Chapter 5 Genetic Analysis of Apomixis - cimmyt

90 o.lel ~II-, Jo. lob.., aod Diego Gaucile,......le••Cloning the Apomixis Gene(s) UsingMolecular Genetics ToolsA major difficulty encountered by thoseinterested in cloning "apomixis genes" issimply defining what they are. Introducingapomixis into crops implies that specific genesare transferred or altered and expressed in thetarget crops. Most likely, not all of the genesinvolved in the apomictic process should betargeted: most, if not all, of them shouldalready be present and playing a role insexually reproducing plants. The issue then iswhich alleles of pertinent genes must betransmitted or manipulated for the inductionand successful development of apomicticembryos and seeds. To date, all efforts to tagapomixis genes, including those presented inthis paper, have focused on the mechanism ofnonreduction, mainly because it is an excellentindicator of apomictic development and it isprobably the easiest one to score. Nevertheless,it should be remembered that apomixis isprobably more complex than the Simpleprocess of nonreduction. The importance ofthis constraint will likely emerge whenattempts are made to synthesize de novoapomicts in sexual organisms"Map-based" cloning in apomictic species.Once a gene has been located on a genetic map,subsequent efforts to specify its position canultimately lead to its isolation (for the firstsu~cessful efforts in plants, see Giraudat et al.1992; Martin et al. 1994). The recentdevelopment of powerful new approaches forphysically mapping chromosome segmentscombined with the ability to clone large DNAfragments (Burke et al. 1987; Shizuya et al.1992), and progress in genome sequencingtechniques have created new and higherstandards for positional cloning in plants. It isstill a laborious and risky task outside of a fewwell-characterized model genomes, but thenumber of genes cloned in this manner arerapidly increasing. However, positionalcloning for apomixis is not very promisingbecause most, if not all, of the candidatespecies for a map-based cloning project arehighly heterozygous tetraploids, for whichlittle genomic characterization exists.Furthermore, when attempting positionalcloning, the first step is to identify achromosomal region, defined by two or moremolecular markers, that flanks the gene understudy. The precision of the estimated positionof the gene is therefore limited by the smallestmeasurable recombination unit, meaning onerecombinant in a given mapping population.Hence, the recombination level around theapomixis gene(s) presents another significantchallenge: positional cloning will proveefficient only insofar as recombination can beobserved near the locus of interest. Asmentioned earlier, recombination near theapomictic alleles is very likely restricted, atleast in Pennisetum and Tripsacum.Consequently, the smallest recombination unitdefined by two markers that encompasses theapomixis locus might well be a relatively largeamount of DNA.Transposon tagging of apomixis genes. Somemodel plants, such as maize, rice, tomato,Arabidopsis, and Petunia have undergoneextensive genome characterization. Specificapproaches are available for gene taggingthese plants that might be considered fortagging apomixis gene(s), provided thatcomponents of apomixis occur in one of theseorganisms.A very promising approach is that oftransposon tagging. Transposable elementsare short DNA sequences that have theproperty to transpose to more or less randomlocations in the genome (see Walbot 1992, fora review). They were discovered in maize, buthave since been identified or introduced invery diverse organisms. They have been usedin a wide range of genetic studies, and havebeen found to be highly effective for genetagging and cloning.