Llavallol, Argentina
Instituto Fitotécnico de Santa Catalina (FCAF, UNLP)
and Centro de Investigaciones Genéticas (UNLP-CONICET-CIC)
Relationships between Zea mays ssp. mays and Zea mays ssp. parviglumis by genomic in situ hybridization (GISH) --Gonzalez, G*, Confalonieri, V*, Comas, C*, Naranjo, CA, Poggio, L*
*also affiliated with Dpto. de Ciencias Biológicas, FCEN, UBA.
Fluorescent in situ hybridization (FISH) is a tool which, when combined with genomic in situ hybridization (GISH), reveals homologies in DNA, mainly in regard to repetitive sequences. These techniques are also very useful in detecting amplifications and divergences of DNA sequences. Moreover, they can be used to discriminate genomes having sequences of allopolyploid origin as well as to detect chromosomes or chromosomal segments derived from introgression. This technique allows us to understand the cryptic polyploid nature of the genus Zea, and the genomic constitution of meiotic configurations in those hybrids where chromosomes from different parents have been distinguished using as a probe total genomic DNA or a repetitive sequence present in one of them (Poggio et al., Cytogenet. Cell Genet. 81:134, 1998; Poggio et al., Genome 42:993-1000, 1999; Poggio et al., Bol. Soc. Argent. Bot. 35:297-304, 2000; and Poggio et al., in press).

Maize and related wild species are of polyploid origin, but which species should be considered the ancestral ones is still unresolved. GISH studies conducted by our group and other research workers using DNA of co-generic species and even related genera as probes, failed to reveal genomic differences. In the present work, we have applied GISH with washing at very high stringency after the hybridization reaction and using unlabeled total genomic DNA as blocking agent in order to reveal cryptic genomic differences.

The species analysed here were Zea mays ssp. parviglumis (Balsas, cult. 6836, IFSC) and Zea mays ssp. mays (knobless line cultivated in IFSC). Chromosome preparations and GISH were carried out according to Poggio et al. (1999). The blocking procedure (Anamthawat-Jonsson et al., Theor. Appl. Genet. 79:721-728, 1990) was applied by adding unlabeled DNA from Zea mays ssp mays and labeled DNA from Zea mays ssp. parviglumis in a 10:1 proportion respectively, on chromosome preparations of Zea mays ssp. parviglumis. This procedure allowed us to discriminate three regions of differential hybridisation: unlabeled, labeled and highly labeled. The first region contains shared DNA sequences of high homology between both taxa and is located in all the telomeric regions of Zea mays ssp. parviglumis; the second region contains free unblocked DNA sequences that could hybridize with DNA from the same species. This region probably corresponds to repetitive DNA inherent to Zea mays ssp. parviglumis. The last chromosome region, with strong hybridization signals, corresponds to the heterochromatic knobs of Zea mays ssp. parviglumis which were not blocked by heterologous DNA from maize because the line used for blocking does not have "knob sequences". These "knob" hybridized regions were taken into account as positive controls of the whole blocking experiment. The experiment reported here demonstrates that there are divergent chromosome regions between these two taxa that are considered subspecies of Zea mays by Doebley and Iltis (Amer. J. Bot. 67:982-993, 1980) and Iltis and Doebley (Amer. J. Bot. 67:994-1004, 1980).

The meiotic analysis of the hybrid Zea mays ssp. mays x Zea mays ssp. parviglumis (2n=20) reveals that 10 bivalents (II) are observed in 80% of the analysed cells in Metaphase I, the remaining 20% showed 9 II + 1 I or 8 II + 2 I. Besides, the mean of closed bivalents was 7.2 with terminal chiasmata. These data suggest that Zea mays ssp. mays and Zea mays ssp. parviglumis have genomes homologous enough for normal pairing to occur.

It is interesting to point out that in situ hybridization used under conditions of high stringency and with blocking agents, can provide valuable information about the type and localization of repetitive sequences in Zea mays and related species, being an adequate complement to the data obtained by classical cytogenetic analysis.

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