Yanzhang Gong

Identification of Genes Related to White and Black Plumage Formation by RNA-Seq from White and Black Feather Bulbs in Ducks

To elucidate the genes involved in the formation of white and black plumage in ducks, RNA from white and black feather bulbs of an F2 population were analyzed using RNA-Seq. A total of 2,642 expressed sequence tags showed significant differential expression between white and black feather bulbs. Among these tags, 186 matched 133 annotated genes that grouped into 94 pathways. A number of genes controlling melanogenesis showed differential expression between the two types of feather bulbs. This differential expression was confirmed by qPCR analysis and demonstrated that Tyr (Tyrosinase) and Tyrp1 (Tyrosinase-related protein-1) were expressed not in W-W (white feather bulb from white dorsal plumage) and W-WB (white feather bulb from white-black dorsal plumage) but in B-B (black feather bulb from black dorsal plumage) and B-WB (black feather bulb from white-black dorsal plumage) feather bulbs. Tyrp2 (Tyrosinase-related protein-2) gene did not show expression in the four types of feather bulbs but expressed in retina. C-kit (The tyrosine kinase receptor) expressed in all of the samples but the relative mRNA expression in B-B or B-WB was approximately 10 fold higher than that in W-W or W-WB. Additionally, only one of the two Mitf isoforms was associated with plumage color determination. Downregulation of c-Kit and Mitf in feather bulbs may be the cause of white plumage in the duck.

Cloning, identification, and expression analysis at the stage of gonadal sex differentiation of chicken miR-363 and 363*.

miRNAs (microRNAs) are small, functional, non-coding RNAs and have been proved to implicate in regulation of diverse biological processes ranging from cell differentiation to organism development. With the purpose of exploring the roles of miRNAs on chicken embryo sexual determination and gonadal differentiation, we cloned and identified the stem-loop precursor structure (GenBank accession no. GU597370) of chicken miR-363 and 363* followed by studying their temporal and spatial expression patterns in chicken embryo at the stage of E3.5-6.5 d (embryonic days 3.5-6.5) by semi-quantitative RT-PCR and WISH (whole-mount in situ hybridization) in this study. The results showed that miR-363* located in cloned sequence of unknown segment in chicken genome, and flanking sequence of miR-363 and 363* according to the structural features of miRNAs precursor. Significantly differential expression (P < 0.05) of gga-miR-363 between female and male chicken embryonic gonads was found at E4.5 and 6.5 d, but the differential expression of gga-miR-363* from E3.5 to 6.5 d between both sexes fell short of significant level. The results of WISH indicated that expression signals of gga-miR-363 mainly appeared at limb bud, notochord, ectoderm, brain in E4.5 d chicken embryo, and urogenital systems (UGSs) at E6.5 d, and the expression level of E6.5 d was higher in the female than that in the male. It can be speculated that gga-miR-363 would involve in the gonadal development and gga-miR-363* might have transient regulatory functions during the early stages of chicken embryo development.

Cloning and expression profiling of the VLDLR gene associated with egg performance in duck (Anas platyrhynchos)

BackgroundThe very low density lipoprotein receptor gene (VLDLR), a member of the low density lipoprotein receptor (LDLR) gene family, plays a crucial role in the synthesis of yolk protein precursors in oviparous species. Differential splicing of this gene has been reported in human, rabbit and rat. In chicken, studies showed that the VLDLR protein on the oocyte surface mediates the uptake of yolk protein precursors into growing oocytes. However, information on the VLDLR gene in duck is still scarce.MethodsFull-length duck VLDLR cDNA was obtained by comparative cloning and rapid amplification of cDNA ends (RACE). Tissue expression patterns were analysed by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR). Association between the different genotypes and egg performance traits was investigated with the general linear model (GLM) procedure of the SAS® software package.ResultsIn duck, two VLDLR transcripts were identified, one transcript (variant-a) containing an O-linked sugar domain and the other (variant-b) not containing this sugar domain. These transcripts share ~70 to 90% identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences showed that duck VLDLR proteins were closely related with those of chicken and zebra finch. The two duck VLDLR transcripts are differentially expressed i.e. VLDLR-a is mainly expressed in muscle tissue and VLDLR-b in reproductive organs. We have localized the duck VLDLR gene on chromosome Z. An association analysis using two completely linked SNP sites (T/C at position 2025 bp of the ORF and G/A in intron 13) and records from two generations demonstrated that the duck VLDLR gene was significantly associated with egg production (P < 0.01), age of first egg (P < 0.01) and body weight of first egg (P < 0.05).ConclusionsDuck and chicken VLDLR genes probably perform similar function in the development of growing oocytes and deposition of yolk lipoprotein. Therefore, VLDLR could be a candidate gene for duck egg performance and be used as a genetic marker to improve egg performance in ducks.

Grey plumage colouration in the duck is genetically determined by the alleles on two different, interacting loci.

Based on the observation of a grey phenotype in the F(1) generation from a cross between two white plumage duck varieties, the white Kaiya and the white Liancheng, we hypothesized a possible interaction between two autosomal loci that determine grey plumage. Using the parental and F(1) individuals, seven testing combinations including five different F(1) intercrosses (F(2)) and two different backcrosses (BC(1) and BC(2)) were designed to test our hypothesis. It was demonstrated by chi-squared analysis that six test matings produced offspring in the expected ratios between the grey and white, with P-values ranging from 0.50 to 0.99. Another mating, where all white offspring were expected, produced 33 white individuals. These results verified that the interaction between two loci produced the grey phenotype. The C locus, which carries the recessive allele (c), was previously thought to be the only gene responsible for white plumage in the duck. This is the first report that an allele (t), carried by the white Liancheng at a different autosomal locus, also determines white plumage in ducks. Furthermore, the dominant alleles at both loci can interact with each other to produce the grey phenotype, and a new dark phenotype, observed in some F(2) individuals, can be attributed to the dosage effect of the T allele.

Endothelin Receptor B2 (EDNRB2) Gene Is Associated with Spot Plumage Pattern in Domestic Ducks (Anas platyrhynchos).

Endothelin receptor B subtype 2 (EDNRB2) is a seven-transmembrane G-protein coupled receptor. In this study, we investigated EDNRB2 gene as a candidate gene for duck spot plumage pattern according to studies of chicken and Japanese quail. The entire coding region was cloned by the reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis showed that duck EDNRB2 cDNA contained a 1311bp open reading frame and encoded a putative protein of 436 amino acids residues. The transcript shared 89%-90% identity with the counterparts in other avian species. A phylogenetic tree based on amino acid sequences showed that duck EDNRB2 was evolutionary conserved in avian clade. The entire coding region of EDNRB2 were sequenced in 20 spot and 20 non-spot ducks, and 13 SNPs were identified. Two of them (c.940G>A and c.995G>A) were non-synonymous substitutions, and were genotyped in 647 ducks representing non-spot and spot phenotypes. The c.995G>A mutation, which results in the amino acid substitution of Arg332His, was completely associated with the spot phenotype: all 152 spot ducks were carriers of the AA genotype and the other 495 individuals with non-spot phenotype were carriers of GA or GG genotype, respectively. Segregation in 17 GA×GG and 22 GA×GA testing combinations confirmed this association since the segregation ratios and genotypes of the offspring were in agreement with the hypothesis. In order to investigate the underlying mechanism of the spot phenotype, MITF gene was used as cell type marker of melanocyte progenitor cells while TYR and TYRP1 gene were used as cell type markers of mature melanocytes. Transcripts of MITF, TYR and TYRP1 gene with expected size were identified in all pigmented skin tissues while PCR products were not obtained from non-pigmented skin tissues. It was inferred that melanocytes are absent in non-pigmented skin tissues of spot ducks.

Adipogenesis in ducks interfered by small interfering ribonucleic acids of peroxisome proliferator-activated receptor γ gene

ABSTRACT Peroxisome proliferator-activated receptor γ (PPARγ) participates in adipocyte differentiation and maintenance, including the promotion of lipid storage in mammals. In the present study, 3 duck PPARγ small interfering RNA (siRNA) expression plasmids were constructed to investigate the effect of downregulating the expression of PPARγ on adipogenesis and fat accumulation in ducks. The results indicate that the 3 siRNA specific for conserved regions of PPARγ can effectively inhibit expression of PPARγ. It was demonstrated that the expression of lipoprotein lipase and adipocyte fatty acid-binding protein in duck adipose tissue is repressed when the expression of PPARγ is downregulated by siRNA. At the same time, the weight of abdominal fat at 21 and 35 d of age is decreased significantly (P < 0.05) compared with the control. However, the triglyceride levels in serum and muscle are not affected when the mRNA of PPARγ is repressed. The current study indicates that the suppression of PPARγ reduces abdominal fat deposition and regulates adipogenesis in ducks.

Analysis of the Offspring Sex Ratio of Chicken by Using Molecular Sexing

Abstract The overall sex ratio of offspring (dead embryos and hatch chicks) from all the fertilized eggs of 140 hens collected for 30 days was studied using duplex PCR of certain fragments of sex chromosomes. Additional 894 dead embryos over a period of 21 days of incubation were also investigated to verify the sex ratio of the dead embryos. The sex of the early dead embryos was identified using this molecular sexing technique. The sex ratio of the hatch chicks and the total offspring of the hens investigated in this experiment did not differ from the expected sex ratio (i.e., 1:1)., However, the number of female dead embryos was significantly more than that of males. The data indicated that the different physiologic function of males and females contributed to female-biased mortality during incubation. It was also found by further analysis that the sex ratios of the offspring of some hens were significantly biased to female or male over the period investigated, which suggested that the sex ratio of offspring might be influenced by the maternal condition to some degrees.

Effects of polymorphisms and haplotypes within the MSTN gene on duck growth trait

Abstract 1. Polymorphisms of the duck MSTN gene were investigated in 413 individuals by DNA sequencing and polymerase chain reaction restriction fragment length polymorphism. Four single nucleotide polymorphisms (G129A, C324T, A981G and C1002A), with A981G and C1002A completely linked, were found in the coding region. 2. Association analysis showed that different genotypes of all the identified SNPs were significantly associated with duck growth rate from week 5, 6 and 2 for G129A, C324T and A981G (C1002A), respectively. The greatest difference in body weight was 180 g at week 9, 106 g at week 8 and 123 g at week 8, respectively, for the three SNP’s. 3. Linkage disequilibrium (LD) analysis indicated that C324T, A981G and C1002A were in strong LD. Nine main diplotypes from the reconstructed five main haplotypes were observed, and different diplotypes were significantly associated with growth rate from week 1. Birds with the h1h1 diplotype exhibited the largest body weight from week 1 onwards. 4. It was concluded that the duck MSTN gene was associated with body weight and is an important candidate gene for duck growth. traits and marker-assisted selection.

Molecular cloning, expression and association study with reproductive traits of the duck LRP8 gene

Abstract 1. Two splice variants of duck LRP8 were identified, one containing 8 ligand-binding repeats (LRP8-1) and the other containing only 7 repeats (LRP8-2). The two transcripts share ~71–91% nucleic acid identity and ~65–94% amino acid identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences shows that duck LRP8 proteins are closely related to those of chicken, turkey and zebra finch. 2. The semi-quantitative reverse transcription polymerase chain reaction (RT-PCR )analysis indicates that the two transcripts are expressed in all the examined tissues, and the LRP8-1 transcript is more highly expressed in hypothalamus, ovary and pituitary gland than in other detected tissues. 3. Six single nucleotide polymorphisms (SNPs) were identified in the coding region. Association analysis demonstrated that the c.528C > T genotypes were associated with egg production (EP) (EP210d, EP300d and EP360d), age at laying the first egg (AFE) and body weight at sexual maturity (BWSM). The c.1371A > G genotypes were associated with egg production (EP210d, EP300d and EP360d). 4. The haplotypes of c.528C > T and c.1371A > G were associated with EP (EP210d, EP300d and EP360d), yolk weight (YW), albumen weight (AW), egg weight (EW), BWSM and the first egg weight (FEW). 5. Duck LRP8 gene was associated with some reproductive traits and is an important candidate gene for the genetic selection of improved reproductive traits.

Genome-wide association analysis identifies potential regulatory genes for eumelanin pigmentation in chicken plumage

Plumage color in chicken is determined by the proportion of eumelanin and pheomelanin pigmentation. As the main ingredient in plumage melanin, eumelanin plays a key role in the dark black, brown and grey coloration. However, very few studies have been performed to identify the related genes and mutations on a genome-wide scale. Herein, a resource family consisting of one backcross population and two F2 cross populations between a black roster and Yukou Brown I parent stockbreed was constructed for identification of genes related to eumelanin pigmentation. Chickens with eumelanin in their plumage were classified as the case group, and the rest were considered the control group. A genome-wide association study of this phenotype and genotypes using Affymetrix 600K HD SNP arrays in this F2 family revealed 13 significantly associated SNPs and in 10 separate genes on chromosomes 1, 2, 3 and 5. Based on previous studies in model species, we inferred that genes, including NUAK family kinase 1 (NUAK1) and sonic hedgehog (SHH), may play roles in the development of neural crest cells or melanoblasts during the embryonic period, which may also affect the eumelanin pigmentation. Our results facilitate the understanding of the genetic basis of eumelanin pigmentation in chicken plumage.