Discrimination between Bn and Mn plants, as well as between Bm hybrids originating from unreduced male or unreduced female gametes, would be possible by crossing parents with different ploidy, or using pollinators with homozygous dominant markers. However, the prerequisites for this approach, including the availability and the crossability of suitable parents, are not always realized. Seed mortality frequently occurs in interploidy crosses, and for detailed analyses, a combination of different techniques would be necessary.
After interploidy crosses, Bn plants are characterized by an intermediate 2C-value of the parents or by a dominant marker of the father, and Mn plants can be recognized by a 2C-value identical to that of the mother or by the lack of a marker belonging to the father. Discrimination between these two types of progeny is an essential prerequisite for apomixis research.
Since obligate sexual plants are frequently missing in apomictic species, crosses between related sexual and apomictic species with different chromosome numbers have been used for quantification of the different reproductive pathways in facultative apomictic plants, and for analyses of the inheritance of apomixis. Such approaches may be problematic, because free recombination can be depressed by the selection of functional gametes or seeds after interspecific pollination, and by meiotic barriers in the Fj plants (Bicknell and Koltunow 2004; Matzk et al. 2005).
Five pathways of embryo formation were found by FCM ploidy analyses in Hi-eracium rubrum (hexaploid) after crossing with the related tetraploid H. pilosella (Asteraceae; Krahulcova et al. 2004). A comparison of these data with results obtained by a novel screening method (cf. Section 6.3) for the same apomictic plants is in progress (see Krahulcova et al. 2004). Even discrimination between maternal and paternal Bm hybrids was possible in Hypericum using interploidy crosses and chromosome counting by light microscopy (Lihova et al. 2000), and with FCM (Brutovska et al. 1998).
Interploidy crosses with subsequent FCM ploidy analyses were also applied in order to screen for unreduced pollen producers in Dactylis glomerata (Poaceae; Maceira et al. 1992), Vaccinium (Ericaceae; Ortiz et al. 1992), and Lotus (Fabaceae; Negri et al. 1995) species. A high percentage of tetraploid, non-maternal progenies originated from a cross between a 4x mother and a 2x father plant, indicating that the male parent has a high capacity for unreduced pollen formation.
Plants with the homozygous dominant alleles for blue aleurone were used as markers to indicate normal sexual reproduction in wheat (Morrison et al. 2004). By means of markers, which are at present frequently replaced by molecular markers, parthenogenetic development can be excluded or maternal identity confirmed.
DNA fingerprinting (RAPD and AFLP markers) was used in alfalfa to verify the rare occurrence of the complete apomictic pathway in a mutant with a high ca pacity for diplospory (Barcaccia et al. 1997). In Hypericum perforatum the maternal plants (MII) were discriminated from segregants (Bn, BIII and MI) by AFLP, RAPD and RFLP fingerprints (Arnholdt-Schmitt 2000; Haluskova and CeMrova 1997). By combination of DNA fingerprinting with light microscopic chromosome counting it has also been possible to differentiate between BII and BIII hybrids or MI and MII plants in H. perforatum (Mayo and Langridge 2003). FCM ploidy analyses, in combination with RAPD fingerprinting, were applied for a detailed determination of the genetic origin of aberrant progenies derived from facultatively apomictic mother plants of Poa pratensis (Huff and Bara 1993). Such detailed studies were laborious, since molecular markers had to be developed, and a relatively complicated procedure for FCM (involving young, fully expanded leaves which were subjected to washing, chopping in buffer, centrifugation and re-suspension in staining buffer) was used for analyzing both mother and progeny plants.
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