXRα-induced adipogenesis.

Functional Intronic Variant in the Retinoblastoma 1 Gene Underlies Broiler Chicken Adiposity by Altering Nuclear Factor-kB and SRY-Related HMG Box Protein 2 Binding Sites.

The present study aimed to search for chicken abdominal fat deposition-related polymorphisms within RB1 and to provide functional evidence for significantly associated genetic variants. Association analyses showed that 11 single nucleotide polymorphisms (SNPs) in intron 17 of RB1, were significantly associated with both abdominal fat weight (P < 0.05) and abdominal fat percentage (P < 0.05). Functional analysis revealed that the A allele of g.32828A>G repressed the transcriptional efficiency of RB1 in vitro, through binding nuclear factor-kappa B (NF-KB) and SRY-related HMG box protein 2 (SOX2). Furthermore, RB1 mRNA expression levels in the abdominal fat tissue of individuals with the A/A genotype of g.32828A>G were lower than those of individuals with the G/G genotype. Collectively, we propose that the intronic SNP g.32828A>G of RB1 is an obesity-associated variant that directly affects binding with NF-KB and SOX2, leading to changes in RB1 expression which in turn may influence chicken abdominal fat deposition.

Genome-wide association study for plasma very low-density lipoprotein concentration in chicken.

The plasma very low-density lipoprotein (VLDL) concentration is an effective blood biochemical indicator that could be used to select lean chicken lines. In the current study, we used Genome-wide association study (GWAS) method to detect SNPs with significant effects on plasma VLDL concentration. As a result, 38 SNPs significantly associated with plasma VLDL concentration were identified using at least one of the three mixed linear model (MLM) packages, including GRAMMAR, EMMAX and GEMMA. Nearly, all these SNPs with significant effects on plasma VLDL concentration (except Gga_rs16160897) have significantly different allele frequencies between lean and fat lines. The 1-Mb regions surrounding these 38 SNPs were extracted, and twelve important regions were obtained after combining the overlaps. A total of 122 genes in these twelve important regions were detected. Among these genes, LRRK2, ABCD2, TLR4, E2F1, SUGP1, NCAN, KLF2 and RAB8A were identified as important genes for plasma VLDL concentration based on their basic functions. The results of this study may supply useful information to select lean chicken lines.

A Novel Regulator of Preadipocyte Differentiation, Transcription Factor TCF21, Functions Partially Through Promoting LPL Expression.

The transcription factor TCF21 has been previously shown to be specifically expressed in white preadipocytes in mice. However, the exact biological function of TCF21 in the context of adipogenesis remains unknown. In the current study, we used chicken lines selected based on their abdominal fat content, and observed a significant decrease in TCF21 mRNA and protein levels in the abdominal fat of lean broilers relative to fat broilers. Moreover, TCF21 expression increased throughout preadipocyte differentiation in vitro. We also found that TCF21 knockdown and over-expression attenuated and promoted preadipocyte differentiation, respectively, as evidenced by appropriate changes in lipid droplet accumulation and altered expressions of C/EBPa, LPL, and A-FABP. Additional chromatin immunoprecipitation analyses and luciferase assays demonstrated that TCF21 promotes the transcription of LPL by directly binding to the E-box motif in the LPL promoter. Together, these results show that TCF21 is a novel regulator of preadipocyte differentiation, in part by directly promoting LPL expression.

Production of transgenic broilers by non-viral vectors via optimizing egg windowing and screening transgenic roosters.

The generation of transgenic chickens is of both biomedical and agricultural significance, and recently chicken transgenesis technology has been greatly advanced. However, major issues still exist in the efficient production of transgenic chickens. This study was designed to optimize the production of enhanced green fluorescence protein (EGFP)-transgenic broilers, including egg windowing at the blunt end (air cell) of egg, and the direct transfection of circulating primordial germ cells by microinjection of the Tol2 plasmid-liposome complex into the early embryonic dorsal aorta. For egg windowing, we discovered that proper manipulation of the inner shell membrane at the blunt end could improve the rate of producing G0 transgenic roosters. From 27 G0 roosters, we successfully collected semen with EGFP-positive sperms from 16 and 19 roosters after direct fluorescence observation and fluorescence-activated cell sorting analyses (13 detected by both methods), respectively. After artificial insemination using the G0 rooster with the highest number of EGFP fluorescent sperm, one G1 EGFP transgenic broiler (1/81, 1.23%) was generated. Our results indicate that appropriate egg windowing and screening of potentially transgene-positive roosters can improve the production of germline-transmitted transgenic birds.

Effect of IGFBP2 Overexpression on the Expression of Fatty Acid Synthesis Genes in Primary Cultured Chicken Hepatocytes.

The effects of insulin-like growth factor binding protein 2 (IGFBP2) on the expression of fatty acid synthesis regulators and triglyceride production were investigated in primary cultured chicken hepatocytes. The full-length chicken IGFBP2 coding region was synthesized by overlap extension PCR and cloned into the pcDNA3.1 vector. An in situ digestion method was used to prepare the chicken hepatocytes. Primary chicken hepatocytes were maintained in monolayer culture. Real-time PCR was used to detect changes in the expression of IGFBP2, PPARG, IGF1, IGF1R, APOAI, and LFABP, after the overexpression of IGFBP2 in chicken hepatocytes. Triglyceride production and glucose content were also evaluated using triglyceride and glucose analysis methods. The expression level of IGFBP2 increased after transfection of the IGFBP2-containing vector. The expression levels of PPARG, IGF1, and IGF1R also increased in cultured chicken hepatocytes after the overexpression of IGFBP2, whereas the expression of LFABP and APOAI decreased. Triglyceride production in primary cultured chicken hepatocytes increased after the overexpression of IGFBP2. These results suggest that IGFBP2 is involved in lipogenesis, increasing both the expression of fatty acid synthesis regulators, and triglyceride production in primary cultured chicken hepatocytes.

Structural Characterization and Association of Ovine Dickkopf-1 Gene with Wool Production and Quality Traits in Chinese Merino.

Dickkopf-1 (DKK1) is an inhibitor of canonical Wnt signaling pathway and regulates hair follicle morphogenesis and cycling. To investigate the potential involvement of DKK1 in wool production and quality traits, we characterized the genomic structure of ovine DKK1, performed polymorphism detection and association analysis of ovine DKK1 with wool production and quality traits in Chinese Merino. Our results showed that ovine DKK1 consists of four exons and three introns, which encodes a protein of 262 amino acids. The coding sequence of ovine DKK1 and its deduced amino acid sequence were highly conserved in mammals. Eleven single nucleotide polymorphisms (SNPs) were identified within the ovine DKK1 genomic region. Gene-wide association analysis showed that SNP5 was significantly associated with mean fiber diameter (MFD) in the B (selected for long wool fiber and high-quality wool), PW (selected for high reproductive capacity, high clean wool yield and high-quality wool) and U (selected for long wool fiber with good uniformity, high wool yield and lower fiber diameter) strains (p < 4.55 × 10-3 = 0.05/11). Single Nucleotide Polymorphisms wide association analysis showed that SNP8 was significantly associated with MFD in A strain and fleece weight in A (selected for large body size), PM (selected for large body size, high reproductive capacity and high meat yield) and SF (selected for mean fiber diameter less than 18 µm and wool fiber length between 5 and 9 cm) strains (p < 0.05), SNP9 was significantly associated with curvature in B and U strains (p < 0.05) and SNP10 was significantly associated with coefficient of variation of fiber diameter in A, PW and PM strains and standard deviation of fiber diameter in A and PM strains (p < 0.05). The haplotypes derived from these 11 identified SNPs were significantly associated with MFD (p < 0.05). In conclusion, our results suggest that DKK1 may be a major gene controlling wool production and quality traits, also the identified SNPs (SNPs5, 8, 9 and 10) might be used as potential molecular markers for improving sheep wool production and quality in sheep breeding.

Differential expression of six genes and correlation with fatness traits in a unique broiler population.

Previous results from genome wide association studies (GWASs) in chickens divergently selected for abdominal fat content of Northeast Agricultural University (NEAUHLF) showed that many single nucleotide polymorphism (SNP) variants were associated with abdominal fat content. Of them, six top significant SNPs at the genome level were located within SRD5A3, SGCZ, DLC1, GBE1, GALNT9 and DNAJB6 genes. Here, expression levels of these six candidate genes were investigated in abdominal fat and liver tissue between fat and lean broilers from the 14th generation population of NEAUHLF. The results showed that expression levels of SRD5A3, SGCZ and DNAJB6 in the abdominal fat and SRD5A3, DLC1, GALNT9, DNAJB6 and GBE1 in the liver tissue differed significantly between the fat and lean birds, and were correlated with abdominal fat traits. The findings will provide important references for further function investigation of the six candidate genes involved in abdominal fat deposition in chickens.

Functional analysis of the upstream regulatory region of chicken miR-17-92 cluster.

miR-17-92 cluster plays important roles in cell proliferation, differentiation, apoptosis, animal development and tumorigenesis. The transcriptional regulation of miR-17-92 cluster has been extensively studied in mammals, but not in birds. To date, avian miR-17-92 cluster genomic structure has not been fully determined. The promoter location and sequence of miR-17-92 cluster have not been determined, due to the existence of a genomic gap sequence upstream of miR-17-92 cluster in all the birds whose genomes have been sequenced. In this study, genome walking was used to close the genomic gap upstream of chicken miR-17-92 cluster. In addition, bioinformatics analysis, reporter gene assay and truncation mutagenesis were used to investigate functional role of the genomic gap sequence. Genome walking analysis showed that the gap region was 1704 bp long, and its GC content was 80.11%. Bioinformatics analysis showed that in the gap region, there was a 200 bp conserved sequence among the tested 10 species (Gallus gallus, Homo sapiens, Pan troglodytes, Bos taurus, Sus scrofa, Rattus norvegicus, Mus musculus, Possum, Danio rerio, Rana nigromaculata), which is core promoter region of mammalian miR-17-92 host gene (MIR17HG). Promoter luciferase reporter gene vector of the gap region was constructed and reporter assay was performed. The result showed that the promoter activity of pGL3-cMIR17HG (-4228/-2506) was 417 times than that of negative control (empty pGL3 basic vector), suggesting that chicken miR-17-92 cluster promoter exists in the gap region. To further gain insight into the promoter structure, two different truncations for the cloned gap sequence were generated by PCR. One had a truncation of 448 bp at the 5'-end and the other had a truncation of 894 bp at the 3'-end. Further reporter analysis showed that compared with the promoter activity of pGL3-cMIR17HG (-4228/-2506), the reporter activities of the 5'-end truncation and the 3'-end truncation were reduced by 19.82% and 60.14%, respectively. These data demonstrated that the important promoter region of chicken miR-17-92 cluster is located in the -3400/-2506 bp region. Our results lay the foundation for revealing the transcriptional regulatory mechanisms of chicken miR-17-92 cluster.

Differential expression of six chicken genes associated with fatness traits in a divergently selected broiler population.

A genome-wide association study has shown a number of chicken (Gallus gallus) single nucleotide polymorphism (SNP) markers to be significantly associated with abdominal fat content in Northeast Agricultural University (NEAU) broiler lines selected divergently for abdominal fat content (NEAUHLF). The six significant SNPs are located in the kinase insert domain receptor (KDR), tumor suppressor candidate 3 (TUSC3), phosphoribosyl pyrophosphate amidotransferase (PPAT), exocyst complex component 1 (EXOC1), v-myb myeloblastosis viral oncogene homolog (avian)-like 2 (MYBL2) and KIAA1211 (undefined) genes. In this study, the expression levels of these genes were investigated in both abdominal fat and liver tissues using 32 14th generation chickens from the NEAUHLF. The levels of expression of KDR in abdominal fat and KDR and TUSC3 in liver differed significantly between the two lines. The expression level of KDR in the abdominal fat was significantly correlated with the abdominal fat weight (AFW) and abdominal fat percentage (AFP). The expression levels of KDR, TUSC3 and PPAT in liver were significantly correlated with AFW and AFP, indicating that the six genes, especially KDR and TUSC3, could be associated with fat traits in domestic chickens. This study could provide insight into the mechanisms underlying the formation of abdominal fat in chickens.