Studies on the Molecular Genetic Markers Associated with Reproduction of Ducks Using DNA Microarray

• Main Authors: Hsiu-Lin Huang, 黃秀琳
• Other Authors: 許文輝
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/79118185253280940630

博士 === 國立中興大學 === 分子生物學研究所 === 99 === DNA microarray technology was used for the studies on differentially-expressed genes in oviduct of Tsaiya ducks in Taiwan. The genotypes associated with reproductive performance for marker-assisted selection were also investigated. The magnum and utero-vaginal junction (UV-J) in oviduct were employed for the hatchability and the fertile-period experiments, respectively. In the hatchability study, transcriptome analysis using home-made cDNA microarray was performed to identify differentially-expressed genes that are correlated with hatchability, and a new PCR-RFLP marker of high hatchability among the identified genes was observed. The cDNA microarray technique was used for gene expression profiling of the magnum epithelium of laying Tsaiya ducks, and several regulated genes associated with hatchability were found. The results of real-time PCR and Western blotting analysis confirmed that the mRNA and protein levels of ovomucoid in the magnum epithelium of animals in the low-hatchability group were significantly higher than the levels in the high-hatchability group (p<0.05). Primers TovF1and TovR1 designed according to the ovomucoid EST sequence were used to amplify genomic DNA samples of different individual Tsaiya ducks, and sequence analysis of the amplified DNA products showed deletion among the ducks from the low-hatchability group. Primers TovF2 andTovR2 then probed inside the amplified DNA products and were used to perform PCR-RFLP analysis to classify the ducks into +/+, +/- and -/- genotypes. The animals of +/+ and +/- genotypes were identified to have a significantly higher hatchability than those of the -/- genotype (p<0.05). In contrast, no differences were observed between genotypes in terms of fertility, fertile period, egg weight or total number of eggs. The results indicated that a novel PCR-RFLP marker of high hatchability, the ovomucoid gene polymorphisms, can be used as a genetic marker for marker-assisted selection to improve hatchability in Tsaiya ducks. In the fertile-period study, prolonged fertile period increases the efficiency of fertilization. Here, RNA was extracted from long- and short-fertile-period groups, converted to double cDNA which was then in vitro transcripted to complementary RNA (cRNA). Affymetrix chips were hybridized with these cRNA, three biological repeats were performed, and 27 transcripts were identified as being differentially-expressed. Interestingly, by searching the annotated pathway databases, the results demonstrated that Neuropeptide Y (NPY), the RNA expression of which was increased by 2.96-fold in the short-fertile-period group as compared with the long-fertile-period group in the experiment described in this thesis, has been shown to reduce blood flow and substance supply to local tissues. Enah/Vasp-like (EVL), the RNA expression of which was significantly increased by 1.77-fold in the short-fertile-period group as compared with the long-period group, has been demonstrated to be important in activated T-cells. In contrast, trafficking kinesin-binding protein 1 (TRAK1), the expression of which was increased by 2.33-fold in the long-period group as compared with the counterparts, has been suggested to inhibit precocious activation of sperm and prolong sperm life in female sperm reservoir.