Sirui Chen

A Genome-Wide SNP Scan Reveals Novel Loci for Egg Production and Quality Traits in White Leghorn and Brown-Egg Dwarf Layers

Availability of the complete genome sequence as well as high-density SNP genotyping platforms allows genome-wide association studies (GWAS) in chickens. A high-density SNP array containing 57,636 markers was employed herein to identify associated variants underlying egg production and quality traits within two lines of chickens, i.e., White Leghorn and brown-egg dwarf layers. For each individual, age at first egg (AFE), first egg weight (FEW), and number of eggs (EN) from 21 to 56 weeks of age were recorded, and egg quality traits including egg weight (EW), eggshell weight (ESW), yolk weight (YW), eggshell thickness (EST), eggshell strength (ESS), albumen height(AH) and Haugh unit(HU) were measured at 40 and 60 weeks of age. A total of 385 White Leghorn females and 361 brown-egg dwarf dams were selected to be genotyped. The genome-wide scan revealed 8 SNPs showing genome-wise significant (P<1.51E-06, Bonferroni correction) association with egg production and quality traits under the Fishers combined probability method. Some significant SNPs are located in known genes including GRB14 and GALNT1 that can impact development and function of ovary, but more are located in genes with unclear functions in layers, and need to be studied further. Many chromosome-wise significant SNPs were also detected in this study and some of them are located in previously reported QTL regions. Most of loci detected in this study are novel and the follow-up replication studies may be needed to further confirm the functional significance for these newly identified SNPs.

Copy number variations identified in the chicken using a 60K SNP BeadChip

Copy number variation (CNV) is considered an important genetic variation, contributing to many economically important traits in the chicken. Although CNVs can be detected using a comparative genomic hybridization array, the high-density SNP array has provided an alternative way to identify CNVs in the chicken. In the current study, a chicken 60K SNP BeadChip was used to identify CNVs in two distinct chicken genetic lines (White Leghorn and dwarf) using the PENNCNV program. A total of 209 CNV regions were identified, distributing on chromosomes 1-22 and 24-28 and encompassing 13.55 Mb (1.42%) of chicken autosomal genome area. Three of seven selected CNVs (73.2% individuals) were completely validated by quantitative PCR. To our knowledge, this is the first report in the chicken identifying CNVs using a SNP array. Identification of 190 new identified CNVs illustrates the feasibility of the chicken 60K SNP BeadChip to detect CNVs in the chicken, which lays a solid foundation for future analyses of associations of CNVs with economically important phenotypes in chickens.

Methylation status of cMHM and expression of sex-specific genes in adult sex-reversed female chickens.

The objective of the current study was to analyze the methylation status of the chicken male hypermethylation (cMHM) region and mRNA expression levels of sex-dependent genes in adult female-to-male sex-reversed chickens. Sex reversal from genetic females into phenotypic males was induced by injection of 1.0 mg fadrozole, an aromatase inhibitor, into fertilized eggs at 3.0 days of incubation. Birds aged 30 weeks were classified into 4 groups according to the histological structure of their gonads and the natural logarithm of the ratio of testosterone to estradiol in serum, namely standard females, slightly sex-reversed females with left ovotestes, highly sex-reversed females with left testes, and standard males. The results showed that methylation of the cMHM amplicon was lowest in the ovaries of standard females and highest in testes of standard males. Methylation levels were significantly higher in the left testes of highly sex-reversed females than in the left ovotestes of slightly sex-reversed females (p < 0.05). Expression analysis of 9 sex-specific genes in the gonad indicated that DMRT1 and HINTZ had a similar expression pattern, with significantly higher levels in standard males as compared to standard females, slightly and highly sex-reversed females (p < 0.05). Expression of FOXL2, AMH, P450arom, SF1, and ERα was obviously higher in standard females than in standard males, slightly and highly sex-reversed females (p < 0.05). Expression of SOX9 in standard females was similar to that in slightly sex-reversed females and lower than in highly sex-reversed females and standard males (p < 0.05).

Genetic parameters of feed efficiency traits in laying period of chickens

Laying records on 1,534 F2 hens, derived from a reciprocal cross between White Leghorns and Dongxiang blue-shelled chickens, were used to estimate genetic parameters for residual feed intake (RFI), feed conversion ratio (FCR), daily feed intake (FI), metabolic BW (MBW), BW gain (BWG), and daily egg mass (EM) at 37 to 40 (T1) and 57 to 60 wk age (T2), respectively. Genetic analysis was subsequently conducted with the AI-REML method using an animal model. Estimates for heritability of RFI, FCR, and FI were 0.21, 0.19, and 0.20 in T1, and 0.29, 0.13, and 0.26 in T2, respectively. In T1 and T2, RFI showed high and positive genetic correlations with FCR (0.51, 0.43) and FI (0.72, 0.84), whereas the genetic correlation between FI and FCR was very low (−0.09, 0.11). Genetically, negative correlations were found between RFI and its component traits (−0.01 to −0.47). In addition, high genetic correlations, from 0.76 to 0.94, were observed between T1 and T2 for RFI, FCR, and FI, suggesting that feed efficiency traits in the 2 stages had a similar genetic background. The results indicate that selection for low RFI could reduce FI without significant changes in EM, while selection on FCR will increase EM. The present study lays the foundation for genetic improvement of feed efficiency during the laying period of chickens.

Developmental phenotypic-genotypic associations of tyrosinase and melanocortin 1 receptor genes with changing profiles in chicken plumage pigmentation

The tyrosinase (TYR) and melanocortin 1 receptor (MC1R) genes have been accepted as major genes involved in the plumage pigmentation of chickens. The co-segregation of plumage coloration and sequence polymorphism in TYR and MC1R genes were investigated using an intercross between black and white plumage color types of the Dongxiang blue-shelled chicken. Profiles of plumage color changing and genes expression levels of TYR and MC1R were observed from hatch to 112 d of age using quantitative real-time reverse transcription-PCR. Intercrossed offspring were classified by phenotypes of plumage colors. The phenotypes of black and amber chicks with genotypes of E_C_ exhibited a black feather pattern, whereas white, gray, and buff chicks with genotypes of E_cc and eecc belonged to the white feather pattern. Although TYR in cooperation with MC1R determined the coloration feather patterns, the different phenotypes did not correspond completely with the genotypes. During the period studied, plumage phenotype changed dramatically, and the buff and gray down were gradually replaced by whiteness feathers. Real-time reverse transcription-PCR studies showed that 1) expression levels of TYR declined dramatically with age, and expression at hatch was highest (P<0.01) during the ages studied; 2) expression level of MC1R was higher at 28 d than at younger and older ages; and 3) expression of TYR in chickens carrying E/E and E/e alleles on MC1R loci were higher than those carrying e/e alleles from hatch to 28 d.

Polymorphisms in Wnt signaling pathway genes are significantly associated with chicken carcass traits

The Wnt signaling pathway plays a crucial role during embryogenesis in vertebrates. In this study, 124 SNP in 31 Wnt signaling pathway genes were selected to genotype 764 individuals in an F(2) resource population by reciprocally crossing Silkie fowls and Cornish broilers, and 102 SNP were polymorphic. Pairwise linkage disequilibrium among the SNP within each gene was calculated. Haplotypes were reconstructed from the SNP in strong linkage disequilibrium. The associations of SNP and haplotypes with carcass traits were analyzed respectively, and the SNP contributions to phenotypic variance were estimated. The present study showed that 58 SNP in 24 genes and 8 haplotype blocks within 7 genes were significantly (P < 0.05) associated with at least one carcass trait. Fourteen SNP (among the 58 SNP) explained >2% phenotypic variance, 12 of which had significantly (P < 0.01) additive or dominant effects. Furthermore, both rs15865526 (Wnt9A) and rs14066777 (MAPK9) as well as their corresponding haplotype blocks were significantly associated with shank circumference and wing weight, respectively. In addition, 5 muscle-weight-related SNP explained >7% phenotypic variance, which was much higher than those of others. It was found that the Wnt signaling pathway was strongly associated with chicken carcass traits, and 7 genes were particularly important, namely RHOA and CHP for breast muscle weight, Wnt3A for breast muscle weight percentage over carcass weight, RAC1 for thigh weight percentage and thigh muscle weight percentage over carcass weight, Wnt11 for thigh weight percentage over carcass weight, Wnt9A for shank length, and MAPK9 for shank circumference. It is evident that Wnt signaling plays a major role in regulating carcass characteristics important for production traits in chickens.

Genome‐wide copy number profiling using high‐density SNP array in chickens

Phenotypic diversity is a direct consequence resulting mainly from the impact of underlying genetic variation, and recent studies have shown that copy number variation (CNV) is emerging as an important contributor to both phenotypic variability and disease susceptibility. Herein, we performed a genome-wide CNV scan in 96 chickens from 12 diversified breeds, benefiting from the high-density Affymetrix 600 K SNP arrays. We identified a total of 231 autosomal CNV regions (CNVRs) encompassing 5.41 Mb of the chicken genome and corresponding to 0.59% of the autosomal sequence. The length of these CNVRs ranged from 2.6 to 586.2 kb with an average of 23.4 kb, including 130 gain, 93 loss and eight both gain and loss events. These CNVRs, especially deletions, had lower GC content and were located particularly in gene deserts. In particular, 102 CNVRs harbored 128 chicken genes, most of which were enriched in immune responses. We obtained 221 autosomal CNVRs after converting probe coordinates to Galgal3, and comparative analysis with previous studies illustrated that 153 of these CNVRs were regarded as novel events. Furthermore, qPCR assays were designed for 11 novel CNVRs, and eight (72.73%) were validated successfully. In this study, we demonstrated that the high-density 600 K SNP array can capture CNVs with higher efficiency and accuracy and highlighted the necessity of integrating multiple technologies and algorithms. Our findings provide a pioneering exploration of chicken CNVs based on a high-density SNP array, which contributes to a more comprehensive understanding of genetic variation in the chicken genome and is beneficial to unearthing potential CNVs underlying important traits of chickens.

A genome-wide association study identifies novel single nucleotide polymorphisms associated with dermal shank pigmentation in chickens

Shank color of domestic chickens varies from black to blue, green, yellow, or white, which is controlled by the combination of melanin and xanthophylls in dermis and epidermis. Dermal shank pigmentation of chickens is determined by sex-linked inhibitor of dermal melanin (Id), which is located on the distal end of the long arm of Z chromosome, through controlling dermal melanin pigmentation. Although previous studies have focused on the identification of Id and the linear relationship with barring and recessive white skin, no causal mutations have yet been identified in relation to the mutant dermal pigment inhibiting allele at the Id locus. In this study, we first used the 600K Affymetrix Axiom HD genotyping array, which includes ~580,961 SNP of which 26,642 SNP were on the Z chromosome to perform a genome-wide association study on pure lines of 19 Tibetan hens with dermal pigmentation shank and 21 Tibetan hens with yellow shank to refine the Id location. Association analysis was conducted by the PLINK software using the standard chi-squared test, and then Bonferroni correction was used to adjust multiple testing. The genome-wide study revealed that 3 SNP located at 78.5 to 79.2 Mb on the Z chromosome in the current assembly of chicken genome (galGal4) were significantly associated with dermal shank pigmentation of chickens, but none of them were located in known genes. The interval we refined was partly converged with previous results, suggesting that the Id gene is in or near our refined genome region. However, the genomic context of this region was complex. There were only 15 SNP markers developed by the genotyping array within the interval region, in which only 1 SNP marker passed quality control. Additionally, there were about 5.8-Mb gaps on both sides of the refined interval. The follow-up replication studies may be needed to further confirm the functional significance for these newly identified SNP.

Comparison of protoporphyrin IX content and related gene expression in the tissues of chickens laying brown-shelled eggs

Protoporphyrin IX (PpIX), an immediate precursor of heme, is the main pigment resulting in the brown coloration of eggshell. The brownness and uniformity of the eggshell are important marketing considerations. In this study, 9 chickens laying darker brown shelled eggs and 9 chickens laying lighter brown shelled eggs were selected from 464 individually caged layers in a Rhode Island Red pureline. The PpIX contents were measured with a Microplate Reader at the wavelength of 412 nm and were compared in different tissues of the 2 groups. Although no significant difference in serum, bile, and excreta was found between the 2 groups, PpIX content in the shell gland and eggshell of the darker group was higher than in those of the lighter group, suggesting that PpIX was synthesized in the shell gland. We further determined the expression levels of 8 genes encoding enzymes involved in the heme synthesis and transport in the liver and shell gland at 6 h postoviposition by quantitative PCR. The results showed that expression of aminolevulinic acid synthase-1 (ALAS1) was higher in the liver of hens laying darker brown shelled eggs, whereas in the shell gland the expression levels of ALAS1, coproporphyrinogen oxidase (CPOX), ATP-binding cassette family members ABCB7 and ABCG2, and receptor for feline leukemia virus, subgroup C (FLVCR) were significantly higher in the hens laying darker brown shelled eggs. Our results demonstrated that hens laying darker brown shelled eggs could deposit more PpIX onto the eggshell and the brownness of the eggshell was dependent on the total quantity of PpIX in the eggshell. More heme was synthesized in the liver and shell gland of hens laying darker brown shelled eggs than those of hens laying lighter brown shelled eggs. High expression level of ABCG2 might facilitate the accumulation of PpIX in the shell gland.

Polymorphisms in the transforming growth factor β3 gene and their associations with feed efficiency in chickens

Growth and feeding traits such as BW, BW gain (BWG), feed intake (FI), and feed conversion ratio (FCR) are of economic importance in poultry production. In this study, 8 SNP of the transforming growth factor β3 (TGF-β3) gene, which are located in the proximity of quantitative trait loci affecting BW and FCR, were selected to be genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in 2 yellow meat-type chicken populations with 724 birds in total. Body weights at 49 (BW49) and 70 (BW70) d of age and FI in the interval were recorded, and respective BWG and FCR were calculated for each bird. Two SNP with a very low minor allele frequency (<1%) were discarded from further analysis. The results showed that both rs13586818 and rs14535174 had significant effects (P < 0.05) on BWG and FCR, and that rs14535177 was significantly (P < 0.05) associated with BW49, BW70, and FI. Furthermore, birds with the GA genotype of rs13586818 showed strongly higher FCR and lower BWG (P < 0.05) compared with AA individuals. The TT birds had significantly larger BWG (P < 0.05) than GT birds in rs14535174, whereas FCR was the opposite. Individuals with the GC genotype of rs14535177 had significantly higher BW49, BW70, FCR, and FI (P < 0.05) than those with the GG genotype. Additionally, haplotypes based on 3 SNP of rs13586818, rs14535174, and rs14535177 were significantly associated with FCR (P < 0.05). The SNP and analyzed haplotypes identified in this study might be used as potential genetic markers in meat-type chicken breeding.