Gene expression profiling by cDNA-AFLP and identification of differentially expressed transcripts of bovine pituitary gland in growing bulls of dairy breeds
Journal Title: Animal Science Papers and Reports - Year 2012, Vol 30, Issue 2
Abstract
The study utilized age-dependent breed-specific gene expression profiling by cDNA-AFLP technique in identification of differentially expressed (DE) candidate genes for growth and development trait. Results revealed 15579 transcript derived fragments (TDFs) expressed in bovine pituitary gland tissue using 96 unique PCR reactions with primer combinations (PC) of TaqI-MseI for Polish Holstein (PH) and Polish Red (PR) dairy breeds. Gene expression profiling by cDNA-AFLP identified 1451, 2877 and 4084 identically displayed (iDD), differentially displayed (DD) and single displayed (sDD) TDFs, respectively. In all transcript profiles, frequencies of DD TDFs were higher than that of iDD TDFs. A total of 60 DD TDFs bands were excised for PH (n=40) and PR (n=20) . Direct sequencing results revealed identification of 24 and 8 DE-TDFs sequences in Polish HF and PR. Based on the significance of BLAST and sequence alignment score, analysis 12 and 4 DE-TDFs sequences of PH and PR were excluded. Overall, 12 Polish HF and 4 PR breed specific DE-TDFs sequence expressed in bovine pituitary gland tissue were identified. The identified DE-TDFs sequences were represented in all bovine chromosomes, except BTA3, 4, 6, 7, 8, 9, 11, 12, 17, 18, 21, 22, 23, 25 and 26. TDF annotation results identified eight sequences that have BLAST hits to known annotated bovine genes and eight sequences to unannotated contig regions in latest gene ensemble database Btau4,0. Two breed-specific target genes i.e., bovine glycophorin C (PH) and bovine arachidonate 5-lipoxygenase (Alox5) isoform 1 (PR) were validated by qRT-PCR. Within breed the age-dependent qRT-PCR analysis revealed that expression levels were differed significantly high (P<0.0001) with nine folds higher expression in young bulls at the age of 6 month (glycophorin C) and 9 month (Alox 5 isoform 1). Between breed the qRT-PCR analyses revealed that the expression levels were highly significant for glycophorin C gene in PR and Alox 5 isoform 1 gene in both breeds. It was concluded that gene expression profiling by cDNA-AFLP is a reliable technique for identification of trait-associated DE candidate genes, and helpful in elucidating and understanding molecular basis of postnatal growth and development of cattle.
Authors and Affiliations
Chandra Shekhar Pareek, Mariusz Pierzchała, Joanna Michno, Sonia Szymańska, Rafał Smoczyński, Natalia Jasik, Marcin Gołębiewski, Jarosław Tyburski, Paweł Urbański, Dorota Goluch, Jolanta Oprządek, Lech Zwierzchowski, Ponsuksili Siriluck, Klaus Wimmers
Keywords
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