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.

Artificial selection footprints in indigenous and commercial chicken genomes.

BACKGROUND: Although many studies have been done to reveal artificial selection signatures in commercial and indigenous chickens, a limited number of genes have been linked to specific traits. To identify more trait-related artificial selection signatures and genes, we re-sequenced a total of 85 individuals of five indigenous chicken breeds with distinct traits from Yunnan Province, China. RESULTS: We found 30 million non-redundant single nucleotide variants and small indels (< 50 bp) in the indigenous chickens, of which 10 million were not seen in 60 broilers, 56 layers and 35 red jungle fowls (RJFs) that we compared with. The variants in each breed are enriched in non-coding regions, while those in coding regions are largely tolerant, suggesting that most variants might affect cis-regulatory sequences. Based on 27 million bi-allelic single nucleotide polymorphisms identified in the chickens, we found numerous selective sweeps and affected genes in each indigenous chicken breed and substantially larger numbers of selective sweeps and affected genes in the broilers and layers than previously reported using a rigorous statistical model. Consistent with the locations of the variants, the vast majority (~ 98.3%) of the identified selective sweeps overlap known quantitative trait loci (QTLs). Meanwhile, 74.2% known QTLs overlap our identified selective sweeps. We confirmed most of previously identified trait-related genes and identified many novel ones, some of which might be related to body size and high egg production traits. Using RT-qPCR, we validated differential expression of eight genes (GHR, GHRHR, IGF2BP1, OVALX, ELF2, MGARP, NOCT, SLC25A15) that might be related to body size and high egg production traits in relevant tissues of relevant breeds. CONCLUSION: We identify 30 million single nucleotide variants and small indels in the five indigenous chicken breeds, 10 million of which are novel. We predict substantially more selective sweeps and affected genes than previously reported in both indigenous and commercial breeds. These variants and affected genes are good candidates for further experimental investigations of genotype-phenotype relationships and practical applications in chicken breeding programs.

Integrative analysis of transcriptomics and metabolomics to reveal the melanogenesis pathway of muscle and related meat characters in Wuliangshan black-boned chickens.

BACKGROUND: Melanin is an important antioxidant in food and has been used in medicine and cosmetology. Chicken meat with high melanin content from black-boned chickens have been considered a high nutritious food with potential medicinal properties. The molecular mechanism of melanogenesis of skeletal muscle in black-boned chickens remain poorly understood. This study investigated the biological gene-metabolite associations regulating the muscle melanogenesis pathways in Wuliangshan black-boned chickens with two normal boned chicken breeds as control. RESULTS: We identified 25 differentially expressed genes and 11 transcription factors in the melanogenesis pathways. High levels of the meat flavor compounds inosine monophosphate, hypoxanthine, lysophospholipid, hydroxyoctadecadienoic acid, and nicotinamide mononucleotide were found in Wuliangshan black-boned chickens. CONCLUSION: Integrative analysis of transcriptomics and metabolomics revealed the dual physiological functions of the PDZK1 gene, involved in pigmentation and/or melanogenesis and regulating the phospholipid signaling processes in muscle of black boned chickens.

Regulation of myostatin expression is associated with growth and muscle development in commercial broiler and DMC muscle.

Myostatin is a negative regulator of skeletal muscle growth. Muscle tissue is the largest tissue in the body and influences body growth. Commercial Avian broiler chickens are selected for high growth rate and muscularity. Daweishan mini chickens are a slow growing small-sized chicken breed. We investigated the relations between muscle (breast and leg) myostatin mRNA expression and body and muscle growth. Twenty chickens per breed were slaughtered at 0, 30, 60, 90, 120, and 150 days of age. Body and muscle weights were higher at all times in Avian chickens. Breast muscle myostatin expression was higher in Avian chickens than in Daweishan mini chickens at day 30. Myostatin expression peaked at day 60 in Daweishan mini chickens and expression remained higher in breast muscle. Daweishan mini chickens myostatin expression correlated positively with carcass weight, breast and leg muscle weight from day 0 to 60, and correlated negatively with body weight from day 90 to 150, while myostatin expression in Avian chickens was negatively correlated with carcass and muscle weight from day 90 to 150. The results suggest that myostatin expression is related to regulation of body growth and muscle development, with two different regulatory mechanisms that switch between days 30 and 60.

Biological mechanisms discriminating growth rate and adult body weight phenotypes in two Chinese indigenous chicken breeds.

BACKGROUND: Intensive selection has resulted in increased growth rates and muscularity in broiler chickens, in addition to adverse effects, including delayed organ development, sudden death syndrome, and altered metabolic rates. The biological mechanisms underlying selection responses remain largely unknown. Non-artificially-selected indigenous Chinese chicken breeds display a wide variety of phenotypes, including differential growth rate, body weight, and muscularity. The Wuding chicken breed is a fast growing large chicken breed, and the Daweishan mini chicken breed is a slow growing small chicken breed. Together they form an ideal model system to study the biological mechanisms underlying broiler chicken selection responses in a natural system. The objective of this study was to study the biological mechanisms underlying differential phenotypes between the two breeds in muscle and liver tissues, and relate these to the growth rate and body development phenotypes of the two breeds. RESULTS: The muscle tissue in the Wuding breed showed higher expression of muscle development genes than muscle tissue in the Daweishan chicken breed. This expression was accompanied by higher expression of acute inflammatory response genes in Wuding chicken than in Daweishan chicken. The muscle tissue of the Daweishan mini chicken breed showed higher expression of genes involved in several metabolic mechanisms including endoplasmic reticulum, protein and lipid metabolism, energy metabolism, as well as specific immune traits than in the Wuding chicken. The liver tissue showed fewer differences between the two breeds. Genes displaying higher expression in the Wuding breed than in the Daweishan breed were not associated with a specific gene network or biological mechanism. Genes highly expressed in the Daweishan mini chicken breed compared to the Wuding breed were enriched for protein metabolism, ABC receptors, signal transduction, and IL6-related mechanisms. CONCLUSIONS: We conclude that faster growth rates and larger body size are related to increased expression of genes involved in muscle development and immune response in muscle, while slower growth rates and smaller body size are related to increased general cellular metabolism. The liver of the Daweishan breed displayed increased expression of metabolic genes.

Effect of Stewing Time on the Small Molecular Metabolites, Free Fatty Acids, and Volatile Flavor Compounds in Chicken Broth.

Chicken broth has a taste of umami, and the stewing time has an important effect on the quality of chicken broth, but there are fewer studies on the control of the stewing time. Based on this, the study was conducted to analyze the effects of different stewing times on the sensory, small molecular metabolites, free fatty acids, and volatile flavor compounds contents in chicken broths by liquid chromatography-quadrupole/time-of-flight mass spectrometry, gas chromatography-mass spectrometry, headspace solid-phase microextraction, and gas chromatography-mass spectrometry. Eighty-nine small molecular metabolites, 15 free fatty acids, and 86 volatile flavor compounds were detected. Palmitic and stearic acids were the more abundant fatty acids, and aldehydes were the main volatile flavor compounds. The study found that chicken broth had the best sensory evaluation, the highest content of taste components, and the richest content of volatile flavor components when the stewing time was 2.5 h. This study investigated the effect of stewing time on the quality of chicken broth to provide scientific and theoretical guidance for developing and utilizing local chicken.

Effect of different salt additions on the taste and flavor-related compounds in chicken soup.

Chicken soup is popular among consumers because of its delicious taste, strong flavor, and abundant nutritional value. Twenty-four Yunnan local hens were stewed by adding different amounts of NaCl [1.5, 2, 2.5, 3%, m/m, calculated based on chicken carcass weight; chicken: water = 1:2 (m/m)] to study the effect of salt addition on taste- and flavor-related compounds in chicken soup. Sensory evaluation results showed that the 2 and 2.5% NaCl treatment groups had higher scores. Water-soluble small molecule compounds were detected by LC-Q/TOF-MS based metabolomics approach, among which amino acids and their derivatives, nucleic acids, and small peptides were the main components. The concentration of Water-soluble small molecule substances in chicken soup samples with different salt additions showed a clear trend of separation and reached the highest in the 2.5% NaCl treatment group. Volatile flavor compounds in the chicken soup were analyzed by HS-SPME-GC-MS, including aldehydes, and alcohols, and the relative concentration of flavor compounds in the 2.5% salt treatment group was the highest. In summary, the addition of salt could improve the overall flavor of chicken broth, and the optimal salt addition of NaCl in chicken soup is 2.5%.

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.

Effects of breeds and dietary protein levels on the growth performance, energy expenditure and expression of avUCP mRNA in chickens.

The physiological mechanisms of thermogenesis, energy balance and energy expenditure are poorly understood in poultry. The aim of this study was designed to investigate the physiological roles of avian uncoupling protein (avUCP) regulating in energy balance and thermogenesis by using three chicken breeds of existence striking genetic difference and feeding with different dietary protein levels. Three chicken breeds including broilers, hybrid chickens, and non-selection Wuding chickens were used in this study. Total 150 chicks of 1 day of age, with 50 from each breed were reared under standard conditions on starter diets to 30 days. At 30 days of age, forty chicks from each breed chicks were divided into two groups. One group was fed low protein diet (LP, 17.0 %), and the other group was fed high protein diet (HP, 19.5 %) for 60 days. Wuding chickens showed the lowest feed conversion efficiency (FCE) and the highest expressions of avUCP mRNA association with high plasma T3 and insulin concentrations. Hybrid chickens showed the lowest expressions of avUCP mRNA association with high FCE and energy efficiency. Expressions of avUCP mRNA association with diet-induced thermogenesis (DIT) were only observed in broiler and hybrid chickens. The expressions of avUCP mRNA were positive association with plasma insulin, T3 and NEFA concentrations. Age influence on the expression of avUCP mRNA were observed only for hybrid and broiler chickens. It seems that both roles of avUCP regulation thermogenesis and lipid utilisation as fuel were observed in the present study response to variation in dietary protein and breeds.

Effects of aqueous extracts of raw pu-erh tea and ripened pu-erh tea on proliferation and differentiation of 3T3-L1 preadipocytes.

Pu-erh tea has shown anti-obesity effects but little is known about its effect on proliferation and differentiation of preadipocytes. This study investigated the effects of the aqueous extracts of raw pu-erh tea and ripened pu-erh tea on proliferation and differentiation of murine 3T3-L1 preadiopocytes. We examined dose and time effects of both aqueous extracts on proliferation of 3T3-L1 preadipocytes. The contents of triglycerides in cytoplasm and the mRNA expression of critical transcriptional factors involved in differentiation were determined. Cytotoxicity and apoptosis rate of preadipocytes by pu-erh tea extracts treatment were test for toxic and pro-apoptotic effects. Both aqueous extracts of pu-erh tea inhibited the proliferation of 3T3-L1 preadipocytes at the selected time points. At lower concentration of raw pu-erh tea extracts (less than 300 µg/ml) and ripened pu-erh tea extracts (less than 350 µg/ml), no significant cytotoxic and pro-apoptotic were observed. Ripened pu-erh tea was more effective with lower IC50 than raw pu-erh tea. Both extracts suppressed the differentiation and down-regulated the gene expression of peroxisome proliferator-activated receptor-γ and CCAAT/enhancer binding proteins-α. Therefore, these results indicate that both aqueous extracts of pu-erh tea can inhibit proliferation and differentiation with ripened pu-erh tea more potent. Polyphenol rich in both extracts may play a role in the inhibition of proliferation and differentiation of 3T3-L1 preadipocytes.

Identification of umami peptides from Wuding chicken by Nano-HPLC-MS/MS and insights into the umami taste mechanisms.

Wuding chicken is popular with consumers in China because of its umami taste. This study aimed to identify novel umami peptides from Wuding chicken and explore the taste mechanism of umami peptides. The molecular masses and amino acid compositions of peptides in Wuding chicken were identified by nano-scale liquid chromatography-tandem mass spectrometry (Nano-HPLC-MS/MS). The taste characteristics of the peptides synthesized by the solid-phase method were evaluated by sensory evaluation combined with electronic tongue technology. The secondary structure of the peptides was further analyzed by circular dichroism (CD), and the relationship between the structure and taste of the peptides was elucidated by molecular docking. The results showed that eight potential umami peptides were identified, among which FVT (FT-3), LDF (LF-3), and DLAGRDLTDYLMKIL (DL-15) had distinct umami tastes, and FT-3 had the highest umami intensity, followed by LF-3 and DL-15. The relative contents of ß-sheets in the three umami peptides were 55.20%, 57.30%, and 47.70%, respectively, which were the key components of Wuding chicken umami peptides. In addition to LF-3 embedded in the cavity-binding domain of the TIR1, both FT-3 and DL-15 were embedded in the venus flytrap domain (VFTD) of the T1R3 to bind the umami receptor T1R1/T1R3. The main binding forces between the umami peptides and the umami receptor T1R1/T1R3 relied on hydrogen bonds and hydrophobic interactions, and the key amino acid residues of the combination of umami peptides and the umami receptor T1R1/T1R3 were Glu292, Asn235, and Tyr262.

Combined application of high-throughput sequencing and LC-MS/MS-based metabolomics to evaluate the formation of Zn-protoporphyrin in Nuodeng ham.

In order to deepen the understanding of the formation mechanism of Zn-protoporphyrin (ZnPP), Nuodeng ham was used as the research object, and the effects of bacterial communities and small molecule metabolites on the formation of ZnPP during the processing of Nuodeng ham were analyzed by high-throughput sequencing and LC-MS/MS-based metabolomics. With the prolongation of processing time, the ZnPP content increased significantly, while the heme content decreased significantly. Compound curing agent significantly inhibited the formation of ZnPP and significantly decreased the heme content. The bacterial communities changed dynamically and the five bacterial genera were significantly positively correlated with ZnPP content. Forty-seven differential metabolites were obtained through screening, of which seventeen differential metabolites were significantly positively correlated with ZnPP content. Correlation analysis showed a strongly positive correlation between Staphylococcus, Delftia, Acinetobacter and these seventeen differential metabolites. These findings can provide a theoretical basis for further establishing the color control measures of Nuodeng ham.

Effect of pu-erh tea on body fat and lipid profiles in rats with diet-induced obesity.

The antiobesity and antihyperlipidaemic effects of pu-erh tea in rats with high fat diet (HFD)-induced obesity were investigated. Male Sprague-Dawley rats were randomly divided into five groups and fed varying diets for an 8-week period: control diet, HFD, and HFD supplemented with low, moderate or high doses of pu-erh tea extract (0.5 g, 2 g and 4 g/kg BW/day, respectively). Pu-erh tea significantly reduced the total body weight and the weight of various adipose pads. Pu-erh tea administration also significantly lowered plasma total cholesterol, triglyceride concentrations and low-density lipoprotein-cholesterol levels in rats with HFD-induced obesity, but did not affect high-density lipoprotein-cholesterol levels. Moreover, pu-erh tea significantly increased lipoprotein lipase, hepatic lipase and hormone-sensitive lipase activities in epididymal fat tissue in rats with HFD-induced obesity. Analysis of real-time reverse transcription-polymerase chain reaction results indicated that pu-erh tea significantly enhanced mRNA levels of hormone-sensitive lipase in rats with HFD-induced obesity. These results suggest that pu-erh tea attenuated visceral fat accumulation and improved hyperlipidemia in a rat model of HFD-induced obesity.

Purification, characterization and immunomodulatory activity of polysaccharides from Leccinum crocipodium (Letellier.) Watliag.

The polysaccharide obtained from edible fungi has been regarded as the major bioactive component related to the nutritional and human's health. In the present study, three purified polysaccharides (LCP-1, LCP-2, and LCP-3) were obtained from Leccinum crocipodium (Letellier.) Watliag. The characterizations of LCP-1, LCP-2, and LCP-3 were determined by high-performance liquid chromatography (HPLC), UV, FT-IR spectrometrys and 1H NMR spectrum. LCP-1 had a molecular weight of 2.303 × 105 Da and 7.519 × 103 Da, and was composed of mannose (Man), ribose (Rib), rhamnose (Rha), glucuronic acid (GluA), galacturonic acid (GalA), glucose (Glu), galactose (Gal), xylose (Xyl), arabinose (Ara) and fucuronic (Fuc). The molecular weight of LCP-2 was 2.655 × 105 Da, and its monosaccharide constituents were Man, Rib, Rha, GluA, Glu, Gal, Xyl, Ara and Fuc. The molecular weight of LCP-3 was 3.783 × 105 Da, and its monosaccharide constituents were Man, Rib, Rha, GluA, GalA, Glu, Gal, Xyl, Ara and Fuc. For the in vitro immunomodulatory experiments demonstrated that three purified polysaccharides could enhance immunomodulatory activities on macrophage RAW 264.7 cells, moreover, LCP-2 and LCP-3 showed stronger immunomodulatory activity than LCP-1. The results indicated that the LCP-1, LCP-2, and LCP-3 could be further developed as functional food or medicine.

Profiles of expression pattern and tissue distribution of host defense peptides genes in different chicken (Gallus gallus) breeds related to body weight.

Host defense peptides (HDPs) are an important first line of defense with antimicrobial and immunomodulatory properties. Selection for increased body weight is hypothesized to be related to reduced immune response. We studied the relationships among body weight, age, and the HDP expression patterns in intestine and immune organs. We used chickens with marked differences of body sizes. The non-selected Daweishan mini chickens showed the highest indexes of immune organs and the lowest concentrations of the plasma immune parameters C3, C4, IgA, and IgY, while the commercial Avian broiler showed the opposite results. The Daweishan mini chickens showed the highest mRNA expressions of HDP genes in small intestine followed by the semi-selected Wuding chickens. Compared with local breeds, broiler chickens showed higher mRNA expression of HDP genes in spleen, thymus, and bursa. Body weight and HDP expression levels were negatively correlated in the intestine and positively in the immune organs. Our results indicated that the HDP immune regulatory roles in small intestine acted as first line of defense in innate immunity in local breeds, and as an adaptive immunity in broiler chickens. Selection was associated with different expression expressions of HDP genes in breed-, age-, and organ-specific manners.

Selection for growth rate and body size have altered the expression profiles of somatotropic axis genes in chickens.

The growth hormone / insulin-like growth factor-1 (GH/IGF-1) pathway of the somatotropic axis is the major controller for growth rate and body size in vertebrates, but the effect of selection on the expression of GH/IGF-1 somatotropic axis genes and their association with body size and growth performance in farm animals is not fully understood. We analyzed a time series of expression profiles of GH/IGF-1 somatotropic axis genes in two chicken breeds, the Daweishan mini chickens and Wuding chickens, and the commercial Avian broilers hybrid exhibiting markedly different body sizes and growth rates. We found that growth rate and feed conversion efficiency in Daweishan mini chickens were significantly lower than those in Wuding chickens and Avian broilers. The Wuding and Daweishan mini chickens showed higher levels of plasma GH, pituitary GH mRNA but lower levels of hepatic growth hormone receptor (GHR) mRNA than in Avian broilers. Daweishan mini chickens showed significantly lower levels of plasma IGF-1, thigh muscle and hepatic IGF-1 mRNA than did Avian broilers and Wuding chickens. These results suggest that the GH part of the somatotropic axis is the main regulator of growth rate, while IGF-1 may regulate both growth rate and body weight. Selection for growth performance and body size have altered the expression profiles of somatotropic axis genes in a breed-, age-, and tissue-specific manner, and manner, and alteration of regulatory mechanisms of these genes might play an important role in the developmental characteristics of chickens.

Upconversion fluorescence metal-organic frameworks thermo-sensitive imprinted polymer for enrichment and sensing protein.

A novel fluorescence material with thermo-sensitive for the enrichment and sensing of protein was successfully prepared by combining molecular imprinting technology with upconversion nanoparticles (UCNPs) and metal-organic frameworks (MOFs). Herein, the UCNPs acted as signal reporter for composite materials because of its excellent fluorescence property and chemical stability. MOFs were introduced to molecularly imprinted polymer (MIP) due to its high specific surface area which increases the rate of mass transfer relative to that of traditional bulk MIP. The thermo-sensitive imprinted material which allows for swelling and shrinking with response to temperature changes was prepared by choosing Bovine hemoglobin (BHB) as the template, N-isopropyl acrylamide (NIPAAM) as the temperature-sensitive functional monomer and N,N-methylenebisacrylamide (MBA) as the cross-linker. The recognition characterizations of imprinted material-coated UCNPs/MOFs (UCNPs/MOFs/MIP) were evaluated, and the results showed that the fluorescence intensity of UCNPs/MOFs/MIP reduced gradually with the increase of BHB concentration. The fluorescence material was response to the temperature. The adsorption capacity was as much as 167.6 mg/g at 28°C and 101.2mg/g at 44°C, which was higher than that of traditional MIP. Therefore, this new fluorescence material for enrichment and sensing protein is very promising for future applications.