Melanin-based coloration in the meat of black-boned chicken is a major economic issue in China. Variation in the pigmentation (hypopigmentation) of chicken muscle causes direct economic losses every year. To determine the molecular mechanisms involved in the melanogenesis of muscle tissue, this study used high-throughput sequencing to compare differences in the transcriptome between black (BM) and white (WM) chicken breast muscles. We constructed 6 cDNA libraries from BM and WM groups in Muchuan black-boned chickens. A comparison between the BM and WM groups revealed 264 differentially expressed genes, of which 152 were upregulated, whereas 112 were downregulated in black muscle. Gene ontology and a Kyoto Encyclopedia of Genes and Genomes pathway analysis identified several differentially enriched biological functions and processes of the 2 muscles. Seven promising candidate genes [PMEL, Ras-related protein RAB29, and 5 solute carrier superfamily genes: SLC6A9, SLC38A4, SLC22A5, SLC35F3, and SLC16A3] may play an important role in the melanogenesis of chicken muscle. Our data provide a valuable resource for identifying genes whose functions are critical for muscle melanogenesis, and will assist studies of the molecular mechanisms of melanogenesis regulation in chicken muscle.

Background/Aims: Melanin is a major and ubiquitous component of plumage colouration, and patterns of melanin pigmentation in birds are extremely varied. However, the molecular mechanism of pigmentation in avian plumage is still largely unknown. Methods: To elucidate the molecular mechanisms involved in the formation of black and white plumage, this study takes advantage of high-throughput sequencing technology to compare differences in the transcriptome between black and white chicken feather bulbs. In total, we constructed six cDNA libraries from black (Group B) and white (Group W) feather bulbs in the dorsal plumage of Muchuan black-boned chickens. Results: A comparison between Groups B and W revealed 61 differentially expressed genes, with 47 displaying higher, and 14 displaying lower, levels of expression in white feather bulbs. Our results revealed a set of candidate genes and two potential metabolic pathways involved in black-bone chicken plumage melanogenesis. These include four homeobox genes (HOXB9, HOXC8, HOXA9, and HOXC 9), two glutathione (GSH) metabolism-related genes (CHAC1 and GPX3), and the transforming growth factor beta (TGF-β) signalling pathway. Two known genes, TYR and MITF, were also shown to play a role in melanin formation. Conclusion: our data provide a valuable resource for discovering genes important in plumage melanin formation and will help further elucidate the molecular mechanisms for black and white plumage.

circular rnas (circrnas) regulate several physiological and pathological processes, but their role in visceral lipid deposition has not been explored. in the present study, human preadipocytes from visceral fat tissue (HPa-v) were induced to form adipocytes, and the circrna expression profiles in HPa-v and adipocytes were detected using circrna microarrays. The microarray data revealed that 2,215 and 1,865 circRNAs were significantly up-and downregulated, respectively, in adipocytes compared with HPa-v. Moreover, the parental genes of differentially expressed circrnas were associated with fatty acid metabolism based on Kyoto encyclopedia of Genes and Genomes analysis. Three circrnas (hsa_circ_0136134, hsa_circ_0017650, and hsa-circrna9227-1) were selected for quantitative Pcr (qPcr) validation, and the qPcr results were consistent with the microarray results. Furthermore, Miranda software was used to predict the micrornas (mirnas) potentially targeting the top 10 up-and downregulated circrnas, and 14 mirnas with more than two mirna response elements targeting these circRNAs. This is the first study of the expression profiles of circrnas in HPa-v and adipocytes and may suggest potential therapeutic targets for the visceral obesity.

The tyrosinase (TYR) gene is the major melanogenesis-related gene for skin (fur) or plumage color in mammals and birds. Genetic variation in the promoter region of a gene may affect gene expression and phenotypes. This study compared the TYR promoter region between pooled DNA (n = 8) of chickens (Gallus gallus) with black and white skin using direct sequencing. The single nucleotide polymorphism (SNP) c.-2228A>T was found to have the opposite allele distribution in the two groups. The results of genotyping in a larger population (n = 188) revealed that SNP c.-2228A>T was associated with the skin color of the black-boned chicken. Individuals with genotypes AA and AT had greater TYR expression than those with genotype TT. A luciferase assay of the promoter activity revealed that genotype AA had greater activity than genotype TT. Transcription factor binding site analyses showed that the c.-2228A allele has a putative binding site for transcription factor AT-rich interaction domain 3a (Arid3a), while the c.-2228T allele has sites for GS homeobox 2 (GSX2), homeobox D9 (Hoxd9), and mix paired-like homeobox (MIXL1). We concluded that the TYR promoter polymorphism affects skin color. SNP c.-2228A>T could be used as a genetic marker for marker-assisted selection of skin color in the black-boned chicken.

Laying performance is an important economical trait of goose production. As laying performance is of low heritability, it is of significance to develop a marker-assisted selection (MAS) strategy for this trait. Definition of sequence variation related to the target trait is a prerequisite of quantitating MAS, but little is presently known about the goose genome, which greatly hinders the identification of genetic markers for the laying traits of geese. Recently developed restriction site-associated DNA (RAD) sequencing is a possible approach for discerning large-scale single nucleotide polymorphism (SNP) and reducing the complexity of a genome without having reference genomic information available. In the present study, we developed a pooled RAD sequencing strategy for detecting geese laying-related SNP. Two DNA pools were constructed, each consisting of equal amounts of genomic DNA from 10 individuals with either high estimated breeding value (HEBV) or low estimated breeding value (LEBV). A total of 139,013 SNP were obtained from 42,291,356 sequences, of which 18,771,943 were for LEBV and 23,519,413 were for HEBV cohorts. Fifty-five SNP which had different allelic frequencies in the two DNA pools were further validated by individual-based AS-PCR genotyping in the LEBV and HEBV cohorts. Ten out of 55 SNP exhibited distinct allele distributions in these two cohorts. These 10 SNP were further genotyped in a goose population of 492 geese to verify the association with egg numbers. The result showed that 8 of 10 SNP were associated with egg numbers. Additionally, liner regression analysis revealed that SNP Record-111407, 106975 and 112359 were involved in a multiplegene network affecting laying performance. We used IPCR to extend the unknown regions flanking the candidate RAD tags. The obtained sequences were subjected to BLAST to retrieve the orthologous genes in either ducks or chickens. Five novel genes were cloned for geese which harbored the candidate laying-related SNP, including membrane associated guanylate kinase (MAGI-1), KIAA1462, Rho GTPase activating protein 21 (ARHGAP21), acyl-CoA synthetase family member 2 (ACSF2), astrotactin 2 (ASTN2). Collectively, our data suggests that 8 SNP and 5 genes might be promising candidate markers or targets for marker-assisted selection of egg numbers in geese.

1. Microphthalmia-associated transcription factor (MITF) plays a pivotal role in melanocyte development by regulating the transcription of major pigmentation enzymes (e.g. TYR, TYRP1 and DCT). A single-nucleotide polymorphism (SNP), c.-638T>C, was identified in the MITF promoter, and genotyping of a population (n = 426) revealed that SNP c.-638T>C was associated with skin colour in black-boned chickens. 2. Individuals with genotypes CC and TC exhibited greater MTIF expression than those with genotype TT. Luciferase assays also revealed that genotype CC and TC promoters had higher activity levels than genotype TT. Expression of melanogenesis-related gene (TYR) was higher in the skin of chickens with the CC and CT genotype compared to TT chickens (P < 0.05). 3. Transcription factor-binding site analyses showed that the c.-638C allele contains a putative binding site for transcription factor sterol regulatory element-binding transcription factor 2, aryl hydrocarbon receptor nuclear translocator, transcription factor binding to IGHM enhancer 3 and upstream transcription factor 2. In contrast, the c.-638T allele contains binding sites for Sp3 transcription factor and Krüppel-like factor 1. 4. It was concluded that MITF promoter polymorphisms affected chicken skin colour. SNP c.-638T>C could be used for the marker-assisted selection of skin colour in black-boned chicken breeding.

ACSF2 (encoded by acyl-CoA synthetase family member 2) belongs to the acyl-CoA synthetase (ACS) family, activating fatty acids by forming a thioester bond with CoA. In our previous study, a SNP residing in the intron of ACSF2 was identified to be linked to goose egg-laying performance. But the structure of goose ACSF2 as well as its role in reproduction remains unknown. In this study, we cloned and characterized ACSF2 in Yangzhou geese. A total of four alternative splice variants, designated as ACSF2-1, ACSF2-2, ACSF2-3 and ACSF2-4 respectively, were identified in the ovary. The coding regions of the four variants are 1770, 1692, 1599 and 1917 bp in length, respectively encoding 589, 563, 532 and 638 amino acids with conserved AMP-binding sites. All ACSF2 variants were widely expressed in 11 tested tissues in geese, except that the ACSF2-2 transcript was not detected in hypothalamus, pituitary gland and granulosa cells. Subcellular localization revealed that ACSF2 is a mitochondrial matrix protein. ACSF2 mRNA level was compared between high egg production (HEP; n = 8) and low egg production (LEP; n = 10) groups and showed a lower (P < 0.05) mRNA level in the HEP group. Further experiments indicated that overexpressing ACSF2 resulted in a significant increase of caspase-3 mRNA levels and that ACSF2 knockdown triggered a decrease in the caspase-3 mRNA level in granulosa cells. Similarly, the lower caspase-3 mRNA levels were identified in ovaries of the HEP group with lower ACSF2 mRNA levels. The research showed that the ACSF2 mRNA levels had a positive correlation with caspase-3 mRNA levels in vivo (R = 0.86, P < 0.01). Our results suggest that lower ACSF2 expression promotes the laying performance of goose possibly by inhibiting granulosa cell apoptosis and facilitating follicular development.

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