| Poultry reproduction traits, including the day at first laying, egg number, egg weight, and so on, are economically important in modern poultry production. Identification of genes associated with reproduction traits could give an insight into the genetic mechanism of the performance of egg laying and be helpful for poultry breeding with better quality and higher output.Candidate gene approach is one of the strategies in mapping quantitative traits, and some major genes have been mapped successfully by this method, e.g. sex-linkage dwarf gene of chicken and stress syndrome gene of swine. Differential display RT-PCR (DDRT-PCR) is one of the important methods in screening differentially expressed genes.To investigate the genes associated with fowl reproduction, we selected FSHR, BMPR-â… B, and IGFBP-4 as candidate genes to identify polymorphisms and analyze the association of these polymorphisms with the egg laying trait of Luqin broiler line.The levels of FSHR, BMPR-â… B, and IGFBP-4 mRNA in ovary were compared between Jiningbairi and Luoman chickens by semiquantitative RT-PCR, and DDRT-PCR was used to identify differentially expressed genes in ovaries of these two breeds.In experiment one, a fragment of about 1.8 kb of the promoter region of chicken FSHR was cloned from Jiningbairi, Wenchang, Xianju, Hailan, Zang, and deposited to GenBank (GenBank accession No. : DQ303232~DQ3032327). Altogether 48 nucleotide variations were observed among these sequences, of which 39 were single nucleotide substitutions, one was three nucleotide substitution and 8 were indels. The substitution at -1046 involves three nucleotides of A, G and T. Of the 39 single nucleotide substitutions, 24 belongs to transitions and 14 belongs to transversions. At -868 site, an insertion of about 200 bp in size was found with a extremely low mutation rate (0.0698) among Luqin population. The insert of -868 site was detected by PCR. An indel of"TG"at -71 site was revealed by 12% denaturing polyacrylamide electrophoresis and a T/A transversion at -237 site was verified by PCR-RFLP. The effect of the -868, -71 and -237 polymorphisms on egg production was simultaneously estimated for both E33 and E42. The results suggested that the effect of genotype on E33 or E42 egg production did not approach a statistically significant level according to total typeâ…¢probability. The population was in a Hardy-Weinberg equilibrium at these three sites. About 24 bp of the 200bp inserted fragment is conserved to human SMARCAL1 gene, which might be related to dynamics of chromatin.In experiment two, BMPR-â… B exon six to seven was cloned from Jiningbairi, Wenchang, Xianju, Hailan, Zang, and GenBank accession No. is EF530593. Altogether 11 single nucleotide substitutions were observed among these sequences, of which nine belong to transitions and two belongs to transversions. The transitions of C/T at site 35 and G/A at site 287 were verified by PCR-SSCP and PCR-RFLP, respectively. The polymorphisms at these two sites had no significant effect on egg production.In experiment three, the levels of FSHR, BMPR-â… B, and IGFBP-4 mRNA in ovary were identified between Jiningbairi and Luoman by semiquantitative RT-PCR. Among two populations, FSHR mRNA level was significantly different between ovaries and follicles at 15 wks and 20 wks. FSHR mRNA expression in yellow follicle are significantly higher than that of F3 or F5 (P<0.05). At 15 wks the levels of BMPR-â… B mRNA in ovaries and YF of Luoma chickens were higher than those of Jiningbairi chickens (P<0.01). IGFBP-4 mRNA expression in follicles was lower than in ovaries among Jiningbairi chickens (P<0.05). However, the levels did not vary among Luomanhe.In experiment four, By DDRT-PCR, seven differentially expressed genes were identified between the ovaries of two populations at different stages. TPD52L and TNRC6A mRNA expression was up-regulated. Otherwise, DNAJC8, LOC771135,ChEST756m4 and LOC772269 mRNA expression decreased significantly (P<0.01). LDHB gene was down-regulated in Luomahe chickens (P<0.01), but no significant change in Jiningbairi chickens.Sexual maturation in poultry is controlled by a network of genes. By selecting FSHR,BMPR-â… B and IGFBP-4 as candidate genes, the present study analyzed their sequences, polymorphisms and expressions and the associations of these polymorphisms with egg production in a synthetic broiler line. Seven differentially expressed genes were identified in ovary tissues during sexual maturation by using differential display RT-PCR approach. The results may provide clues for further determination of chicken reproduction-related genes, especially those involved in early sexual maturation, and may be helpful for molecular breeding of laying and broiler chickens for high production and quality.
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