Plant & Animal Genomes XIII Conference

January 15-19, 2005
Town & Country Convention Center
San Diego, CA

The FAD2-1 Gene Is Tandemly Duplicated And Silenced By RNA Interference In High Oleic Acid Sunflower

G. Felix Schuppert¹ , B. Shaun Bushman⁴ , Kristen D. Kasschau² , Mary B. Slabaugh¹ , JC Carrington³ , Steven J. Knapp⁴

¹  Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331 USA

²  Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331 USA

³  Center for Gene Research and Biotechnology, Oregon State University, Corvallis, OR 97331 USA

⁴  Center for Applied Genetic Technologies, 111 Riverbend Road, The University of Georgia, Athens, GA 30602 USA

High oleic acid sunflower (Helianthus annuus L.) lines carry a negative dominant mutation (Ol) that is necessary but usually not sufficient for producing high oleic acid seed oils. Ol impairs the expression of a seed specific oleate desaturase (FAD2-1). FAD2-1 is duplicated in the mutant. Genetic mechanisms underlying the FAD2-1 mutation were elucidated. By amplifying the 3.1 kb intergenic region spanning the 3’ and 5’-UTRs of the duplicated copies, FAD2-1 was found to be tandemly duplicated in the mutant. The 5’-UTR of the first copy contains a 1.7 kb intron. The novel joint was discovered in the 5’-UTR intron of the second copy (1.4 kb of the intron was missing). The FAD2-1 locus was sequenced from the mutant and found to be 7.8 kb long. Reduced accumulation the FAD2-1 transcript in developing kernels of the mutant was predicted to be caused triggering of the RNA interference (RNAi) pathway. Northern analyses of total RNA isolated from developing kernels of wildtype and mutant lines revealed accumulation of 21 nt RNAs in mutant lines only, corresponding to a FAD2-1 specific probe. The FAD2-1 duplication was hypothesized to have eliminated the transcription termination signal in a downstream gene oriented opposite of FAD2-1, thereby yielding an extended transcript complementary to FAD2-1, forming double-stranded RNA, and triggering the RNAi machinery. When tested by bidirectional RT-PCR analyses, mutant lines produced transcripts in sense and antisense directions, whereas wildtype lines only produced transcripts in the sense direction. Hence, FAD2-1 appears to be silenced by RNAi in the mutant.