| Summary: | 博士 === 國立中興大學 === 動物科學系所 === 100 === The purpose of this study was to identify a set of differentially expressed genes with potential involvement in duck egg formation process using cDNA microarray analysis; and to develop the molecular DNA markers by investigating the associations of gene polymorphisms with duck egg production. In experiment 1, the pituitary target RNA were obtained at 0, 4, 8, 12, 16, 20 and 22 h after laying and labeled for hybridization with in-house cDNA microarray (8736 spots). The hybridization signals were amplified by Tyramide signal amplification technology and scanned by fluorescent scanner at 532 nm for image acquisition and quantification. Data normalization, log transformation and statistics were carried out by Avadis software. One hundred sixty-seven differentially expressed cDNAs were observed in fold change ≥ 2.0 at 4, 8, 12, 16, 20, 22 h relative to 0 h (P < 0.05). Raw sequences of the cDNAs were processed through sequence analysis including trimming vector sequence, and removing low quality and short sequence (< 100 bp), thus resulted in 136 expressed sequences tag (EST) sequences. There were 16 contigs assembled along with 71 singletons separated after cluster analysis. The results of cluster analysis were used for sequence indentify and functional annotation. Removing repeat and mitochondrial DNA sequences, 37 known genes with potential involvement in ovarian follicles development and egg formation were acquired based on BLASTN (GeneBlank). These known gene sequences were annotated in Gene Ontology (GO); they included biological process (35.1%), molecular function (54.0%), and cellular component (37.8%). For these known genes that mapped to biological processes, most of them with their functional annotations were related to metabolic processes and translation. For molecular function, most of the known genes were related to macromolecule binding and catalytic activity, and for these cellular component genes were expressed in intracellular region. In experiment 2, the specific-primers were designed for target gene amplification according to the EST and complete sequences obtained from our laboratory and GeneBank database, respectively. Identification of the expressed gene polymorphisms was performed by single strand conformation polymorphism (SSCP) technology, DNA sequencing, and SNaPshot minisequencing. Furthermore, the association between the gene polymorphisms and duck traits was analyzed. Total of 32 single nucleotide polymorphisms (SNPs) within growth hormone (GH), prolactin (PRL), destrin (DSTN), proline-rich nuclear receptor coactivator 1 (PNRC1), cyclin-dependent kinase 2-associated protein 1 (CDK2AP1) genes were found, of which 1, 3, 20 and 8 SNPs were located in 5’-untranslated region (UTR), exons, introns and 3’-UTR, respectively. The SNP-trait association analysis showed that the GH gene diplotypes constructed on C3169T, C3700T, and C5058G were associated with maximum duration of fertility (MDF) (P < 0.05), and the H1H1 diplotype was advantage for duck fertility. In PRL gene, the diplotypes constructed on T213C, T295C, G309T, C381A, G3941T, and A3975C were associated with egg weight at 40 wk of age (EW40) and MDF (P < 0.05). Within all diplotypes, the ducks with H1H1 diplotype had the highest MDF, and the H1H2 diplotype had the highest EW40. In DSTN gene, the H6H7 diplotype was dominant for EW40, and the H3H3, H3H8, and H6H6 diplotypes were dominant for MDF within all diplotypes constructed on T92C, C93A, G100T, and T106C. In PNRC1 gene, the G98T genotype effects were line-specific that the ducks with GG genotype had higher egg weight at first egg than ducks with GT genotype in control line, whereas the ducks with GG had lower hatchability rate than ducks with GT genotype in selected line (P < 0.05). Moreover, the ducks with －/A genotype had higher MDF than ducks with －/－ genotype in CDK2AP1 gene (P < 0.05). The results showed that these SNP markers will be helpful for marker-assisted selection in the breeding program of Tsaiya ducks.
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