Plant & Animal Genomes XIV Conference

January 14-18, 2006
Town & Country Convention Center
San Diego, CA

cDNA-AFLP Profiling And Quantitative Expression Analysis Of Low-Temperature-Induced Transcripts And Genes In Winter And Spring Wheats

SEEDHABADEE GANESHAN , Carli Holderness , Jenna Drinkwater , Allen Limin , Brian Fowler , Ravindra Chibbar

  Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan, S7N 5A8, Canada

The objective of this study was to determine expression profiles using cDNA-AFLP and quantitative expression using real-time PCR (QPCR) of transcripts and genes during temporal cold-acclimation of wheat plants. A winter hardy cultivar, Norstar, a spring cultivar, Manitou and two sets of near-isogenic lines (NILs) were used based on the Vrn-A1 (spring) and vrn-A1 (winter) alleles in each of the Norstar and Manitou genetic backgrounds, respectively (winter Norstar-spring Norstar, winter Manitou-spring Manitou). Plants were grown at 6 oC and leaves were collected at specific time intervals during early stages (0, 2, 7, 14, 21 and 28 days) of cold acclimation. For cDNA-AFLP total RNA was extracted, mRNA isolated and reverse transcribed prior to making double stranded cDNA. cDNA was digested with EcoRI and MseI, and after ligation of EcoRI and MseI adaptors, pre-amplification was performed. After selective amplification, PCR products were resolved on a PAGE gel. Differentially expressed transcripts were excised, re-amplified and sub-cloned for sequencing. A uniquely expressed transcript of 450 bp was found to hybridize to a fragment of a wheat CBF1 gene used as probe. For real-time PCR, first strand cDNA was synthesized using gene specific primers. Some of the genes studied to date include Wcs120, Wcor410 and wheat CBF1. The expression of Wcs120 was variable among the four lines and was found to be associated with LT50.