Chapter 5 Genetic Analysis of Apomixis - cimmyt

F"'II\ Sexoallty ,. Apolllixl" MoIeaolar.d G...tk Approadoe. 193based on their pattern ofexpression rather thanon a mutant phenotype (Bellenet al. 1989; Bieret al. 1989; Grossniklaus et al. 1989; Wilson etal. 1989). Enhancer detector screens have beenextremely successful in Drosophiladevelopmental genetics and similarapproaches were rapidly adapted to othermodel systems. Because of the largeintergenomic regions in mice, gene traps weredeveloped that depend on a modification ofthis approach involving the generation oftranscriptional fusions to the reporter gene(Gossleretal.1990;Skarnes 1990; Friedrich andSoriano 1991). In Arabidopsis, similar systemsbased on T-DNA insertional mutagenesis(Topping et al. 1991; Fobert et al. 1991;Kertbundit et al. 1991) or the AciDstransposable element system from maize(Sundaresan et al. 1995; Springer et al. 1995;Smith and Fedoroff 1995) have beendeveloped.Enhancer detection and gene trap systemsoffer an added benefit: they allow theidentification of genes that are not readilyamenable to classical genetic analyses (Bellenet al. 1989; Bier et al. 1989; Grossniklaus et al.1989; Wilson et al. 1989). They have beenespecially useful in studying developmentalprocesses occurring late in development, i.e.,after the effective lethal phase of acorresponding mutation. For example, a genethat is required for essential steps during bothembryo and ovule development would beidentified as an embryo lethal mutation andits function during ovule development wouldbe masked. Enhancer detection allowedidentification of the first embryo lethal genesin Drosophila that are also required foroogenesis or eye development (Grossniklauset al. 1989; Mlodzik et al. 1990). The dissectionof processes characterized by functionalredundancy and high complexity is alsogreatly facilitated by enhancer detection(Wilson et al. 1990; Bellen et al. 1990).Enhancer detection has some importantadvantages over classical genetic screens forthe identification of genes required in thegametophytic phase of the life cycle: (i) manygenes that encode components of the basiccellular machinery display a gametophytelethal phenotype if disrupted. Essential genesare expected to show widespread although notnecessarily ubiquitous expression, whereasexpression in particular cell types of themegagametophyte suggests a function in cellspecification and differentiation; (ii) genesrequired for both micro- and megagametogenesiscan be isolated, because a largepercentage of enhancer detector insertions donot disrupt gene function. Mutants affectingboth male and female gametophytes canusually only be recovered as rare partiallypenetrant mutations or in genomic regions thatcan be covered by a duplication (Vollbrecht1997); (iii) enhancer detection is the onlyefficient technique that allows theidentification of genes expressed in very fewor even single cells. By focusing on the cellsand tissues where a gene is expressed, subtlephenotypes can be identified that may noteasily be recognized in phenotypic screens;and (iv) most importantly, enhancer detectorand gene trap transposons greatly facilitate themolecular cloning of genomic sequencesflanking the insertion site. In addition, theyallow a detailed genetic analysis of the detectedgene through remobilization and the recoveryof additional alleles, regional chromosomalrearrangements, and revertant sectors (e.g.,Grossniklaus et al. 1992; Springer et al. 1995;Tsugeki et al. 1996; Grossniklaus et al. 1998b).Generation of Transposants andOngoing ScreensTo identify genes involved in femalegametogenesis, we have generated nearly4,300 lines carrying randomly insertedenhancer detector or gene trap transposons (U.Grossniklaus, J. Moore, W. Gagliano, J-P. VielleCalzada, unpublished data). We are using the