Chapter 5 Genetic Analysis of Apomixis - cimmyt

114 ROil A. 8idlooll(Sherwood et al. 1994); Brachiaria sp. (Valle andSavidan 1996); Hi.:racium (Gadella 1991;Bicknell et al. 2000); and diplospory inTripsaClim dactylaides (Leblanc et al. 1995b;Grimanelli et al. 1998); TaraxaClim (van Dijk etal. 1999); and Erigeron (Noyes and Rieseberg2000). Earlier work by Dopp (1939) indicatesthat it may even be true for the fern Dryapteris.Simple inheritance would be particularlyvaluable for analyzing the molecular biologyof the trait.For the advancement of a molecular researchprogram, it is particularly advantageous if amodel system can be genetically transformed.This permits the introduction of marker genesto follow segregation and recombination andmutagenic sequences such as T-DNA tags andtransposons to assist in the cloning ofsequences associated with the expression ofapomixis. Furthermore, transformationpermits expression studies about putativeregulatory elements by their fusion to markersequences and the functional testing ofputative control genes by their introductioninto sexual or mutant genotypes.It would also be preferable to use a specieswith a small genome size, such as Arabidapsis,to facilitate the identification of critical loci.Similarly, an ideal system would already becharacterized with respect to genomicsequence and mapped morphological andmolecular markers (deletions, translocations,RFLPs, RAPDs, transposons, etc.) for thelocalization of loci. Finally, it would beadvantageous, although not essential, to usea crop species to assist in the transfer ofresearch findings into practical outcomes.Experimental MethodsThe success of any program on apomixis willdepend on both the natural attributes of thespecies used and on the power of thetechniques available to manipulate itexperimentally. For some species, techniqueshave already been documented, while forothers they would need to be established.Tissue culture and transformation techniquesare particularly valuable. They permit therapid micropropagation of selected types, theretention of somatic genotypes through invitro shoot regeneration, and the maintenanceof valuable genotypes through long-term coldstorage. It is also possible to recoverinterspecific hybrids using embryo rescue,and to manipulate ploidy in culture throughthe use of anther or microspore culture toisolate reduced genotypes and by colchicineapplication for chromosome doubling.As the initiation of the embryo sac is the firstdecisive event of the female reproductivephase, a thorough embryological analysis isan indispensable base for an investigation ofapomixis, while embryology continues to benecessary at all stages of the research. Reliablecytological techniques are therefore essentialfor this work. Fortunately, this is one area ofapomixis research that is well documented,particularly with respect to the use ofversatile, routine clearing techniques (Leblancet al. 1995a) that enable the use of thicksections to visualize the complex internalstructure of the ovule.Quantifying ApomixisMost studies of apomixis require a methodfor determining the presence of apomixis, andwhen possible, for quantifying its extent,either with respect to prevalence in apopulation or level of expression in a singlegenotype. Measuring apomixis, however, isa difficult task. When considered as a geneticevent, apomixis is reproduction through seedwithout either allelic segregation orrecombination. Assessment, therefore, shouldbe strictly based on whether allelicrearrangement occurs. This is seldompractical experimentally and most systems forassessing apomixis use a correlated character.The reliability of these correlations is clearly