Yanping Feng

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.

Sex Ratio Bias in Early-Dead Embryos of Chickens Collected During the First Week of Incubation

According to Mendelian heredity laws, the sex ratio of a given chicken population during hatching is expected to be 1:1. In this study, we collected 432 chicken embryos that died during the first week of incubation from 5 different breeds. The sexes of the early-dead embryos were determined by using the previously described molecular sexing technique of double PCR. The female-to-male sex ratio was analyzed for departure from the expected 1:1 sex ratio by chi(2) testing. These results showed that the number of female dead embryos was significantly greater than that of males in the Hubei local breeding stock, Zhusi, and Hy-line Variety Brown (P < 0.05, P < 0.01, P < 0.01 respectively), with observed female-to-male sex ratios of 1.40:1, 2.03:1, and 2.22:1, respectively. Two other Chinese local breeds (the Yellow chicken and the Aijiaohuang chicken) also showed altered sex ratios, although the differences were not significant. Altogether, these results indicated that female chickens were more likely than male chickens to die at the early stages of incubation.

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.

Expression analysis of differentially expressed miRNAs in male and female chicken embryos

MicroRNAs (miRNAs) are small non-coding RNA molecules that play key roles in the regulation of development processes of many tissues and organs at the post-transcriptional level. However, little is known about how they affect chicken gonadal development. We examined the expression of four miRNAs (miR-218, -200b, -196, and -206) in chicken embryonic gonads at embryonic days 3.5-6.5. Their target genes were predicted by miRDB, TargetScan and PicTar algorithms. The expression levels of these four miRNAs differed with sex to varying degrees; miR-200b was expressed at a significantly higher level in female gonads during the entire interval. The whole mount in situ hybridization result showed considerably higher expression of miR-200b in females than in males in E5.5 embryos. The miRNA target scanning results indicated several genes with functions in gonad development and gonad function. We conclude that miR-200b is involved in the regulation of gonad development and sexual differentiation of chicken embryos.

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.

miRNAs expression profile in bast of ramie elongation phase and cell wall thickening and end wall dissolving phase

MicroRNAs (miRNAs) are 20–24 nucleotide long non-coding RNAs that play critical regulatory roles during plant development, organ morphogenesis, and cell fate determination and differentiation. In this study, miRNA microarray chips were used to explore the expression profile of ramie miRNAs between the bast of fiber elongation phase and those of cell wall thickening and end wall dissolving phase. There are 150 and 148 credible miRNAs in the bast of fiber elongation phase and cell wall thickening and end wall dissolving phase, respectively. These miRNAs distributed in 27 species and mainly concentrated in nine species. Analysis showed that 51 miRNAs were differentially expressed: 27 up-regulated (miR166, miR172, miR396, miR482, miR894 and miR2911 families) and 24 down-regulated (miR156, miR159, miR164, miR319 and miR1450 families) in the bast of fiber elongation phase compared with the bast of cell wall thickening and end wall dissolving phase. To further confirm our results, we examined the expression of three miRNAs (zma-miR172b*, pvu-miR482 and vvi-172a) by quantitative real-time reverse transcriptase-PCR. Our results will provide a molecular basis for future research miRNA function on ramie genetics and breeding.

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.