Key parameters of physiological responses to acute heat stress in two commercial layers determined by Fisher discriminant analyses.

Given the escalating global warming and the intense nature of modern poultry production, layers are becoming increasingly susceptible to heat stress. This stress disrupts the physiological processes of layers, which leads to reduced productivity and welfare. To address this issue, it is crucial to first evaluate the stress response systematically. However, such evaluations are still lacking in this field. The objective of this study was to accurately monitor the impact of thermal stress and identify common and key indicators that would support decision-making to maintain layer welfare and productivity under stress. We constructed two heat stress models to reflect moderate (32 °C) to severe (36 °C) stress effects and obtained a comprehensive profile of blood physiological parameters associated with the layers' responses to heat stress. We found that genetic differences had limited influence on their physiological responses to heat stress after 32 °C heat challenges. Using 8 selected and significantly changed parameters, layers' physiological status under heat stress could be accurately determined (judgmental accuracy of 98%). As ambient temperature increased to 36 °C, birds suffered more severe challenges that parameters changed in larger percentages. Additionally, breed variations of the physiological responses became apparent, a Fisher discriminant function based on 5 selected parameters could distinguish heat stress effects at 32 °C or 36 °C with 80% accuracy. The results obtained from this study provide two discriminant models for assessing heat stress and shed lights on developing effective and widely applicable heat stress mitigation strategies targeting these indicators.

Genetic structure and characteristics of Tibetan chickens.

Tibetan chicken is one of the most common and widely distributed highland breeds, and is often used as a model organism for understanding genetic adaptation to extreme environments in Tibet. Despite its apparent geographical diversity and large variations in plumage patterns, the genetic differences within breed were not accounted for in most studies and have not been systematically investigated. In order to reveal and genetically differentiate the current existing TBC sub-populations that might have major implications for genomic research in TBCs, we systematically evaluated the population structure and demography of current TBC populations. Based on 344 whole-genome sequenced birds including 115 Tibetan chickens that were mostly sampled from family-farms across Tibet, we revealed a clear separation of Tibetan chickens into 4 sub-populations that broadly aligns with their geographical distribution. Moreover, population structure, population size dynamics, and the extent of admixture jointly suggest complex demographic histories of these sub-populations, including possible multiple origins, inbreeding, and introgressions. While most of the candidate selected regions found between the TBC sub-populations and Red Jungle fowls were nonoverlapping, 2 genes RYR2 and CAMK2D were revealed as strong selection candidates in all 4 sub-populations. These 2 previously identified high altitude associated genes indicated that the sub-populations responded to similar selection pressures in an independent but functionally similar fashion. Our results demonstrate robust population structure in Tibetan chickens that will help inform future genetic analyses on chickens and other domestic animals alike in Tibet, recommending thoughtful experimental design.

Identification of candidate genes related to highland adaptation from multiple Chinese local chicken breeds by whole genome sequencing analysis.

Understanding the genetic mechanism of highland adaptation is of great importance for breeding improvement of Tibetan chickens (TBC). Some studies of TBC have identified some candidate genes and pathways from multiple subgroups, but the related genetic mechanisms remain largely unknown. Different genetic backgrounds and the independent genetic basis of highland adaptation make it difficult to identity the selective region of highland adaptation with all TBC samples. In this study, we conducted pre-analysis in a large-scale population to select a TBC subgroup with the purest and highest level of highland-specific lineage for the further analysis. Finally, the 37 samples from a TBC subgroup and 19 Lahsa White chickens were used to represent the highland group for further analysis with 80 samples from five Chinese local lowland breeds as controls. Population structure analysis revealed that highland adaptation significantly affected population stratification in Chinese local chicken breeds. Genome-wide selection signal analysis identified 201 candidate genes associated with highland adaptation of TBC, and these genes were significantly enriched in calcium signaling, vascular smooth muscle contraction and the cellular response to oxidative stress pathways. Additionally, we identified a narrow 1.76 kb region containing an overlapping region between HBZ and an active enhancer, and our identified region showed a highly significant signal. The highland group selected the haplotype with high activity to improve the oxygen-carrying capacity, thus being adapted to a hypoxic environment. We also found that STX2 was significantly selected in the highland group, thus potentially reducing the oxidative stress caused by hypoxia, and that STX2 exhibited the opposite effects on highland adaptation and reproductive traits. Our findings advance our understanding of extreme environment adaptation of highland chickens, and provide some variants and genes beneficial to TBC genetic breeding improvement.

Genome-wide run of homozygosity analysis reveals candidate genomic regions associated with environmental adaptations of Tibetan native chickens.

BACKGROUND: In Tibet, the two most important breeds are Tibetan chicken and Lhasa white chicken, and the duo exhibit specific adaptations to the high altitude thereby supplying proteins for humans living in the plateau. These breeds are partly included in the conservation plans because they represent important chicken genetic resources. However, the genetic diversity of these chickens is rarely investigated. Based on whole-genome sequencing data of 113 chickens from 4 populations of Tibetan chicken including Shigatse (SH), Nyemo (NM), Dagze (DZ) and Nyingchi (LZ), as well as Lhasa white (LW) chicken breed, we investigated the genetic diversity of these chicken breeds by genetic differentiation, run of homozygosity (ROH), genomic inbreeding and selection signature analyses. RESULTS: Our results revealed high genetic diversity across the five chicken populations. The linkage disequilibrium decay was highest in LZ, while subtle genetic differentiation was found between LZ and other populations (Fst ranging from 0.05 to 0.10). Furthermore, the highest ROH-based inbreeding estimate (FROH) of 0.11 was observed in LZ. In other populations, the FROH ranged from 0.04 to 0.06. In total, 74, 111, 62, 42 and 54 ROH islands containing SNPs ranked top 1% for concurrency were identified in SH, NM, DZ, LZ and LW, respectively. Genes common to the ROH islands in the five populations included BDNF, CCDC34, LGR4, LIN7C, GLS, LOC101747789, MYO1B, STAT1 and STAT4. This suggested their essential roles in adaptation of the chickens. We also identified a common candidate genomic region harboring AMY2A, NTNG1 and VAV3 genes in all populations. These genes had been implicated in digestion, neurite growth and high-altitude adaptation. CONCLUSIONS: High genetic diversity is observed in Tibetan native chickens. Inbreeding is more intense in the Nyingchi population which is also genetically distant from other chicken populations. Candidate genes in ROH islands are likely to be the drivers of adaptation to high altitude exhibited by the five Tibetan native chicken populations. Our findings contribute to the understanding of genetic diversity offer valuable insights for the genetic mechanism of adaptation, and provide veritable tools that can help in the design and implementation of breeding and conservation strategies for Tibetan native chickens.

Multiple ancestral haplotypes harboring regulatory mutations cumulatively contribute to a QTL affecting chicken growth traits.

In depth studies of quantitative trait loci (QTL) can provide insights to the genetic architectures of complex traits. A major effect QTL at the distal end of chicken chromosome 1 has been associated with growth traits in multiple populations. This locus was fine-mapped in a fifteen-generation chicken advanced intercross population including 1119 birds and explored in further detail using 222 sequenced genomes from 10 high/low body weight chicken stocks. We detected this QTL that, in total, contributed 14.4% of the genetic variance for growth. Further, nine mosaic precise intervals (Kb level) which contain ancestral regulatory variants were fine-mapped and we chose one of them to demonstrate the key regulatory role in the duodenum. This is the first study to break down the detail genetic architectures for the well-known QTL in chicken and provides a good example of the fine-mapping of various of quantitative traits in any species.

A quantitative trait locus on chromosome 2 was identified that accounts for a substantial proportion of phenotypic variance of the yellow plumage color in chicken.

Chicken plumage color is an important economical trait in poultry breeding, as triple-yellow indigenous broilers are preferred over western commercial broilers in the Chinese market. However, the studies on the pigmentation of plumage coloration are relatively rare at present. Here, we performed a genome-wide mapping study on an F2 intercross, whose 2 founders were one hybrid commercial line "High Quality chicken Line A" that originated from the Anak red chicken and one indigenous line "Huiyang Beard" chicken that is a classical "triple-yellow" Chinese indigenous breed. Moreover, we used an automatic colorimeter that can quantitatively assess the colorations in L∗, a∗, and b∗ values. One major quantitative trait locus (QTL) on chromosome 2 was thus identified by both genome-wide association and linkage analyses, which could explain 10 to 20% of the total phenotypic variance of the b∗ measurements of the back plumage color. Using linkage analysis, 2 additional QTL on chromosome 1 and 20 were also found to be significantly associated with the plumage coloration in this cross. With additional samples from Anak red and Huiyang Beard chickens as well as pooled resequencing data from the 2 founders of this cross, we then further narrowed down the QTL regions and identified several candidate genes, such as CABLES1, CHST11, BCL2L1, and CHD22. As the effects of QTL found in this study were substantial, quantitatively measuring the coloration rather than the descriptive measurements provides stronger statistical power for the analyses. In addition, this major QTL on chromosome 2 that was associated with feather pigmentation at the genome-wide level will facilitate the future chicken breeding for yellow plumage color. In conclusions, we mapped 3 associated QTL on chromosome 1, 2, and 20. The candidate genes identified in this study shed light in the genetic basis of yellow plumage color in chicken.

A comprehensive analysis of the genomic organization, expression and phylogeny of immunoglobulin light chain genes in pigeon (Columba livia).

Previous studies on immunoglobulin light chain (IgL) genes in avian species are limited to Galloanseres, and few studies have investigated IgL genes in Neoaves, which includes most living birds. Based on published genome data, we demonstrate that the pigeon (Columba livia) IgL locus spans approximately 24 kb of DNA and contains twenty Vλ segments located upstream of a single pair of Jλ-Cλ. Among the identified Vλ gene segments, four segments are structurally intact and all four segments are able to recombine with Jλ. Moreover, the four functional Vλ segments are preferentially utilized in VλJλ recombination. Phylogenetic analysis suggests that the presence of the four functional Vλ segments in pigeon was likely generated by gene duplication that occurred after the divergence of pigeon and other birds. Our study provides insight into IgL gene evolution and evolutionary diversity of Ig genes in birds.

Polygenic Basis and Variable Genetic Architectures Contribute to the Complex Nature of Body Weight -A Genome-Wide Study in Four Chinese Indigenous Chicken Breeds.

Body weight (BW) is one of the most important economic traits for animal production and breeding, and it has been studied extensively for its phenotype-genotype associations. While mapping studies have mostly aimed at finding as many loci as possible that contributed to the variation in BW, the role of other factors in its genetic architecture, including their frequencies in the population and their interactions, have been largely overlooked. To comprehensively characterized the genetic architecture of BW, we performed a genome-wide association study (GWAS) both at the single-marker and haplotype level on birds from four indigenous Chinese chicken breeds (Chahua, Silkie, Langshan, and Beard), rather than studying crosses between two founder lines. Additionally, samples from two more breeds (Red Junglefowl and Recessive White) were included to better reflect variable genetic characteristics across populations. Six loci were mapped in this study, revealing the polygenic basis underlying BW. Moreover, by further examining the frequencies of the significantly associated haplotypes in each subpopulation and their effect sizes, most of the loci were found to affect BW in the Beard chicken breed alone. Two loci in GGA9 and GGA27, however, had a common effect on BW across subpopulations, showing that different underlying genetic mechanisms contribute to the phenotypic variability. These findings, particularly the variable genetic architectures found in different loci, improve our understanding of the overall genetic contributions to the large variability in BW among Chinese indigenous chicken breeds. These findings thus will have important implications for future chicken breeding.

A copy number variation generated by complicated organization of PCDHA gene cluster is associated with egg performance traits in Xinhua E-strain.

In recent years, a mass of duplicated and deleted DNA sequences have been found in human and animal genomes following the prevalence of employing high-throughput sequencing and SNP array. However, few copy number variation (CNV) studies have been performed on egg performance traits of chicken. In this study, 17 loci reported in previous studies were selected for CNV detection in the Xinhua E-strain by using the CNVplex kit, and the detection results showed that locus14 exhibited CNV. Further association analysis indicated the copies of locus14 could be significantly associated with age at first egg (AFE; P < 0.0086) and egg number at 250 d (250EN; P < 0.036). DNA sequence amplification showed the loss of a 260-bp-long fragment in the upstream of locus14, which mainly occurred in normal or copy-gain individuals. The qPCR results showed that subjects with gain of copies could promote the total expression level of the PCDHA gene cluster in the pituitary gland of adult individuals. Additionally, PCR amplification with randomly combined primers revealed a larger number of chicken variable exons than that previously reported, indicating the complexity of the organization of the PCDHA gene cluster. Those variable exons are divergent in their distribution among the populations of Xinhua E-strain, Chahua, Tibetan, and Tulufan Game Chicken, and most individuals only possess part of variable exons. Overall, the copies of locus14 reflect the variable exon dosage effects on the total expression level of the PCDHA gene cluster, which may regulate the layer egg production by affecting the development of the neural system.

Artificial Selection Response due to Polygenic Adaptation from a Multilocus, Multiallelic Genetic Architecture.

The ability of a population to adapt to changes in their living conditions, whether in nature or captivity, often depends on polymorphisms in multiple genes across the genome. In-depth studies of such polygenic adaptations are difficult in natural populations, but can be approached using the resources provided by artificial selection experiments. Here, we dissect the genetic mechanisms involved in long-term selection responses of the Virginia chicken lines, populations that after 40 generations of divergent selection for 56-day body weight display a 9-fold difference in the selected trait. In the F15 generation of an intercross between the divergent lines, 20 loci explained >60% of the additive genetic variance for the selected trait. We focused particularly on fine-mapping seven major QTL that replicated in this population and found that only two fine-mapped to single, bi-allelic loci; the other five contained linked loci, multiple alleles or were epistatic. This detailed dissection of the polygenic adaptations in the Virginia lines provides a deeper understanding of the range of different genome-wide mechanisms that have been involved in these long-term selection responses. The results illustrate that the genetic architecture of a highly polygenic trait can involve a broad range of genetic mechanisms, and that this can be the case even in a small population bred from founders with limited genetic diversity.

Genome-wide standing variation facilitates long-term response to bidirectional selection for antibody response in chickens.

BACKGROUND: Long-term selection experiments provide a powerful approach to gain empirical insights into adaptation, allowing researchers to uncover the targets of selection and infer their contributions to the mode and tempo of adaptation. Here we implement a pooled genome re-sequencing approach to investigate the consequences of 39 generations of bidirectional selection in White Leghorn chickens on a humoral immune trait: antibody response to sheep red blood cells. RESULTS: We observed wide genome involvement in response to this selection regime. Many genomic regions were highly differentiated resulting from this experimental selection regime, an involvement of up to 20% of the chicken genome (208.8 Mb). While genetic drift has certainly contributed to this, we implement gene ontology, association analysis and population simulations to increase our confidence in candidate selective sweeps. Three strong candidate genes, MHC, SEMA5A and TGFBR2, are also presented. CONCLUSIONS: The extensive genomic changes highlight the polygenic genetic architecture of antibody response in these chicken populations, which are derived from a common founder population, demonstrating the extent of standing immunogenetic variation available at the onset of selection.

A Complex Structural Variation on Chromosome 27 Leads to the Ectopic Expression of HOXB8 and the Muffs and Beard Phenotype in Chickens.

Muffs and beard (Mb) is a phenotype in chickens where groups of elongated feathers gather from both sides of the face (muffs) and below the beak (beard). It is an autosomal, incomplete dominant phenotype encoded by the Muffs and beard (Mb) locus. Here we use genome-wide association (GWA) analysis, linkage analysis, Identity-by-Descent (IBD) mapping, array-CGH, genome re-sequencing and expression analysis to show that the Mb allele causing the Mb phenotype is a derived allele where a complex structural variation (SV) on GGA27 leads to an altered expression of the gene HOXB8. This Mb allele was shown to be completely associated with the Mb phenotype in nine other independent Mb chicken breeds. The Mb allele differs from the wild-type mb allele by three duplications, one in tandem and two that are translocated to that of the tandem repeat around 1.70 Mb on GGA27. The duplications contain total seven annotated genes and their expression was tested during distinct stages of Mb morphogenesis. A continuous high ectopic expression of HOXB8 was found in the facial skin of Mb chickens, strongly suggesting that HOXB8 directs this regional feather-development. In conclusion, our results provide an interesting example of how genomic structural rearrangements alter the regulation of genes leading to novel phenotypes. Further, it again illustrates the value of utilizing derived phenotypes in domestic animals to dissect the genetic basis of developmental traits, herein providing novel insights into the likely role of HOXB8 in feather development and differentiation.

Standing genetic variation as a major contributor to adaptation in the Virginia chicken lines selection experiment.

BACKGROUND: Artificial selection provides a powerful approach to study the genetics of adaptation. Using selective-sweep mapping, it is possible to identify genomic regions where allele-frequencies have diverged during selection. To avoid false positive signatures of selection, it is necessary to show that a sweep affects a selected trait before it can be considered adaptive. Here, we confirm candidate, genome-wide distributed selective sweeps originating from the standing genetic variation in a long-term selection experiment on high and low body weight of chickens. RESULTS: Using an intercross between the two divergent chicken lines, 16 adaptive selective sweeps were confirmed based on their association with the body weight at 56 days of age. Although individual additive effects were small, the fixation for alternative alleles across the loci contributed at least 40 % of the phenotypic difference for the selected trait between these lines. The sweeps contributed about half of the additive genetic variance present within and between the lines after 40 generations of selection, corresponding to a considerable portion of the additive genetic variance of the base population. CONCLUSIONS: Long-term, single-trait, bi-directional selection in the Virginia chicken lines has resulted in a gradual response to selection for extreme phenotypes without a drastic reduction in the genetic variation. We find that fixation of several standing genetic variants across a highly polygenic genetic architecture made a considerable contribution to long-term selection response. This provides new fundamental insights into the dynamics of standing genetic variation during long-term selection and adaptation.

Identification of Valid Housekeeping Genes for Real-Time Quantitative PCR Analysis of Collapsed Lung Tissues of Neonatal Somatic Cell Nuclear Transfer-Derived Cattle.

Cloned calves produced by somatic cell nuclear transfer frequently suffer alveolar collapse as newborns. To study the underlying pathophysiological mechanisms responsible for this phenomenon, the expression profiles of numerous genes involved in lung development need to be investigated. Quantitative real-time PCR is commonly adopted in gene expression analysis. However, selection of an appropriate reference gene for normalization is critical for obtaining reliable and accurate results. Seven housekeeping genes-ß-glucuronidase (GUSB), phosphoglycerate kinase 1 (PGK1), ß-2-microglobolin (B2M), peptidylprolyl isomerase A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA-box binding protein (TBP), and 5.8S ribosomal RNA (5.8S rRNA)-were selected and evaluated as candidates. Their gene expression levels in the collapsed lungs of deceased neonate cloned calves and normal lung derived from normal calves were assessed. The ranking of gene expression stability was estimated by the geNorm, NormFinder, and BestKeeper programs. 5.8S rRNA and PPIA were determined to be the most stable reference genes by geNorm and BestKeeper, whereas the combination of GAPDH and TBP was suggested as reference genes by NormFinder. Taking these results into account, we conclude that 5.8S rRNA and PPIA could be the most reliable reference genes for studying the genes involved in alveolar collapse. Moreover, 5.8S rRNA could be represented as a uniform reference gene in similar cases.

Transgenic mouse milk expressing human bile salt-stimulated lipase improves the survival and growth status of premature mice.

The lactating human mammary gland and the pancreas both produce bile salt-stimulated lipase (BSSL), a lipolytic enzyme acting on a wide range of substrates, including triglyceride, cholesterol esters, and fat-soluble vitamins esters. Breast milk BSSL has a particularly important role in the digestion of milk fat by newborn infants. We report the generation of transgenic mice that harbored a human BSSL gene controlled by a mammary gland-specific promoter. BSSL levels in transgenic mouse milk were raised to 376.8 µg/ml, corresponding to an activity of 9.15 U/ml. Premature wild-type neonates nursed by transgenic dams exhibited significantly higher survival rate than did the control neonates nursed by wild dams (95 vs. 83.3 % and, P < 0.05). They also showed 43.8 % greater body weight gain and 33.3 % lesser fecal crude fat levels than did the controls. This study provides significant evidence that increased levels of BSSL in milk may reduce mortality and improve the growth and fat absorption in premature mice during neonatal development.

Natural CMT2 variation is associated with genome-wide methylation changes and temperature seasonality.

As Arabidopsis thaliana has colonized a wide range of habitats across the world it is an attractive model for studying the genetic mechanisms underlying environmental adaptation. Here, we used public data from two collections of A. thaliana accessions to associate genetic variability at individual loci with differences in climates at the sampling sites. We use a novel method to screen the genome for plastic alleles that tolerate a broader climate range than the major allele. This approach reduces confounding with population structure and increases power compared to standard genome-wide association methods. Sixteen novel loci were found, including an association between Chromomethylase 2 (CMT2) and temperature seasonality where the genome-wide CHH methylation was different for the group of accessions carrying the plastic allele. Cmt2 mutants were shown to be more tolerant to heat-stress, suggesting genetic regulation of epigenetic modifications as a likely mechanism underlying natural adaptation to variable temperatures, potentially through differential allelic plasticity to temperature-stress.

MAPfastR: quantitative trait loci mapping in outbred line crosses.

MAPfastR is a software package developed to analyze quantitative trait loci data from inbred and outbred line-crosses. The package includes a number of modules for fast and accurate quantitative trait loci analyses. It has been developed in the R language for fast and comprehensive analyses of large datasets. MAPfastR is freely available at: http://www.computationalgenetics.se/?page_id=7.

The duck genome and transcriptome provide insight into an avian influenza virus reservoir species.

The duck (Anas platyrhynchos) is one of the principal natural hosts of influenza A viruses. We present the duck genome sequence and perform deep transcriptome analyses to investigate immune-related genes. Our data indicate that the duck possesses a contractive immune gene repertoire, as in chicken and zebra finch, and this repertoire has been shaped through lineage-specific duplications. We identify genes that are responsive to influenza A viruses using the lung transcriptomes of control ducks and ones that were infected with either a highly pathogenic (A/duck/Hubei/49/05) or a weakly pathogenic (A/goose/Hubei/65/05) H5N1 virus. Further, we show how the duck's defense mechanisms against influenza infection have been optimized through the diversification of its ß-defensin and butyrophilin-like repertoires. These analyses, in combination with the genomic and transcriptomic data, provide a resource for characterizing the interaction between host and influenza viruses.

Genetic dissection of growth traits in a Chinese indigenous × commercial broiler chicken cross.

BACKGROUND: In China, consumers often prefer indigenous broiler chickens over commercial breeds, as they have characteristic meat qualities requested within traditional culinary customs. However, the growth-rate of these indigenous breeds is slower than that of the commercial broilers, which means they have not yet reached their full economic value. Therefore, combining the valuable meat quality of the native chickens with the efficiency of the commercial broilers is of interest. In this study, we generated an F2 intercross between the slow growing native broiler breed, Huiyang Beard chicken, and the fast growing commercial broiler breed, High Quality chicken Line A, and used it to map loci explaining the difference in growth rate between these breeds. RESULTS: A genome scan to identify main-effect loci affecting 24 growth-related traits revealed nine distinct QTL on six chromosomes. Many QTL were pleiotropic and conformed to the correlation patterns observed between phenotypes. Most of the mapped QTL were found in locations where growth QTL have been reported in other populations, although the effects were greater in this population. A genome scan for pairs of interacting loci identified a number of additional QTL in 10 other genomic regions. The epistatic pairs explained 6-8% of the residual phenotypic variance. Seven of the 10 epistatic QTL mapped in regions containing candidate genes in the ubiquitin mediated proteolysis pathway, suggesting the importance of this pathway in the regulation of growth in this chicken population. CONCLUSIONS: The main-effect QTL detected using a standard one-dimensional genome scan accounted for a significant fraction of the observed phenotypic variance in this population. Furthermore, genes in known pathways present interesting candidates for further exploration. This study has thus located several QTL regions as promising candidates for further study, which will increase our understanding of the genetic mechanisms underlying growth-related traits in chickens.