Poster: Reproductive Development

Abs # 479: Macroarrays and targeted gene silencing in fern gametophytes: Toward cloning the plant sex determination genes

Presenter:	Tanurdzic, Milos , milos@purdue.edu
Authors	Tanurdzic, Milos  (A) (B)  Rutherford, George  (B)   Banks, Jo A (A) (B)
Affiliations:	(A): Purdue Genetics Program (B): Department of Botany and Plant Pathology, Purdue University

Plant gametophytes provide all land plants with the essential features necessary for sexual reproduction. Given the extreme reduction in size (but not in function) of the angiosperm gametophytes, the process of sex determination is best studied using free-living, haploid, sexually dimorphic gametophytes such as those of the homosporous fern Ceratopteris richardii. While Ceratopteris has proven to be a useful genetic system for dissecting a sex determination pathway, its major limitation is the means to clone the >100 genetically defined sex-determining genes. We present two novel resources that have been developed in our lab that alleviate these limitations. We have developed a normalized cDNA library with more than 350,000 clones. These have been arrayed onto nylon membranes and >35,000 clones have been assayed for differences in expression levels during different stages of male and hermaphrodyte development. To assess their function, we have investigated the feasibility of RNA interference as a reverse genetic tool in Ceratopteris. By bombarding young gametophytes with constructs designed to promote the expression of a double stranded RNA, we observe a "mutant" phenotype that spreads throughout the entire gametophyte in a non-cell autonomous fashion. The successful silencing of three genes by RNAi demonstrates that this approach will allow us to determine the function of gametophytically expressed genes and, ultimately, clone the sex-determining genes.