Lujiang Qu

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.

An EAV-HP insertion in 5' Flanking region of SLCO1B3 causes blue eggshell in the chicken.

The genetic determination of eggshell coloration has not been determined in birds. Here we report that the blue eggshell is caused by an EAV-HP insertion that promotes the expression of SLCO1B3 gene in the uterus (shell gland) of the oviduct in chicken. In this study, the genetic map location of the blue eggshell gene was refined by linkage analysis in an F2 chicken population, and four candidate genes within the refined interval were subsequently tested for their expression levels in the shell gland of the uterus from blue-shelled and non-blue-shelled hens. SLCO1B3 gene was found to be the only one expressed in the uterus of blue-shelled hens but not in that of non-blue-shelled hens. Results from a pyrosequencing analysis showed that only the allele of SLCO1B3 from blue-shelled chickens was expressed in the uterus of heterozygous hens (O*LC/O*N). SLCO1B3 gene belongs to the organic anion transporting polypeptide (OATP) family; and the OATPs, functioning as membrane transporters, have been reported for the transportation of amphipathic organic compounds, including bile salt in mammals. We subsequently resequenced the whole genomic region of SLCO1B3 and discovered an EAV-HP insertion in the 5′ flanking region of SLCO1B3. The EAV-HP insertion was found closely associated with blue eggshell phenotype following complete Mendelian segregation. In situ hybridization also demonstrated that the blue eggshell is associated with ectopic expression of SLCO1B3 in shell glands of uterus. Our finding strongly suggests that the EAV-HP insertion is the causative mutation for the blue eggshell phenotype. The insertion was also found in another Chinese blue-shelled breed and an American blue-shelled breed. In addition, we found that the insertion site in the blue-shelled chickens from Araucana is different from that in Chinese breeds, which implied independent integration events in the blue-shelled chickens from the two continents, providing a parallel evolutionary example at the molecular level.

A systematic analysis of miRNA transcriptome in Marek's disease virus-induced lymphoma reveals novel and differentially expressed miRNAs.

Marek’s disease is a lymphoproliferative neoplastic disease of the chicken, which poses a serious threat to poultry health. Marek’s disease virus (MDV)-induced T-cell lymphoma is also an excellent biomedical model for neoplasia research. Recently, miRNAs have been demonstrated to play crucial roles in mediating neoplastic transformation. To investigate host miRNA expression profiles in the tumor transformation phase of MDV infection, we performed deep sequencing in two MDV-infected samples (tumorous spleen and MD lymphoma from liver), and two non-infected controls (non-infected spleen and lymphocytes). In total, 187 and 16 known miRNAs were identified in chicken and MDV, respectively, and 17 novel chicken miRNAs were further confirmed by qPCR. We identified 28 down-regulated miRNAs and 11 up-regulated miRNAs in MDV-infected samples by bioinformatic analysis. Of nine further tested by qPCR, seven were verified. The gga-miR-181a, gga-miR-26a, gga-miR-221, gga-miR-222, gga-miR-199*, and gga-miR-140* were down-regulated, and gga-miR-146c was up-regulated in MDV-infected tumorous spleens and MD lymphomas. In addition, 189 putative target genes for seven differentially expressed miRNAs were predicted. The luciferase reporter gene assay showed interactions of gga-miR-181a with MYBL1, gga-miR-181a with IGF2BP3, and gga-miR-26a with EIF3A. Differential expression of miRNAs and the predicted targets strongly suggest that they contribute to MDV-induced lymphomagenesis.

Laying performance and egg quality of blue-shelled layers as affected by different housing systems

Blue-shelled eggs are gaining popularity as the consumption demand diversifies in some countries. This study was carried out to investigate the laying performance and egg quality of the blue-shelled egg layers as well as the effects of different housing systems on egg production and quality traits. One thousand pullets from Dongxiang blue-shelled layers were divided into 2 even groups and kept in different housing systems (outdoor vs. cage). Daily laying performance was recorded from 20 to 60 wk of age. External and internal egg quality traits were examined at 26, 34, 42, and 50 wk. Yolk cholesterol concentration and whole egg cholesterol content were measured at 40 wk of age. Average laying rate from 20 to 60 wk for the cage (54.7%) was significantly higher than that of outdoor layers (39.3%). Among all of the egg quality traits, only eggshell color was affected by housing system. Interaction between housing system and layer age was found in egg weight, eggshell color, eggshell ratio, yolk color, and yolk weight. Meanwhile, cholesterol concentration in yolk was 8.64 +/- 0.40 mg/g in the outdoor eggs, which was significantly lower than that of eggs from the cage birds (10.32 +/- 0.48 mg/g; P < 0.05). Whole egg cholesterol content in the outdoor eggs (125.23 +/- 6.32 mg/egg) was also significantly lower than that of eggs from the caged layers (158.01 +/- 8.62 mg/egg). The results demonstrated that blue-shelled layers have lower productivity in the outdoor system than in the cage system. Blue-shelled layers have lower egg weight, larger yolk proportion, and lower cholesterol content compared with commercial layers. In a proper marketing system, lower productivity could be balanced by a higher price for the better quality of blue-shelled eggs.

Effect of myofiber characteristics and thickness of perimysium and endomysium on meat tenderness of chickens

The objective of the present study was to evaluate the role of myofiber characteristics and the thickness of 2 major muscle membranes, perimysium and endomysium, in determining the breast meat tenderness of chickens. Birds from 2 breeds (White Leghorn and a line of broiler) were chosen. Chicks were sexed and wing-banded at hatch and were grown in separate cages in a single house. Sixty broilers and 60 White Leghorns were harvested at 6 wk of age, respectively, whereas another 60 White Leghorns were slaughtered at 18 wk of age. An equal number of males and females was maintained for each group. Body weight, breast muscle weight, pH, drip loss, cooking loss, Warner-Bratzler shear force value (SFV), total energy of shear force, fiber diameter, sarcomere length, myofiber density, and the thickness of endomysium and perimysium of the breast were determined for each bird. At 6 wk of age, histological examination indicated that the size of myofiber and thickness of endomysium and perimysium of broilers were larger than that those of White Leghorns (P < 0.01), whereas the SFV, drip loss, and cooking loss of broilers were smaller (P < 0.01). A comparison between the White Leghorns at 18 wk and the broilers at 6 wk, which were at similar BW but different ages, showed that the breast muscle weight of broilers was larger (P < 0.01) than that of White Leghorns. For breast muscle, the endomysium of broilers at 6 wk was thicker than that of White Leghorns at 18 wk (P < 0.01), whereas the perimysium was thinner (P < 0.01). The SFV, drip loss, and the cooking loss of broilers were smaller than those of White Leghorns at similar BW (P < 0.01). Meat tenderness was negatively correlated with myofiber density (-0.27) and the thickness of endomysium (-0.29) and positively correlated with the thickness of perimysium (0.20). It is suggested that muscle membrane should be considered in evaluating meat tenderness of the chicken.

Positive diversifying selection in avian Mx genes

Mx proteins are interferon-induced GTPases that confer antiviral activities against RNA viruses. We analysed the molecular evolution of the Mx gene in birds using data on interspecific divergence in anseriform and galliform birds, and on intraspecific diversity in commercial chicken lines, local Chinese chicken breeds as well as in the mallard. The overall ratio of non-synonymous to synonymous substitution was unusually high, 0.80, indicating relaxed constraint or positive selection. Evidence for the latter was provided by that a total of 11–18 codons were found to have evolved under positive selection. The great majority of these codons are located in a region unique to birds at the N-terminal end of the Mx protein. We found an excess of non-synonymous polymorphisms relative to synonymous variants in all comparisons. This, together with positive Tajima’s D values in the local Chinese chicken breeds and in the mallard suggests that balancing selection is acting in avian Mx genes. As such, Mx mimics the major histocompatibility complex system, indicating that heterozygous individuals are better off withstanding pathogen attack.

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).

Genome-Wide Characterization of Insertion and Deletion Variation in Chicken Using Next Generation Sequencing

Insertion and deletion (INDEL) is one of the main events contributing to genetic and phenotypic diversity, which receives less attention than SNP and large structural variation. To gain a better knowledge of INDEL variation in chicken genome, we applied next generation sequencing on 12 diverse chicken breeds at an average effective depth of 8.6. Over 1.3 million non-redundant short INDELs (1–49 bp) were obtained, the vast majority (92.48%) of which were novel. Follow-up validation assays confirmed that most (88.00%) of the randomly selected INDELs represent true variations. The majority (95.76%) of INDELs were less than 10 bp. Both the detected number and affected bases were larger for deletions than insertions. In total, INDELs covered 3.8 Mbp, corresponding to 0.36% of the chicken genome. The average genomic INDEL density was estimated as 0.49 per kb. INDELs were ubiquitous and distributed in a non-uniform fashion across chromosomes, with lower INDEL density in micro-chromosomes than in others, and some functional regions like exons and UTRs were prone to less INDELs than introns and intergenic regions. Nearly 620,253 INDELs fell in genic regions, 1,765 (0.28%) of which located in exons, spanning 1,358 (7.56%) unique Ensembl genes. Many of them are associated with economically important traits and some are the homologues of human disease-related genes. We demonstrate that sequencing multiple individuals at a medium depth offers a promising way for reliable identification of INDELs. The coding INDELs are valuable candidates for further elucidation of the association between genotypes and phenotypes. The chicken INDELs revealed by our study can be useful for future studies, including development of INDEL markers, construction of high density linkage map, INDEL arrays design, and hopefully, molecular breeding programs in chicken.

Expression profiles of genes within a subregion of chicken major histocompatibility complex B in spleen after Marek’s disease virus infection

Major histocompatibility complex has previously been shown to influence the resistance of chicken to Mareks disease virus (MDV). However, little is known about expression of other genes in the MHC-I and II pathway after MDV infection. This study aimed at investigating 8 immune-related genes in the MHC core region that affects host responses to MDV. Spleens of infected and age-matched uninfected chickens were removed at 4, 7, 14, 21, and 28 d postinfection for gene expression detection using real-time PCR. Different expression patterns of MHC-I and II pathway genes were observed in the spleen. In the MHC-I pathway, the expression of transporter of antigen protein 1 (TAP1), transporter of antigen protein 2 (TAP2), and transporter of antigen protein-binding protein (TAPBP) genes was significantly increased in the spleen of MDV-infected than that of uninfected chickens. It indicated that host antivirus responses were generated to enhance antigen presentation. However, MHC-II pathway genes showed contrary trends. Classical MHC-II β chain major gene (BLB2) and nonclassical class II genes [DM α chain gene (DMA), DM β chain gene-1 (DMB1), and DM β chain gene-2 (DMB2)] had consistent lower transcripts in spleens of MDV-infected than that of uninfected chickens, which reflected that MDV interfered with multiple components of the MHC-II pathway. Overall, expression of most genes in the MHC core region was altered; moreover, the genes in endogenous and exogenous antigen presentation pathways had different expression patterns in the spleen after MDV infection.

gga-miR-26a targets NEK6 and suppresses Marek’s disease lymphoma cell proliferation

MicroRNA (miRNA) are a class of highly conserved, small noncoding RNA that emerge as key posttranscriptional regulators in various neoplastic transformations. Our previous study profiling the miRNA transcriptome in Mareks disease virus (MDV)-induced lymphoma revealed many novel and differentially expressed miRNA, including gga-miR-26a, which was downregulated in MDV-infected spleens of chickens. In this study, differential expression of gga-miR-26a between MDV-infected and noninfected spleens at 4, 7, 14, 21, and 28 d postinfection was analyzed by real-time PCR. The results showed gga-miR-26a were downregulated in MDV-infected spleens at cytolytic infection, latency, and tumor transformation phases. Subsequent cell proliferation assay revealed cell viability was lower in gga-miR-26a mimic transfection group than that in negative controls. Target genes of gga-miR-26a were identified by luciferase reporter gene assay. The results showed significant interaction between gga-miR-26a and Never In Mitosis Gene A (NIMA)-related kinase 6 (NEK6) gene. Subsequent gain of function experiment and Western blot assay showed that mRNA and protein levels of NEK6 were downregulated after gga-miR-26 mimic was transfected into MDV-transformed lymphoid cell line (MSB-1), indicating that NEK6 was modulated by gga-miR-26a. The expression of NEK6 showed a higher trend in MDV-infected samples including tumorous spleen and MD lymphoma from liver than that in noninfected controls. The results suggested that gga-miR-26a inhibited MSB-1 cell proliferation. Gga-miR-26a and its direct target, NEK6, might play important roles in MDV infection.