Chapter 5 Genetic Analysis of Apomixis - cimmyt

f..... s....Ii,y 10 Apalllhls: MoIeaoIar .d GHtlk Appr-MS 195genesis and are currently being analyzed inmore detail at the molecular and genetic level(J-P. Vielle-Calzada and U. Grossniklaus,unpublished data).For the rapid isolation and sequencing ofgenomic regions flanking the Os insertion weadapted a PCR-based procedure, TAIL-PCR(Liu et al. 1995), to be used in conjunction withOs elements (see also Tsugeki et al. 1996).Using a set of Os-specific primers(GrossnikJaus et al. 1998a) in combination witharbitrary primers, we isolated at least oneflanking fragment for more than 150 lines thatwe identified in various screens. Using twosets of arbitrary primers, the success rate wasgreater than 95% (GrossnikJaus et al. 1998a).We have identified insertions into a multitudeof genes that encode essential proteinsinvolved in basic metabolic and cellularprocesses, putative regulatory proteins, andseveral novel sequences of unknown function.Based on sequence information, the majorityof the detected genes appear to be involved ingene regulation and signaling processes.Identification of DevelopmentallyRegulated Genes and Their PromotersVery few genes expressed in themegagametophyte have been described(Nadeau et a1.1996; Belostotsky and Meagher1996), and genes expressed in individual cellsof the embryo sac have not previously beenidentified and characterized. Recently, cDNAlibraries obtained from isolated egg cell andin vitro fertilized zygotes of maize have beengenerated, leading to the identification ofgenes expressed in the embryo sac and embryo(Dresselhaus et al. 1994, 1996, 1999a,b).Although no cell type-specific genes have beenisolated yet, this approach holds great promisefor the identification of embryo sac-specificgenes. We use enhancer detector and gene traptransposons carrying the uidA reporter geneencoding b-g1ucuronidase (GUS) Oefferson etal. 1986; Jefferson 1987). The expression of GUScan be visualized by histochemical stainingOefferson et al. 1987; Kavanagh et al. 1988). Toidentify genes expressed during ovuledevelopment and female gametogenesis, weanalyzed GUS expression in maturing ovulesof about 2,300 transposants (R. Baskar, J.Moore, W. Gagliano, U. Grossniklaus,unpublished data). Between 9% and 10% ofthe enhancer trap lines and 2% to 3% of thegene trap lines show spatially restricted GUSexpression in mature ovules. Approximatelyhalf of these enhancer detector lines showexpression restricted to sporophyte andgametophyte, respectively, whereas very fewshow regional expression in bothgametophytic and sporophytic tissues.Although many Arabidopsis promoters havebeen found to be highly compact (e.g., Dwyeret al. 1994; Thoma et al. 1994; Xia et al. 1996),enhancers that drive reporter gene expressioncould be at a considerable distance from thesite of insertion. This makes the isolation ofthe detected gene and its promoter morelaborious, requiring that the expressionpattern of an isolated gene be confirmed. Todate, we have analyzed the expression of threegenes expressed in the megagametophyte; insitu hybridization indicates that they areindeed expressed as expected (Vielle-Calzadaet al. 2000; R. Baskar, J-P. Vielle-Calzada andU. GrossnikJaus, unpublished data). It wouldbe preferable to identify gene trap insertionswithcell type-specific expression because theyhave to be inserted within the transcriptionunit in order to function. However, becausegene traps must integrate within the gene inthe correct orientation (Sundaresan et al. 1995)and GUS activity is often weak, the frequencyat which highly specific expression patternsare recovered is very low. The screeningprocess for cell-type specific expression in theovule and megagametophyte is extremelylaborious, requiring preparations for highresolutionlight microscopy. Therefore, we