Annotations of four high-quality indigenous chicken genomes identify more than one thousand missing genes in subtelomeric regions and micro-chromosomes with high G/C contents.

BACKGROUND: Although multiple chicken genomes have been assembled and annotated, the numbers of protein-coding genes in chicken genomes and their variation among breeds are still uncertain due to the low quality of these genome assemblies and limited resources used in their gene annotations. To fill these gaps, we recently assembled genomes of four indigenous chicken breeds with distinct traits at chromosome-level. In this study, we annotated genes in each of these assembled genomes using a combination of RNA-seq- and homology-based approaches. RESULTS: We identified varying numbers (17,497-17,718) of protein-coding genes in the four indigenous chicken genomes, while recovering 51 of the 274 "missing" genes in birds in general, and 36 of the 174 "missing" genes in chickens in particular. Intriguingly, based on deeply sequenced RNA-seq data collected in multiple tissues in the four breeds, we found 571 ~ 627 protein-coding genes in each genome, which were missing in the annotations of the reference chicken genomes (GRCg6a and GRCg7b/w). After removing redundancy, we ended up with a total of 1,420 newly annotated genes (NAGs). The NAGs tend to be found in subtelomeric regions of macro-chromosomes (chr1 to chr5, plus chrZ) and middle chromosomes (chr6 to chr13, plus chrW), as well as in micro-chromosomes (chr14 to chr39) and unplaced contigs, where G/C contents are high. Moreover, the NAGs have elevated quadruplexes G frequencies, while both G/C contents and quadruplexes G frequencies in their surrounding regions are also high. The NAGs showed tissue-specific expression, and we were able to verify 39 (92.9%) of 42 randomly selected ones in various tissues of the four chicken breeds using RT-qPCR experiments. Most of the NAGs were also encoded in the reference chicken genomes, thus, these genomes might harbor more genes than previously thought. CONCLUSION: The NAGs are widely distributed in wild, indigenous and commercial chickens, and they might play critical roles in chicken physiology. Counting these new genes, chicken genomes harbor more genes than originally thought.

High quality assemblies of four indigenous chicken genomes and related functional data resources.

Many lines of evidence indicate that red jungle fowl (RJF) is the primary ancestor of domestic chickens. Although multiple versions of RJF (galgal2-galgal5 and GRCg6a) and commercial chickens (GRCg7b/w and Huxu) genomes have been assembled since 2004, no high-quality indigenous chicken genomes have been assembled, hampering the understanding of chicken domestication and evolution. To fill the gap, we sequenced the genomes of four indigenous chickens with distinct morphological traits in southwest China, using a combination of short, long and Hi-C reads. We assembled each genome (~1.0 Gb) into 42 chromosomes with chromosome N50 90.5-90.9 Mb, amongst the highest quality of chicken genome assemblies. To provide resources for gene annotation and functional analysis, we also sequenced transcriptomes of 10 tissues for each of the four chickens. Moreover, we corrected many mis-assemblies and assembled missing micro-chromosomes 29 and 34-39 for GRCg6a. Our assemblies, sequencing data and the correction of GRCg6a can be valuable resources for studying chicken domestication and evolution.

Polymorphism, Genetic Effect, and Association with Egg-Laying Performance of Chahua Chickens Matrix Metalloproteinases 13 Promoter.

Matrix metalloproteinases are a group of proteases involved in the regulation of ovarian follicular development and ovulation. Among the different MMPs, MMP13 is known to play an important role in reproduction. Therefore, this study aimed to screen the molecular genetic markers of the MMP13 gene that affect the egg-laying performance of Chahua chickens. Polymerase chain reaction (PCR) and sequencing were performed in the 5' regulation region of the MMP13 gene to detect loci significantly related to the egg-laying performance of Chahua chickens. A double fluorescence reporting system, quantitative reverse transcription PCR (RT-qPCR), and Western blotting were used to study whether gene expression was regulated by identified sites, providing a theoretical basis to improve egg production in Chahua chickens. The results revealed six single nucleotide polymorphisms (SNPs; A-1887T, T-1889C, A-1890T, T-2252C, T-2329C, and C-2360A) in the promoter region of the MMP13 gene. Further analysis revealed that hens with T-1890-C-1889-T-1887/T-1890-C-1889-T-1887 (mutant type, MT) had an earlier age at first egg (AFE) than hens with A-1890-T-1889-A-1887/A-1890-T-1889-A-1887 (wild type, WT; p < 0.05). RT-qPCR showed that the relative expression level of the MMP13 gene in the ovarian tissues of individuals with the mutation was higher than that of individuals with the wild gene (p < 0.05). Western blot results confirmed higher levels of the MMP13 protein in MT ovaries compared to those in WT ovaries. Thus, this study suggests that mutation sites on the MMP13 promoter may affect gene expression. In conclusion, the MMP13 gene in Chahua chickens may be significant for egg-laying performance, and the polymorphism in its promoter region could be used as a molecular marker to improve egg-laying performance.

Transcriptome Profile Analysis Identifies Candidate Genes for the Melanin Pigmentation of Skin in Tengchong Snow Chickens.

Tengchong Snow chickens are one of the most precious, black-boned chickens in Yunnan province and usually produce black meat. However, we found a small number of white meat traits in the chicken population during feeding. In order to determine the pattern of melanin deposition and the molecular mechanism of formation in the Tengchong Snow chicken, we measured the luminance value (L value) and melanin content in the skin of black meat chickens (Bc) and white meat chickens (Wc) using a color colorimeter, ELISA kit, and enzyme marker. The results showed that the L value of skin tissues in black meat chickens was significantly lower than that of white meat chickens, and the L value of skin tissues gradually increased with an increase in age. The melanin content of skin tissues in black meat chickens was higher than that of white meat chickens, and melanin content in the skin tissues gradually decreased with an increase in age, but this difference was not significant (p > 0.05); the L value of skin tissues in black meat chickens was negatively correlated with melanin content, and the correlation coefficient was mostly above -0.6. In addition, based on the phenotypic results, we chose to perform the comparative transcriptome profiling of skin tissues at 90 days of age. We screened a total of 44 differential genes, of which 32 were upregulated and 12 were downregulated. These DEGs were mainly involved in melanogenesis, tyrosine metabolism and RNA transport. We identified TYR, DCT, and EDNRB2 as possible master effector genes for skin pigmentation in Tengchong Snow black meat chickens through DEGs analysis. Finally, we measured the mRNA of TYR, DCT, MC1R, EDNRB2, GPR143, MITF, and TYRP1 genes through a quantitative real-time polymerase chain reaction (qPCR) and found that the mRNA of all the above seven genes decreased with increasing age. In conclusion, our study initially constructed an evaluation system for the black-boned traits of Tengchong Snow chickens and found key candidate genes regulating melanin deposition, which could provide an important theoretical basis for the selection and breeding of black-boned chickens.

Advances in Single-Cell Sequencing Technology and Its Application in Poultry Science.

Single-cell sequencing (SCS) uses a single cell as the research material and involves three dimensions: genes, phenotypes and cell biological mechanisms. This type of research can locate target cells, analyze the dynamic changes in the target cells and the relationships between the cells, and pinpoint the molecular mechanism of cell formation. Currently, a common problem faced by animal husbandry scientists is how to apply existing science and technology to promote the production of high-quality livestock and poultry products and to breed livestock for disease resistance; this is also a bottleneck for the sustainable development of animal husbandry. In recent years, although SCS technology has been successfully applied in the fields of medicine and bioscience, its application in poultry science has been rarely reported. With the sustainable development of science and technology and the poultry industry, SCS technology has great potential in the application of poultry science (or animal husbandry). Therefore, it is necessary to review the innovation of SCS technology and its application in poultry science. This article summarizes the current main technical methods of SCS and its application in poultry, which can provide potential references for its future applications in precision breeding, disease prevention and control, immunity, and cell identification.

Hyperpigmentation Inhibits Early Skeletal Muscle Development in Tengchong Snow Chicken Breed.

Tengchong snow, which has white feathers and black meat, is one of the most important black-bone chicken breeds and a genetic treasure of black food in China. Although the black meat traits are dominant, there are some chickens with white meat traits born in the process of folk selection and breeding. The purpose of this study was to compare the differences in skeletal muscle development between Tengchong snow black meat chickens (BS) and white meat chickens (WS), as well as whether excessive melanin deposition has an effect on skeletal muscle development. The BS and WS groups were selected to determine their muscle development difference at stages of 1, 7, 14, 21, and 42 days, using histological stain methods to analyze the development and composing type of breast and leg muscle fibers, as well as the count of melanin in BS muscle fibers. Finally, we were validated key candidate genes associated with muscle development and melanin synthesis. The results showed that BS breast muscle development was inhibited at 7, 14, and 21 days, while the leg muscle was inhibited at 7, 14, 21, and 42 days, compared to WS. Melanin deposition was present in a temporal migration pattern and was greater in the leg muscles than in the breast muscles, and it focused around blood vessels, as well as the epithelium, perimysium, endomysium, and connective tissue. Additionally, melanin produced an inhibitory effect similar to MSTN during skeletal muscle fiber development, and the inhibition was strongest at the stage of melanin entry between muscle fibers, but the precise mechanisms need to be confirmed. This study revealed that melanin has an inhibitory effect on the early development of skeletal muscle, which will provide new insights into the role of melanin in the black-boned chicken and theoretical references for the future conservation and utilization of black-boned chicken.

Influence of Chinese Herbal Formula on Bone Characteristics of Cobb Broiler Chickens.

To evaluate the prevention and treatment effect of a Chinese herbal formula (CHF) on the bone disease of Cobb broiler chickens, compare its efficacy with Bisphosphonates (BPs), and provide a theoretical basis for studying the nutritional regulation technology of CHF to improve the bone characteristics of broiler chickens. In this study, 560 one-day-old Cobb broiler chickens were examined for the influence of Chinese herbal formula (CHF) and Bisphosphonates (BPs). Different doses of CHF and BPs were added to the diet, and the 30- and 60-day-old live weight, tibial bone strength, the microstructure of the distal femur cancellous bone, blood biochemical indexes related to bone metabolism, and genes related to bone metabolism were determined and analyzed. The results showed that the live weight of Cobb broilers fed with CHF and BPs in the diet was as follows: The live weight of the CHF group was higher than that of the normal control (NC) group, while the live weight of the BPs group was lower than that of the NC group; the CHF and BPs improved the bone strength of Cobb broilers and increased the elastic modulus, yield strength, and maximum stress of the tibia. CHF and BPs increased the cancellous bone mineral density (BMD), bone tissue ratio (BV/TV), bone surface area tissue volume ratio (BS/TV), bone trabecular thickness (Tb.Th), and bone trabecular number (Tb.N) in the distal femur, and decreased the bone surface area bone volume ratio (BS/BV) and bone trabecular separation (Tb.Sp). Thus, the microstructure of the bone tissue of the distal femur was improved to a certain extent. Both the CHF and the BPs also increased the serum levels of the vitamin D receptor (VDR), osteoprotegerin (OPG), and alkaline phosphatase (ALP), and decreased the content of osteocalcin (OT). Meanwhile, CHF and BPs upregulated the expression of osteogenic genes (BMP-2, OPG, Runx-2) to promote bone formation and downregulated the expression of osteoclastic genes (RANK, RANKL, TNF-α) to inhibit bone resorption, thus affecting bone metabolism. Conclusion: The CHF could improve the skeletal characteristics of Cobb broilers by upregulating the expression of bone-forming-related genes and downregulating the expression of bone-breaking-related genes, thus preventing and controlling skeletal diseases in Cobb broilers. Its effect was comparable to that of BPs. Meanwhile, the CHF-H group achieved the best results in promoting the growth and improvement of the skeletal characteristics of Cobb broilers based on the live weight and skeletal-characteristics-related indexes.

Physicochemical, Nutritional Properties and Metabolomics Analysis Fat Deposition Mechanism of Chahua Chicken No. 2 and Yao Chicken.

Poultry is an important dietary source of animal protein, accounting for approximately 30% of global meat consumption. Because of its low price, low fat and cholesterol content, and no religious restrictions, chicken is considered a widely available healthy meat. Chahua chicken No. 2 is a synthetic breed of Chahua chicken derived from five generations of specialized strain breeding. In this study, Chahua chicken No. 2 (CH) and Yao chicken (Y) were used as the research objects to compare the differences in physicochemical and nutritional indicators of meat quality between the two chicken breeds, and metabolomics was used to analyze the differences in metabolites and lipid metabolism pathways and to explore the expression of genes involved in adipogenesis. The physical index and nutritional value of CH are better than that of Y, and the chemical index of Y is better than that of CH. However, the chemical index results of CH are also within the normal theoretical value range. Comprehensive comparison shows that the meat quality of CH is relatively good. Metabolomics analysis showed that CH and Y had 85 different metabolites, and the differential metabolites were mainly classified into eight categories. KEGG pathway enrichment analysis revealed 13 different metabolic pathways. The screened PPARG, FABP3, ACSL5, FASN, UCP3 and SC5D were negatively correlated with muscle fat deposition, while PPARα, ACACA and ACOX1 were positively correlated with muscle fat deposition. The meat quality of CH was better than Y. The metabolites and metabolic pathways obtained by metabonomics analysis mainly involved the metabolism of amino acids and fatty acids, which were consistent with the differences in meat quality between the two breeds and the contents of precursors affecting flavor. The screened genes were associated with fatty deposition in poultry.