Characteristics and potential biomarkers of flavor compounds in four Chinese indigenous chicken breeds.

Chinese indigenous chickens have a long history of natural and artificial selection and are popular for their excellent meat quality and unique flavor. This study investigated six meat quality-related traits in Ningdu yellow, Baier yellow, Kangle, and Shengze 901 chickens. Two-dimensional gas chromatography-time-of-flight mass spectrometry was used to detect unique flavors in 24 breast muscle samples from the same phenotyped chickens. Overall, 685, 618, 502, and 487 volatile organic compounds were identified in Ningdu yellow, Baier yellow, Kangle, and Shengze 901 chickens, respectively. The flavor components were separated into eight categories, including hydrocarbons and aldehydes. Multivariate analyses of the identified flavor components revealed some outstanding features of these breeds. For example, the hydrocarbons (22.09%) and aldehydes (14.76%) were higher in Ningdu yellow chickens and the highest content of N, N-dimethyl-methylamine was in Ningdu yellow, Baier yellow, and Shengze 901 chickens, indicating the maximum attribution to the overall flavor (ROAV = 439.57, 289.21, and 422.80). Furthermore, we found that 27 flavor compounds differed significantly among the four Chinese breeds, including 20 (e.g., 1-octen-3-ol), two (e.g., 2-methyl-naphthalene), four (e.g., 2,6-lutidine), and one (benzophenone) flavor components were showed significant enrichment in Ningdu yellow, Baier yellow, Kangle, and Shengze 901 chickens, respectively. The flavor components enriched in each breed were key biomarkers distinguishing breeds and most were significantly correlated with meat quality trait phenotypes. These results provide novel insights into indigenous Chinese chicken meat flavors.

A catalog of microbial genes and metagenome-assembled genomes from the quail gut microbiome.

The gut microbiome plays an important role in quail feed efficiency, immunity, production, and even behavior. Gut microbial gene catalogs and reference genomes are important for understanding the quail gut microbiome. However, quail gut microbes are lacked sequenced genomes and functional information to date. In this study, we report the first catalog of the microbial genes and metagenome-assembled genomes (MAGs) in fecal and cecum luminal content samples from 3 quail breeds using deep metagenomic sequencing. We identified a total of 2,419,425 nonredundant genes in the quail genome catalog, and a total of 473 MAGs were reconstructed through binning analysis. At 95% average nucleotide identity, the 473 MAGs were clustered into 283 species-level genome bins (SGBs), of which 225 SGBs belonged to species without any available genomes in the current database. Based on the quail gene catalog and MAGs, we identified 142 discriminative bacterial species and 244 discriminative MAGs between Chinese yellow quails and Japanese quails. The discriminative MAGs suggested a strain-level difference in the gut microbial composition. Additionally, a total of 25 Kyoto Encyclopedia of Genes and Genomes functional terms and 88 carbohydrate-active enzymes were distinctly enriched between Chinese yellow quails and Japanese quails. Most of the different species and MAGs were significantly interrelated with the shifts in the functional capacities of the quail gut microbiome. Taken together, we constructed a quail gut microbial gene catalog and enlarged the reference of quail gut microbial genomes. The results of this study provide a powerful and invaluable resource for quail gut microbiome-related research.

Whole Genome Resequencing Helps Study Important Traits in Chickens.

The emergence of high-throughput sequencing technology promotes life science development, provides technical support to analyze many life mechanisms, and presents new solutions to previously unsolved problems in genomic research. Resequencing technology has been widely used for genome selection and research on chicken population structure, genetic diversity, evolutionary mechanisms, and important economic traits caused by genome sequence differences since the release of chicken genome sequence information. This article elaborates on the factors influencing whole genome resequencing and the differences between these factors and whole genome sequencing. It reviews the important research progress in chicken qualitative traits (e.g., frizzle feather and comb), quantitative traits (e.g., meat quality and growth traits), adaptability, and disease resistance, and provides a theoretical basis to study whole genome resequencing in chickens.

Gut microbiome and serum metabolome analyses identify biomarkers associated with sexual maturity in quails.

Increasing evidence indicates that the gut microbiome plays an important role in host aging and sexual maturity. However, the gut microbial taxa associated with sexual maturity in quails are unknown. This study used shotgun metagenomic sequencing to identify bacterial taxa associated with sexual maturity in d 20 and d 70 quails. We found that 17 bacterial species and 67 metagenome-assembled genomes (e.g., Bacteroides spp. and Enterococcus spp.) significantly differed between the d 20 and d 70 groups, including 5 bacterial species (e.g., Enterococcus faecalis) enriched in the d 20 group and 12 bacterial species (e.g., Christensenella massiliensis, Clostridium sp. CAG:217, and Bacteroides neonati) which had high abundances in the d 70 group. The bacterial species enriched in d 20 or d 70 were key biomarkers distinguishing sexual maturity and significantly correlated with the shifts in the functional capacities of the gut microbiome. Untargeted serum metabolome analysis revealed that 5 metabolites (e.g., nicotinamide riboside) were enriched in the d 20 group, and 6 metabolites (e.g., D-ribose, stevioside, and barbituric acid) were enriched in the d 70 group. Furthermore, metabolites with high abundances in the d 20 group were significantly enriched for the KEGG pathways of arginine biosynthesis, nicotinate and nicotinamide metabolism, and lysine degradation. However, glutathione metabolism and valine, leucine and isoleucine biosynthesis were enriched in high-abundance metabolites from the d 70 group. These results provide important insights into the effects of gut microbiome and host metabolism on quail sexual maturity.

RNA Sequencing of the Pituitary Gland and Association Analyses Reveal PRKG2 as a Candidate Gene for Growth and Carcass Traits in Chinese Ningdu Yellow Chickens.

Growth and carcass traits are of great economic importance to the chicken industry. The candidate genes and mutations associated with growth and carcass traits can be utilized to improve chicken growth. Therefore, the identification of these genes and mutations is greatly importance. In this study, a total of 17 traits related to growth and carcass were measured in 399 Chinese Ningdu yellow chickens. RNA sequencing (RNA-seq) was performed to detect candidate genes using 12 pituitary gland samples (six per group), which exhibited extreme growth and carcass phenotypes: either a high live weight and carcass weight (H group) or a low live weight and carcass weight (L group). A differential expression analysis, utilizing RNA-seq, between the H and L groups identified 428 differentially expressed genes (DEGs), including 110 up-regulated genes and 318 down-regulated genes. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the identified genes showed a significant enrichment of 158 GO terms and two KEGG pathways, including response to stimulus and neuroactive ligand-receptor interaction, respectively. Furthermore, RNA-seq data, qRT-PCR, and quantitative trait transcript (QTT) analysis results suggest that the PRKG2 gene is an important candidate gene for growth and carcass traits of Chinese Ningdu yellow chickens. More specifically, association analyses of a single nucleotide polymorphism (SNP) in PRKG2 and growth and carcass traits showed that the SNP rs16400745 was significantly associated with 12 growth and carcass traits (P < 0.05), such as carcass weight (P = 9.68E-06), eviscerated weight (P = 3.04E-05), and semi-eviscerated weight (P = 2.14E-04). Collectively, these results provide novel insights into the genetic basis of growth in Chinese Ningdu yellow chickens and the SNP rs16400745 reported here could be incorporated into the selection programs involving this breed.

Gut archaea associated with bacteria colonization and succession during piglet weaning transitions.

BACKGROUND: Host-associated gut microbial communities are key players in shaping the fitness and health of animals. However, most current studies have focused on the gut bacteria, neglecting important gut fungal and archaeal components of these communities. Here, we investigated the gut fungi and archaea community composition in Large White piglets using shotgun metagenomic sequencing, and systematically evaluated how community composition association with gut microbiome, functional capacity, and serum metabolites varied across three weaning periods. RESULTS: We found that Mucoromycota, Ascomycota and Basidiomycota were the most common fungi phyla and Euryarchaeota was the most common archaea phyla across individuals. We identified that Methanosarcina siciliae was the most significantly different archaea species among three weaning periods, while Parasitella parasitica, the only differential fungi species, was significantly and positively correlated with Methanosarcina siciliae enriched in day 28 group. The random forest analysis also identified Methanosarcina siciliae and Parasitella parasitica as weaning-biased archaea and fungi at the species level. Additionally, Methanosarcina siciliae was significantly correlated with P. copri and the shifts of functional capacities of the gut microbiome and several CAZymes in day 28 group. Furthermore, characteristic successional alterations in gut archaea, fungi, bacteria, and serum metabolites with each weaning step revealed a weaning transition coexpression network, e.g., Methanosarcina siciliae and P. copri were positively and significantly correlated with 15-HEPE, 8-O-Methyloblongine, and Troxilin B3. CONCLUSION: Our findings provide a deep insight into the interactions among gut archaea, fungi, bacteria, and serum metabolites and will present a theoretical framework for understanding gut bacterial colonization and succession association with archaea during piglet weaning transitions.

Identifying biomarkers of the gut bacteria, bacteriophages and serum metabolites associated with three weaning periods in piglets.

BACKGROUND: The establishment of the piglet gut microbiome has a prolonged influence on host health, as it sets the stage for establishment of the adult swine microbiome. Substantial changes in host metabolism and immunity around the time of weaning may be accompanied by alterations in the gut microbiome. In this study, we systematically evaluated differences in the gut microbiome and host metabolites among three weaning periods using shotgun metagenomic sequencing and untargeted metabolomic profiling in piglets. RESULTS: We identified that P. copri was the most significantly different species among three weaning periods, and was the key bacterial species for mitigating piglet adaptation during the weaning transition, while Bacillus_phage_BCD7, the only differential bacteriophages, was significantly and positively correlated with P. copri enriched in day 28 group. Additionally, P. copri and Bacillus_phage_BCD7 was significantly correlated with the shifts of functional capacities of the gut microbiome and several CAZymes in day 28 group. Furthermore, the altered metabolites we observed were enriched in pathways matched to the functional capacity of the gut microbiome e.g., aminoacyl-tRNA biosynthesis. CONCLUSION: The results from this study indicate that the bacteria-phage interactions and host-microbial interactions during the weaning transition impact host metabolism, leading to beneficial host changes among three weaning periods.

A mutation in PHKG1 causes high drip loss and low meat quality in Chinese Ningdu yellow chickens.

With increasing societal development and the concurrent improvement in people's quality of life, meat consumption has gradually changed from a focus on "quantity" to "quality". Broiler production is increasingly used as a means to improve meat quality by altering various characteristics, especially its genetic factors. However, until now, little has been known about the genetic variants related to meat quality traits in Chinese purebred chicken populations. To better understand these genetic underpinnings, a total of 17 traits related to meat quality and carcass were measured in 325 Chinese Ningdu yellow chickens. We performed DNA sequencing to detect nucleotide mutations, after which we conducted association studies between PHKG1 gene polymorphisms and traits related to meat quality and carcass. Results indicated a large phenotypic variation in meat quality traits. More specifically, the single nucleotide polymorphism (SNP) rs15845448 was significantly associated with drip loss at 24 h (P = 8.04 × 10-6) and 48 h (P = 5.47 × 10-6), pH (P = 2.39 × 10-3), and meat color L* (P = 9.88 × 10-3). Moreover, the SNP rs15845448 reduced 24 h and 48 h drip loss by 3.62 and 5.97%, respectively. However, no significant associations were found between rs15845448 and carcass traits (P > 0.05). Furthermore, a haplotype block containing 2 adjacent SNPs (rs15845448 and rs15845450) was identified. This block displayed 4 distinct haplotypes that had significant association with drip loss at 24 h and 48 h, pH, and meat color L*. Collectively, these results provide new insights into the genetic basis of meat quality in Chinese Ningdu yellow chickens. Moreover, the significance of SNP rs15845448 could be incorporated into the selection programs involving this breed.

Metagenomic Analysis Identifies Sex-Related Cecal Microbial Gene Functions and Bacterial Taxa in the Quail.

Background: Japanese quail (Coturnix japonica) are important and widely distributed poultry in China. Researchers continue to pursue genetic selection for heavier quail. The intestinal microbiota plays a substantial role in growth promotion; however, the mechanisms involved in growth promotion remain unclear. Results: We generated 107.3 Gb of cecal microbiome data from ten Japanese quail, providing a series of quail gut microbial gene catalogs (1.25 million genes). We identified a total of 606 main microbial species from 1,033,311 annotated genes distributed among the ten quail. Seventeen microbial species from the genera Anaerobiospirillum, Alistipes, Barnesiella, and Butyricimonas differed significantly in their abundances between the female and male gut microbiotas. Most of the functional gut microbial genes were involved in metabolism, primarily in carbohydrate transport and metabolism, as well as some active carbohydrate-degrading enzymes. We also identified 308 antibiotic-resistance genes (ARGs) from the phyla Bacteroidetes, Firmicutes and Euryarchaeota. Studies of the differential gene functions between sexes indicated that abundances of the gut microbes that produce carbohydrate-active enzymes varied between female and male quail. Bacteroidetes was the predominant ARG-containing phylum in female quail; Euryarchaeota was the predominant ARG-containing phylum in male quail. Conclusion: This article provides the first description of the gene catalog of the cecal bacteria in Japanese quail as well as insights into the bacterial taxa and predictive metagenomic functions between male and female quail to provide a better understanding of the microbial genes in the quail ceca.

Gene Expression Profiling in Ovaries and Association Analyses Reveal HEP21 as a Candidate Gene for Sexual Maturity in Chickens.

The age of onset of sexual maturity is an important reproductive trait in chickens. In this study, we explored candidate genes associated with sexual maturity and ovary development in chickens. We performed DGE RNA-sequencing analyses of ovaries of pre-laying (P-F-O1, L-F-O1) and laying (P-F-O2, L-F-O2) hens of two sub-breeds of Ningdu Yellow chicken. A total of 3197 genes were identified in the two comparisons, and 966 and 1860 genes were detected exclusively in comparisons of P-F-O1 vs. P-F-O2 and L-F-O1 vs. L-F-O2, respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that genes involved in transmembrane signaling receptor activity, cell adhesion, developmental processes, the neuroactive ligand-receptor interaction pathway, and the calcium signaling pathway were enriched in both comparisons. Genes on these pathways, including growth hormone (GH), integrin subunit beta 3 (ITGB3), thyroid stimulating hormone subunit beta (TSHB), prolactin (PRL), and transforming growth factor beta 3 (TGFB3), play indispensable roles in sexual maturity. As a gene unique to poultry, hen egg protein 21 kDa (HEP21) was chosen as the candidate gene. Differential expression and association analyses were performed. RNA-seq data and qPCR showed that HEP21 was significantly differentially expressed in pre-pubertal and pubertal ovaries. A total of 23 variations were detected in HEP21. Association analyses of single nucleotide polymorphisms (SNPs) in HEP21 and reproductive traits showed that rs315156783 was significantly related to comb height at 84 and 91 days. These results indicate that HEP21 is a candidate gene for sexual maturity in chickens. Our results contribute to a more comprehensive understanding of sexual maturity and reproduction in chickens.

Base composition is the primary factor responsible for the variation of amino acid usage in zebra finch (Taeniopygia guttata).

In the present study, we carried out an examination of the amino acid usage in the zebra finch (Taeniopygia guttata) proteome. We found that tRNA abundance, base composition, hydrophobicity and aromaticity, protein second structure, cysteine residue (Cys) content and protein molecular weight had significant impact on the amino acid usage of the zebra finch. The above factors explained the total variability of 22.85%, 25.37%, 10.91%, 5.06%, 4.21%, and 3.14%, respectively. Altogether, approximately 70% of the total variability in zebra finch could be explained by such factors. Comparison of the amino acid usage between zebra finch, chicken (Gallus gallus) and human (Homo sapiens) suggested that the average frequency of various amino acid usage is generally consistent among them. Correspondence analysis indicated that base composition was the primary factor affecting the amino acid usage in zebra finch. This trend was different from chicken, but similar to human. Other factors affecting the amino acid usage in zebra finch, such as isochore structure, protein second structure, Cys frequency and protein molecular weight also showed the similar trends with human. We do not know whether the similar amino acid usage trend between human and zebra finch is related to the distinctive neural and behavioral traits, but it is worth studying in depth.

Copy Number Variation in SOX6 Contributes to Chicken Muscle Development.

Copy number variations (CNVs), which cover many functional genes, are associated with complex diseases, phenotypic diversity and traits that are economically important to raising chickens. The sex-determining region Y-box 6 (Sox6) plays a key role in fast-twitch muscle fiber differentiation of zebrafish and mice, but it is still unknown whether SOX6 plays a role in chicken skeletal muscle development. We identified two copy number polymorphisms (CNPs) which were significantly related to different traits on the genome level in chickens by AccuCopy® and CNVplex® analyses. Notably, five white recessive rock (CN = 1, CN = 3) variant individuals and two Xinghua (CN = 3) variant individuals contain a CNP13 (chromosome5: 10,500,294-10,675,531) which overlaps with SOX6. There is a disordered region in SOX6 proteins 265-579 aa coded by a partial CNV overlapping region. A quantitative real-time polymerase chain reaction showed that the expression level of SOX6 mRNA was positively associated with CNV and highly expressed during the skeletal muscle cell differentiation in chickens. After the knockdown of the SOX6, the expression levels of IGFIR1, MYF6, SOX9, SHOX and CCND1 were significantly down-regulated. All of them directly linked to muscle development. These results suggest that the number of CNVs in the CNP13 is positively associated with the expression level of SOX6, which promotes the proliferation and differentiation of skeletal muscle cells by up-regulating the expression levels of the muscle-growth-related genes in chickens as in other animal species.

Hydrophobicity and aromaticity are primary factors shaping variation in amino acid usage of chicken proteome.

Amino acids are utilized with different frequencies both among species and among genes within the same genome. Up to date, no study on the amino acid usage pattern of chicken has been performed. In the present study, we carried out a systematic examination of the amino acid usage in the chicken proteome. Our data indicated that the relative amino acid usage is positively correlated with the tRNA gene copy number. GC contents, including GC1, GC2, GC3, GC content of CDS and GC content of the introns, were correlated with the most of the amino acid usage, especially for GC rich and GC poor amino acids, however, multiple linear regression analyses indicated that only approximately 10-40% variation of amino acid usage can be explained by GC content for GC rich and GC poor amino acids. For other intermediate GC content amino acids, only approximately 10% variation can be explained. Correspondence analyses demonstrated that the main factors responsible for the variation of amino acid usage in chicken are hydrophobicity, aromaticity and genomic GC content. Gene expression level also influenced the amino acid usage significantly. We argued that the amino acid usage of chicken proteome likely reflects a balance or near balance between the action of selection, mutation, and genetic drift.

Differences of Z chromosome and genomic expression between early- and late-feathering chickens.

One duplicated segment on chicken Z chromosome is a causal mutation to the late-feathering phenotype. However, understanding biological process of the late-feathering formation is also of interest to chicken breeding and feather development theory. One hundred and thirty-seven valid single nucleotide polymorphisms (SNPs) from an SNP database were used to perform an association study of the Z chromosome in Xinghua chickens. Two SNPs, which were respectively on 9607480 bp and 10607757 bp, were significantly associated with feathering phenotypes. This result indicated the causal mutation of the late-feathering formation in Xinghua chickens was consistent with the previous report which showed the late-feathering locus ranged 9966364­10142688 bp on Z chromosome. Microarray expressions were implemented for six 1-day-old female Xinghua chicks. Compared to the early-feathering chicks, there were 249 and 83 upregulated and downregulated known genes in the late-feathering chicks. Forty-one genes were expressed in late-feathering chicks, but not in early-feathering ones. At least 14 significantly differentially expressed genes were directly related to keratin. In the region of the sex-linked feathering gene, only prolactin receptor (PRLR) gene was a significantly differentially expressed gene. Expression of PRLR in late-feathering chicks was 1.78-fold as that in early-feathering chicks. Late-feathering Wenchang chicks also had higher expression level of PRLR than early-feathering ones. This study suggested that increasing PRLR expression that resulted from the special variant on chicken Z chromosome caused the late-feathering phenotype.

Mutation bias is the driving force of codon usage in the Gallus gallus genome.

Synonymous codons are used with different frequencies both among species and among genes within the same genome and are controlled by neutral processes (such as mutation and drift) as well as by selection. Up to now, a systematic examination of the codon usage for the chicken genome has not been performed. Here, we carried out a whole genome analysis of the chicken genome by the use of the relative synonymous codon usage (RSCU) method and identified 11 putative optimal codons, all of them ending with uracil (U), which is significantly departing from the pattern observed in other eukaryotes. Optimal codons in the chicken genome are most likely the ones corresponding to highly expressed transfer RNA (tRNAs) or tRNA gene copy numbers in the cell. Codon bias, measured as the frequency of optimal codons (Fop), is negatively correlated with the G + C content, recombination rate, but positively correlated with gene expression, protein length, gene length and intron length. The positive correlation between codon bias and protein, gene and intron length is quite different from other multi-cellular organism, as this trend has been only found in unicellular organisms. Our data displayed that regional G + C content explains a large proportion of the variance of codon bias in chicken. Stepwise selection model analyses indicate that G + C content of coding sequence is the most important factor for codon bias. It appears that variation in the G + C content of CDSs accounts for over 60% of the variation of codon bias. This study suggests that both mutation bias and selection contribute to codon bias. However, mutation bias is the driving force of the codon usage in the Gallus gallus genome. Our data also provide evidence that the negative correlation between codon bias and recombination rates in G. gallus is determined mostly by recombination-dependent mutational patterns.

The influence of recombination on SNP diversity in chickens.

The association between recombination rate and diversity, but not divergence is considered to be driven mainly by natural selection: fixation of positively selected variants and associated hitchhiking effects and/or background selection eliminating deleterious alleles. In the present study, we investigated the relationship between recombination rate, SNP diversity and interspecies divergence for 29 loci in chickens. We found that recombination rate is positively correlated with nucleotide diversity but is not correlated with interspecies divergence. It appears that variation in recombination rate explains over 30% of the variation in levels of diversity among 29 loci. Our data suggested that natural selection is a main factor in shaping SNP diversity in chickens. Since SNP diversity is significantly lower at Z-linked than at autosomal loci, we argued that genetic hitchhiking might be more important than background selection in producing the observed correlation.

[Comparative study of SNP diversity and calculation of the effective size of population in chicken].

A region (200 kb) of Contig. 060226.1 on GGA1 was selected to study the average diversity of Red Jungle Fowl (RJF), Taihe Silk chicken (TS), and White Recessive Rock (WRR), and estimate the effective population size (Ne) of chicken. The mean heterozygosities of RJF, TS and WRR were 0.28533+/-0.034747, 0.32926+/-0.039191 and 0.30168+/-0.040382, respectively. Significant test indicted that there is not significant difference between them (P=0.2368>0.05). The initial chicken effective population size was estimated to be about 20 000-150 000. Chicken has undergone serious population bottleneck effect during the first stage of domestication. However, this bottleneck effect did not result in a substantial loss of diversity as revealed by SNP. The possible explanations for similar SNP diversity between domesticated chicken and Red Jungle Fowl might due to many factors, such as a big Ne in earlier phase of domestication, population expending in breed differentiation, abroad crossing between breeds (especially crossing with RJF), together with high recombination rate in chicken genome.

SNP mapping of QTL affecting growth and fatness on chicken GGA1.

An F2 chicken population was established from a crossbreeding between a Xinghua line and a White Recessive Rock line. A total of 502 F2 chickens in 17 full-sib families from six hatches was obtained, and phenotypic data of 488 individuals were available for analysis. A total of 46 SNP on GGA1 was initially selected based on the average physical distance using the dbSNP database of NCBI. After the polymorphism levels in all F0 individuals (26 individuals) and part of the F1 individuals (22 individuals) were verified, 30 informative SNP were potentially available to genotype all F2 individuals. The linkage map was constructed using Cri-Map. Interval mapping QTL analyses were carried out. QTL for body weight (BW) of 35 d and 42 d, 49 d and 70 d were identified on GGA1 at 351-353 cM and 360 cM, respectively. QTL for abdominal fat weight was on GGA1 at 205 cM, and for abdominal fat rate at 221 cM. Two novel QTL for fat thickness under skin and fat width were detected at 265 cM and 72 cM, respectively.

[Single nucleotide polymorphisms and fine mapping QTL in chickens].

As an important economically animal and a model animal, a great of progress of SNP diversity and QTL mapping in chickens have been made in recent years. The present paper not only summarized these achievements but also commanded an extensive view about the fine mapping of QTL by the use of SNP marker.