Chapter 5 Genetic Analysis of Apomixis - cimmyt

192 Ueli Gro".llae,system to identify mutants that lead to theformation of an embryo sac from an unreducedcell lineage. In addition, full-sized kernels canbe scored for absence of the dominant paternalR-nj marker in the embryo, which indicatesparthenogenetic development.Because obtaining a mutation that causes bothnonreduction and parthenogenesis may beextremely difficult, a second screen aimed atthe isolation of parthenogenetic mutants hasbeen developed. It makes use of the el1mutation, which, when homozygous, producesa large fraction of unreduced embryo sacs. Inel1 homozygotes, independent assortmentduring meiosis I is not affected and theresulting gametes are not genetically identical(Roades and Dempsey 1966). Nevertheless, itprovides a reliable source for unreduced femalegametes. Lines homozygous for rl and el1 anddisplaying high Mutator activity have beenconstructed to serve as female recipients.Kernels derived from crosses with a tetraploidR-nj pollen donor can be scored for rejection ofthe R-nj marker, i.e., for the maternalphenotype, in order to identify embryos thatdeveloped without a paternal contribution.Such embryos will be diploid (through theaction of ell), which greatly facilitatessubsequent genetic characterization. Thesegenetic screens aimed at the isolation ofmutations that lead to nonreduction,parthenogenesis, or a combination of bothaspects wtll provide useful material to furtherour understanding of these processes at themolecular and genetic level.Enhancer Detection as aPowerful Tool to StudySexual Reproduction inArabidopsisThe molecular and genetic bases ofmegasporogenesis and megagametogenesisare poorly understood. To date, only onefemale-specific mutant that affectssporogenesis has been identified (Siddiqi et al.2000; Motamayor et al. 2000) and attempts toisolate genes that regulate the developmentalevents initiating female gametogenesis andembryo development are just beginning. Asan alternative to the isolation of mutations, weidentify genes expressed specifically duringmegasporogenesis and megagametogenesis.The inaccessibility of the developing embryosac and the small number of cells involvedmake this a difficult undertaking usingconventional molecular methods such asdifferential screening techniques. Therefore,we use a novel technology, enhancer detection,which allows the identification ofdevelopmentally regulated genes based ontheir pattern of expression. Enhancer detectionis one of the most powerful tools to identifytissue specifically expressed genes and theirregulatory sequences. Application of thisapproach in angiosperms will lead to theidentification of many genes that controlgametogenesis and cellular differentiation inthe female gametophyte. In addition, it willidentify many cell type- and tissue-specificregulatory regions that will be required toexpress candidate genes in a precise temporaland spatial fashion.Enhancer Detection andGene Trap SystemsEnhancer detection was first developed in thefruit fly Drosophila melanogaster and relies on amobile genetic element carrying a reportergene under the control of a weak constitutivepromoter (O'Kane and Gehring 1987). Thisminimal promoter is usually not active butideally it can be activated in all tissues and atall developmental stages. Ifit comes under thecontrol of genomic cis-regulatory elementssuch as enhancers, the reporter gene isexpressed in a specific temporal and spatialpattern (Figure 12.4, p. 197). This patternreflects the expression of a nearby genecontrolled by the same regulatory elementsand, thus, allows the identification of genes