Chapter 5 Genetic Analysis of Apomixis - cimmyt

lod"1101 of ApomIxi. II S....IPIlII,. ~ MlfatHe.1s 225M 2. This allows the detection of recessive aswell as dominant mutants. Also, it is wellknown that mutations occur in sectors of M]plants, and by screening only a singleinflorescence per plant, many mutants may belost. This loss can be avoided by screening intheM 2Transposon Mutagenesis for theIsolation of Apomictic Mutants ofArabidopsis and PetuniaTransposon mutagenesis, like T-DNA tagging,creates mutations by insertion of thetransposon into a gene. Its advantage is thattagged genes can be isolated by using theinserted sequence as a molecular probe. Also,a large number of mutants can be producedsimply by repeated selfing of the plants. Thisapproach is currently being applied toArabidopsis and Petunia, as described in detailby Ramulu et al. (1997). We shall brieflysummarize the main points.For Arabidopsis, a two-element system, derivedfrom the maize transposable element En- [,was used (Aarts et al. 1995). The maizetransposon has a 13 bp inverted repeat at eachterminus and encodes a transposase requiredfor transposition. The two-element systemconsists of a nonautonomous "wings-elipped"En-transposase under the control of the CaMV355 promoter and a nonautonomous mobileI-element with flanking inverted repeats thathas been inserted into a kanamycin resistance(nptII) gene. Both elements are containedwithin a T-DNA that also carries a hygromycinresistance marker for selecti on oftransformants. Several lines that containedabout 20 I-elements and the En-transposasewere crossed with homozygous cerl and cer6-2mutants, and homozygous male sterile lineswere selected from the segregating F 2population. Propagation of these lines forseveral generations under permissiveconditions is expected to create a large numberof new mutations, which can be screenedunder nonpermissive conditions for apomicticmutants.For transposon mutagenesis in Petunia,Ramulu et al. (1997) are using a two-elementtransposon system found in Petunin, whichconsists of a nonautonomous element, dTphl,and an autonomous element carrying thetransposase, ACTl (Doodeman et al. 1984;Gerats et al. 1990). A line containing more than200 copies of dTphl, which produces a highfrequency of unstable mutations in selfedprogeny, was used to establish a number oftransposon genotypes, which were eachcrossed with a conditional male-sterile plant.Male sterility in this line results from theabsence of flavonols, which is caused by achalcone synthase antisense gene (Ylstra et al.1994). The application of flavonols, which arerequired for pollen tube growth, restoresfertili ty and allows selfing. Plants homozygousfor the malesterilityphenotype will beselectedin F 2populations, and screening for apomicticmutants will be conducted on a large numberofF 3and F 4plants in the absence of flavonols.Branching Out in the BrassicasA benefit of mapping data from severalimportant crop plants and from Arabidopsis hasbeen the discovery that groups of genes withinlarge segments of the chromosomes arearranged in the same linear order betweenrelated species regardless of differences ingenome size (Flavell and Moore 1996). Inmany cases, molecular markers identified foronespecies are found to map to correspondinglocations in a related species. This high levelof synteny can be exploited for the isolationof genes from species with large genomes. Thehomologous gene can first be isolated from arelated species with a small genome, such asArabidopsis, where fine mapping andchromosome walking are feasible; it can thenbe used as a probe for direct isolation of thegene from the species of interest.