Chapter 5 Genetic Analysis of Apomixis - cimmyt

128 or..Ie, leblal( allll Aodrea Mam"',"be useful for progeny tests and shouldtherefore be considered, Progeny tests areusually performed on seedlings or fully-grownplants, but other tissues from earlierdevelopmental stages, such as ovaries,endosperms or seeds, can also be used.1. Analysis of pollinated ovaries or seeds.Determining ploidy levels in pollinated ovariesor seeds (albuminated) provides informationon both reduction (meiosis) and fertilizationevents. Ratios between endosperm andembryos and between female and malecontributions to the endosperm in apomictsoften differs from those in sexual plants exceptfor the Panicurn-type a posporousdevelopment (Figure. 9.1). For many otherapomictic pathways, these ratios differ. Forexample, endosperms found in tetraploiddiplosporous apomicts are higher than in theirsexual tetraploid counterparts for identicalpollen donors (i.e., lOx versus 6x if the pollenis 2x); endosperm/embryo ratio forautonomous apomixis is 2:1 and 5:2 [(4x + 4x)+ 2x / 4x + OJ for tetraploid pseudogamousapomicts (tetraploid pollen donor).Fertilization by unreduced pollen (Chao 1980;Huff and Bara 1993) and endopolyploidization,which sometimes occurs during endospermdevelopment, is also possible and may furthercomplicate analyses. However, endospermploidy level(s) may suggest apomicticreproduction or allow the quantification offacultative apomixis. Nevertheless, it cannotreveal the precise nature of the apomicticmechanisms involved.Ploidy level in fertilized ovaries or immatureseeds cannot easily be determined usingclassical chromosome counting methods, butflow cytometry now permits rapidmeasurement of DNA content in a variety ofplant tissues, including single embryos, youngendosperms, or seeds (Galbraith et at. 1983;Kowles et al. 1990; Hignight et al. 1991).Analyses in numerous apomictic species haveproven flow cytometry to be a rapid andreliable procedure for determining the modeof reproduction (Mazzucato et al. 1994;Brautigam and Brautigam 1996; Grimanelli etal. 1997b; Gupta et al. 1998; Naumova et al.1999; Matzk et al. 2000). Another option forDNA content estimation of the endospermnuclei is to combine staining with 4'-6­diamidino-2-phenylindole (DAPI), Huoresencemicroscopy, and image analysis(Naumova et al. 1993; Sherwood 1995; Cacereset al. 1999).2. Ovule regenerated plants. In tetraploidaccessions of AIlium tuberosum, Kojima andKawaguchi (1989) reported a high frequencyof tetraploid regenerated plants fromunpollinated cultured ovules, suggestingapomixis expression. This indicator could beapplied in screening because, in similarculture media, sexual plants would generatefew (poly)haploids, whereas apomeioticovules would grow mostly into plantlets withthe same number of chromosomes as themother plant.3. Analysis of progeny plants. Progeny testsmust clearly identify either hybrid offspring(n + 0 types are generally poorly represented)or seed production in absence of pollinationwhen pseudogamous apomixis orautonomous apomixis, respectively, aresuspected. Hybrids can be identified using (i)morphological descriptors, (ii) cytologicaldata, and/or (iii) marker analyses, if the originof the progeny is appropriate.Remarks on progeny size. The use ofprogenies from controlled crosses isrecommended. Male parents bearingdiscriminating traits (dominant traits,different chromosome numbers, etc.) shouldbe chosen when available, limiting possibleconfusions between selfed and hybridprogenies. However, open pollinatedprogenies can be used when mother plantsare sufficiently heterozygous to detect