Krüppel-like factor KLF9 inhibits chicken intramuscular preadipocyte differentiation.

1. Poultry meat quality is affected by many factors, among which intramuscular fat (IMF) is predominant. IMF content affects tenderness, juiciness and flavour of meat. Krüppel-like transcriptional factors (KLFs) are important regulators of adipocyte differentiation. However, little is known about the KLF9 gene associated with poultry IMF deposition, especially intramuscular adipocyte differentiation.2. Previous work has shown that chicken KLF9 was differentially expressed during adipogenesis of intramuscular preadipocytes differentiation. In this study, the function of KLF9 in chicken intramuscular preadipocytes differentiation was investigated.3. In the chicken preadipocyte differentiation model, KLF9 expression showed a major increase with adipogenic induction. Overexpression of KLF9 down-regulated the expression of the adipogenic marker gene AP2, and impaired triglyceride accumulation. Knockdown of KLF9 in chicken intramuscular preadipocytes increased the expression of PPARG, CEBPA and AP2. In addition, it was proposed that KLF9 may regulate adipogenesis via lncRNAs NONGGAT002209.2, NONGGAT003346.2, NONGGAT000436.2 and NONGGAT006302.2 in chicken.4. The data supported a novel role of KLF9 in regulating chicken intramuscular preadipocyte differentiation. Such findings may contribute to a more thorough understanding of chicken IMF deposition and the improvement of poultry meat quality.

Effects of different starch sources on glucose and fat metabolism in broiler chickens.

1. The aim of the present study was to investigate the effects of different starch sources (corn, wheat, and rice) on the blood glucose level, glycogen content of liver and muscle, expression of GSK-3ß and FAS mRNA, abdominal fat weight and abdominal fat deposition in broiler chickens. 2. A total of 360, one-day-old AA (Arbor Acres) broiler chickens were randomly assigned to three treatment groups, each with six replicates, consisting of 20 chickens per replicate, and fed either a corn-, wheat- or rice-based diet for 21 days. The chickens were then subdivided into groups A and B, and the chickens in these two subgroups were processed or sampled for 28 days, respectively. 3. The results indicated that post-prandial time significantly affected the glucose concentration, glycogen content in the liver and breast muscle and expression of GSK-3ß and FAS mRNAs (P < 0.05). The expression of the GSK-3ß gene in the chicken liver of the corn-based diet group was higher (P < 0.05) than that in the wheat-based diet group, and the expression of the FAS gene in the corn-based diet group was lower (P < 0.05) than that in the wheat-based and rice-based diet groups. Abdominal fat weight and deposition in the corn-based diet group were lower than those of the wheat-based and rice-based diet groups, but these differences were not significant (P > 0.05). 4. The results suggested that the efficiency of glucose absorption in animals might have an effect on the fat deposition efficiency in the liver and that diets with different starch sources might affect fat deposition in chickens.

Genetic diversity of mtDNA D-loop sequences in four native Chinese chicken breeds.

1. To explore the genetic diversity of Chinese indigenous chicken breeds, a 585 bp fragment of the mitochondrial DNA (mtDNA) region was sequenced in 102 birds from the Xichuan black-bone chicken, Yunyang black-bone chicken and Lushi chicken. In addition, 30 mtDNA D-loop sequences of Silkie fowls were downloaded from NCBI. The mtDNA D-loop sequence polymorphism and maternal origin of 4 chicken breeds were analysed in this study. 2. The results showed that a total of 33 mutation sites and 28 haplotypes were detected in the 4 chicken breeds. The haplotype diversity and nucleotide diversity of these 4 native breeds were 0.916 ± 0.014 and 0.012 ± 0.002, respectively. Three clusters were formed in 4 Chinese native chickens and 12 reference breeds. Both the Xichuan black-bone chicken and Yunyang black-bone chicken were grouped into one cluster. Four haplogroups (A, B, C and E) emerged in the median-joining network in these breeds. 3. It was concluded that these 4 Chinese chicken breeds had high genetic diversity. The phylogenetic tree and median network profiles showed that Chinese native chickens and its neighbouring countries had at least two maternal origins, one from Yunnan, China and another from Southeast Asia or its surrounding area.

Promoter analysis and tissue expression of the chicken ASB15 gene.

1. This study was conducted to explore the promoter region of the chicken ASB15 gene by detecting the activities of the dual luciferase reporter gene and to assess expression profiles of the ASB15 gene in 10 different tissues from Gushi chickens. 2. Five dual luciferase reporter gene vectors were constructed and transfected into DF1 cells. The activities of recombined plasmids were measured and the core promoter was confirmed by bioinformatic analysis. Total RNA was extracted and the relative expression of the ASB15 gene was examined. 3. Data analysis indicated that the promoter was located from -955 to -212 bp. Results showed that the chicken ASB15 gene was expressed in heart, breast muscle and leg muscle. 4. This study has confirmed the promoter region and the expression profile of the chicken ASB15 gene, which provides a foundation for further exploring its transcriptional regulation and function.

Polymorphisms of Flanking Region of the ASB15 Gene and Their Associations with Performance Traits in Chicken.

Research on the identity of genes and their relationship with traits of economic importance in chickens could assist in the selection of poultry. In this study, an F2 resource population of Gushi chickens crossed with Anka broilers was used to detect single-nucleotide polymorphisms (SNPs) in the flanking region of the ASB15 gene by DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). One SNP of -1271 C>T in 5' flanking region of the chicken ASB15 gene and two SNPs of the 10618 A>G and 10716 G>A in 3' flanking region were identified. Furthermore, the 10618 A>G and 10716 G>A in 3' flanking region were in complete linkage. Association analysis results showed that -1271 C>T was not associated with performance traits, while the 10618 A>G and 10716 G>A were significantly associated with BW2, 4, 6, 8, 10, 12, SL12, CD8, CW4, 8, 12, BSL4, 8, 12, and SEW, EW, WW, BMW, LW, CW, SFT. Our results suggest that the ASB15 gene profoundly affects chicken performance traits.

Assessment of correlation between pre-miRNA-1757 polymorphism and chicken performance traits.

Single nucleotide polymorphism in microRNAs (miRNA) may influence their target gene selection and regulation efficiency, leading to animal phenotypic variation. The aim of this study was to evaluate the possible effect of single nucleotide polymorphisms in the miRNA-1757 gene precursor region (pre-mir-1757) on economic-related traits in chicken. Genotyping was performed using Sequenom MassArray® iPLEX GOLD System. Association analysis was performed using SPSS19.0. The data showed that the G/C polymorphism was significantly correlated with semi-evisceration weight, evisceration weight, carcass weight, body weight at 10 weeks of age, shank length at 4 weeks of age, pectoral angle at 8 weeks of age, and body slanting length and pelvis breadth at 12 weeks of age (P < 0.05), and led to the alteration of the RNA secondary structure of pre-mir-1757. Our results provide useful information for further annotation studies of miRNA function.

Identification of single nucleotide polymorphisms in the ASB15 gene and their associations with chicken growth and carcass traits.

ASB15 is a member of the ankyrin repeat and suppressor of cytokine signaling box family, and is predominantly expressed in skeletal muscle. In the present study, an F2 resource population of Gushi chickens crossed with Anka broilers was used to investigate the genetic effects of the chicken ASB15 gene. Two single nucleotide polymorphisms (SNPs) (rs315759231 A>G and rs312619270 T>C) were identified in exon 7 of the ASB15 gene using forced chain reaction-restriction fragment length polymorphism and DNA sequencing. One was a missense SNP (rs315759231 A>G) and the other was a synonymous SNP (rs312619270 T>C). The rs315759231 A>G polymorphism was significantly associated with body weight at birth, 12-week body slanting length, semi-evisceration weight, evisceration weight, leg muscle weight, and carcass weight (P < 0.05). The rs312619270 T>C polymorphism was significantly associated with body weight at birth, 4, 8, and 12-week body weight, 8-week shank length, 12-week breast bone length, 8 and 12-week body slanting length, breast muscle weight, and carcass weight (P < 0.05). Our results suggest that the ASB15 gene profoundly affects chicken growth and carcass traits.

A novel 2-bp indel within Krüppel-like factor 15 gene (KLF15) and its associations with chicken growth and carcass traits.

1. The Krüppel-like factor (KLF) family of zinc-finger transcription factors plays a critical role in cell differentiation, phenotypic modulation and physiologic function. KLF15 has been proposed to regulate adipogenesis and gluconeogenesis. The objective of this study was to establish the association between KLF15 gene polymorphism and chicken growth and carcass traits. 2. An F2resource population of Gushi chickens crossed with Anka broilers was used to investigate the genetic effects of the chicken KLF15 gene. A 2-bp indel mutation (G13781_13782del/insAG) within intron 2 was detected, and a polymerase chain reaction-restriction fragment length polymorphism method was developed to genotype the F2 individuals. 3. Association analysis showed that the Single Nucleotide Polymorphisms (SNP) was significantly associated with chicken growth and carcass traits. The chickens with the insAG/insAG genotype generally had a significantly higher body weight and size than other genotypes. Gene expression for each genotype showed that birds carrying insAG/insAG had a higher expression level than the other genotypes. 4. The results suggested that this polymorphic site may serve as a useful target for marker assisted selection of chicken growth and carcass traits.

Adjacent SNPs in the transcriptional regulatory region of the FADS2 gene associated with fatty acid and growth traits in chickens.

Delta-6 fatty acid desaturases are rate-limiting desaturases involved in metabolic processes of fatty acids, and they are encoded by the FADS2 gene. In the current study, an F2 resource population of Gushi chickens crossed with Anak broilers was used to investigate the genetic effects of the chicken FADS2. Two adjacent single nucleotide polymorphisms (SNPs) (g.4290C>G and g.4291C>A) were identified in the transcriptional regulatory region of the FADS2 gene by means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and created restriction site-PCR-RFLP. Associations between the two SNPs with chicken fatty acid contents and growth traits were determined using linkage disequilibrium, haplotype construction, and association analysis. The two SNPs and their haplotype combinations were significantly associated with linoleic acid (C18:2), α-linolenic acid (C18:3), arachidonic acid (C20:4), body weight (BW)2, BW4, BW6, shank girth (SG)4, and breast bone length 4 (P<0.05). These results suggested that the SNPs of the FADS2 gene affected the content of essential fatty acid in muscle, and played a role in the early-stage growth rate of chickens.

A novel 31-bp indel in the paired box 7 (PAX7) gene is associated with chicken performance traits.

1. A novel 31-bp indel polymorphism in intron 3 of the chicken paired box 7 (PAX7) gene was identified and genotyped in an F2 resource population of Gushi chicken crossed with Anka broiler to analyse its associations with chicken growth, carcass and meat quality traits. 2. Results showed that the 31-bp indel was significantly associated with body weight at 4, 6, 8, 10, 12 weeks of age and body size indices including shank length, shank girth, body slanting length at 8 and 12 weeks of age. Significant associations were found for carcass weight, semi-evisceration weight, evisceration weight, breast muscle fibre diameter, leg muscle fibre diameter, breast muscle fibre density, while no significant association with leg muscle fibre density was observed. 3. It was concluded that the 31-bp indel in intron 3 of the PAX7 gene was associated with chicken growth, carcass and meat quality traits where the 31-bp deletion had a negative effect on chicken growth and carcass traits and positive effect on meat quality traits.

Deposition rule of yolk cholesterol in two different breeds of laying hens.

The objective of this study was to investigate the deposition rule of yolk cholesterol in Lushi Green-shelled and Silky Fowl layers. A total of 90 layers of each breed were selected at an age of 15 to 51 weeks. Productive performance was recorded on a weekly basis, whereas yolk cholesterol was determined at 4-week intervals from 21 to 51 weeks of age. The average yolk cholesterol content of Silky Fowl layers during the laying period was higher than that of Lushi Green-shelled layers (58.16 and 49.67%, P > 0.05). Yolk cholesterol content decreased at 21 to 31 weeks of the laying period, whereas a non-significant increasing trend was observed during 31 to 51 weeks of laying period. In conclusion, yolk cholesterol content is not only dependent on the age of hen but also the breed of layers.

Two novel SNPs of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene associated with growth and meat quality traits in the chicken.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is rate-limiting for metabolism of cholesterol; it plays an important role in endogenous cholesterol biosynthesis. We used DNA sequencing technology and created restriction site PCR-RFLP to detect HMGCR SNPs in an F(2) resource population of Gushi chicken and Anka broilers. We found a G/T mutation (Gln/His) in exon 17 and a T/C mutation (Pro/Pro) in exon 18. Based on association analysis of these HMGCR polymorphisms in 864 Gushi/Anka F(2) hybrids, these two mutations have significant effects on growth, carcass, meat quality, and lipid concentration.

Polymorphisms of the PNPLA3 gene and their associations with chicken growth and carcass traits.

1. An F(2) resource population of Gushi chickens crossed with Anka broilers was used to investigate the genetic effects of the chicken PNPLA3 gene on growth and adipose accumulation. 2. Associations between three SNPs (g.40006G > T, g.42344T > C and g.42404A > T) and broiler traits were determined using linkage disequilibrium, haplotype construction and association analysis. 3. The g.40006G > T mutation was associated with body weights at 4, 6, 8, 10 and 12 weeks of age, carcass weight, evisceration weight and semi-evisceration weight (P < 0.05). 4. Haplotypes of the g.42344T > C and g.42404A > T mutations were associated with body weight at 12 weeks, carcass weight, evisceration weight, and semi-evisceration weight (P < 0·05) and were associated with significant dominance effects. 5. The results suggest that the PNPLA3 gene may be in linkage with the causative mutation or a QTL controlling growth traits in chickens. In contrast to human studies, the polymorphisms were not associated with fat related traits.

Identification and abundance of miRNA in chicken hypothalamus tissue determined by Solexa sequencing.

We used Solexa sequencing technology to identify and determine the abundance of miRNAs and compared the characteristics and expression patterns of miRNA of 1-day-old and 36-week-old chicken hypothalamuses. We obtained 17,825,753 and 10,928,745 high-quality reads from 36-week-old and 1-day-old chickens, respectively. Three hundred and seventy-one conserved miRNAs were expressed in both libraries. Among the conserved miRNAs, 22 miRNAs were up-regulated and 157 miRNAs were down-regulated in the 36-week-old chicken hypothalamus tissues. The abundance of sRNAs between 1-day-old and 36-week-old chickens differed considerably. KEGG pathway analysis suggested that the target genes of highly expressed miRNAs in the chicken hypothalamus are associated with metabolism and development. This information on differential expression of miRNAs in the hypothalamus of 1-day-old and 36-week-old chickens will help us understand the molecular mechanisms of metabolism and development.

Identification of the transcript isoforms and expression characteristics for chicken Lpin1.

Lpin1 was a gene with important effects on controlling lipid/energy metabolism in humans and mice. However, little was known about chicken Lpin1 gene. In the present study, two transcript isoforms of chicken Lpin1 were identified. Lpin1-α was predicted encoding one 902 amino acid protein, whereas Lpin1-δ was predicted encoding one 918 amino acid protein with an insertion of 48-bp fragment from intron 12 of chicken Lpin1-α, and a conservative element was found to be located in intron 12 of chicken Lpin1-α genomic sequence. Ten variants were identified from chicken Lpin1-α coding sequence, and two missense mutations were predicted to affect the protein function of Lpin1. Reverse transcription PCR (RT-PCR) analysis revealed that chicken total Lpin1, Lpin1-α and Lpin1-δ were expressed in all analyzed tissues, and presented clear tissue expression differences. Real-time quantitative RT-PCR revealed that 30% energy restriction significantly elevated the total Lpin1 mRNA expression level in hepatic (P < 0.01) and adipose (P < 0.01) tissues of birds. Chicken total Lpin1 gene mRNA expression level presented a significantly inverse correlation with some traits including abdominal fat rate (P < 0.01), serum high-density lipoprotein (P < 0.05) and total cholesterol (P < 0.05), which would make a foundation for the further study on chicken Lpin1 gene function.

Haplotypic variation and characteristics across the toll-like receptor 3 locus in chickens.

Toll-like receptor 3 (TLR3) has an important protective function against viral infection. The ability of an individual to respond properly to TLR ligands may be impaired by variants located in the TLR genes. By directly PCR sequencing four exons and their flanking sequence of chicken TLR3, a total of 50 nucleotide variants were identified from five breeds. Tibetan chickens and Silkies exhibited more abundant variation sites and rare alleles. Thirty haplotypes were reconstructed, with 31 variants whose minor allelic frequency was above 5% in five breeds, which revealed four divergent clades. Chicken TLR3 was partitioned into three haplotype blocks by the htSNPer program, and six tag SNPs could be used to distinguish these 30 haplotypes. Thirty variants were located in the coding sequence of chicken TLR3, and 16 of them were non-synonymous substitutions. It is predicted that p.Ser180Gly amino substitution could form an N-myristoylation site; the p.Lys240Thr amino substitution in chicken TLR3 could result in the loss of one protein kinase C phosphorylation site. These data provide a basic understanding of chicken TLR3 sequence variation and provide haplotypic markers for disease association studies.

Identification of differentially expressed genes induced by energy restriction using annealing control primer system from the liver and adipose tissues of broilers.

Female Arbor Acre broilers were divided into 2 groups at 18 d of age. One group of chickens had free access to feed (AL), and the other group of chickens had 30% energy restriction (ER). Adipose and hepatic RNA samples were collected at 48 d of age. We employed an accurate reverse-transcription (RT) PCR method that involves annealing control primers to identify the differentially expressed genes (DEG) between ER and AL groups. Using 20 annealing control primers, 43 differentially expressed bands (40 downregulated and 3 upregulated in the ER group) were detected from the hepatic tissue, whereas no differentially expressed bands were detected from the adipose tissue. It seems that energy restriction could induce more DEG in hepatic tissue than that in adipose tissue and could result in more gene-expression downregulation in hepatic tissue. Eight DEG (6 known and 2 unknown genes) were gained from hepatic tissue and confirmed by RT-PCR, which were all supported by released expressed sequence tag sequences. Their expressions were all downregulated by energy restriction in hepatic tissues. Six known genes are RPL7, RPLP1, FBXL12, ND1, ANTXR2, and SLC22A18, respectively, which seem to play essential roles in the protein translation, energy metabolism, and tumor inhibition. The alterations of gene expression in 3 selected genes, including ND1 (P < 0.01), FBXL12 (P < 0.01), and RPLP1 (P < 0.05), were supported by real-time quantitative RT-PCR reaction. Our data provide new insights on the metabolic state of broilers changed by energy restriction.

Association study of polymorphisms inside the miR-1657 seed region with chicken growth and meat traits.

1. Polymorphisms occurring in the seed region of microRNAs (miRNAs) could influence their target gene and lead to phenotypic variation. The purpose of the research was to explore the genetic effects of the rs14934924 (G > A) mutation resident in the conserved seed region of miR-1657 on growth and meat traits of the Gushi-Anka F2 resource population. 2. The NdeI polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and association analysis were used to analyse the polymorphism. 3. The mutation was associated with body weight at 8 weeks of age, shank girth at 12 weeks of age, breast bone length at 12 weeks of age, pelvis breadth at 4 weeks of age and subcutaneous fat thickness (P < 0·05) and was associated with body weight at 4, 6, 10 and 12 weeks of age (P < 0·01). 4. Our results will be a useful resource for a subsequent study in miRNA function, and provide a basis for molecular techniques in chicken breeding.

Novel 9-bp indel in visfatin gene and its associations with chicken growth.

1. The effects of polymorphisms of the visfatin gene on growth performance, carcase traits, meat quality and serum variables were investigated in an F(2) resource population of Gushi chickens crossed with Anka broilers. 2. A 9-bp ('TAACCTGTG') insertion/deletion in intron10 of the visfatin gene was found and a total of 964 individuals were genotyped in the resource population. Genotypes (II, ID and DD) were identified based on the 9-bp insertion (allele I) or deletion (allele D). The insertion/deletion polymorphism was used for analysing associations of the gene with growth traits, carcase traits and meat quality traits in 414 F(2) chickens. 3. The DD genotype was not detected in those 66 F(1) chickens and the I allelic frequency (0·724-0·879) was obviously higher than the D allelic frequency (0·121-0·276) in the birds of three generations. 4. The 9-bp insertion/deletion was associated with the traits of 8-week shank length, 12-week shank length, 4-week pectoral angle and pancreas weight. The relationships with other traits: body weight, carcase traits, meat quality traits and serum variables, were not significant. 5. It was concluded that allele D (9-bp deletion) of the visfatin gene had a negative effect on skeletal growth.

A novel single-nucleotide polymorphism of the visfatin gene and its associations with performance traits in the chicken.

Visfatin is a peptide that is predominantly expressed in visceral adipose tissue and is hypothesized to be related to obesity and insulin resistance. In this study, a novel silent single-nucleotide polymorphism (SNP) was found in exon 7 of the chicken visfatin gene (also known as PBEF1) by single-stranded conformation polymorphism (SSCP) and DNA sequencing. In total, 836 chickens forming an F2 resource population of Gushi chicken crossed with Anka broiler were genotyped by XbaI forced RFLP, and the associations of this polymorphism with chicken growth, carcass characteristics, and meat quality were analyzed. Significant associations were found between the polymorphism and 4-week body weight (BW4), 6-week body weight (BW6), 4-week body slanting length (BSL4), fat bandwidth (FBW), breast muscle water loss rate (BWLR) and breast muscle fiber density (BFD) (P < 0.05), as well as 4-week breastbone length (BBL4) (P < 0.01). These observations suggested that the polymorphism in exon7 of the visfatin gene had significant effects on the early growth traits of chicken.