Abdel-Ali Benslimane

Restriction fragment length polymorphism of the wheat nuclear ribosomal DNA has been studied in several steps of a breeding scheme, including parental genotypes, F1 hybrid, F9 generation, and anther-derived doubled haploid lines obtained from F9. Ribosomal DNA represents a suitable molecular marker in following segregation and possible divergency from expected homozygosity after haploidization of an androgenetic process. It has been shown to undergo variations among the first cycle-doubled haploid lines in the relative amount of two different sizes of ribosomal DNA repeat units. The specificity and peculiar properties of the plant system used allowed us to assign an intrachromosomal location (short arm of the chromosomes 1B, 1R or 6B) to several ribosomal DNA repeat units that differ by the length of their nontranscribed spacer region.

We report here the identification and characterization of two minicircular plasmid-like DNAs isolated from mitochondria of a moroccan date-palm variety. Both molecules were cloned and used as probes in Southern analyses of mitochondrial and total-cellular DNA. Evidence was obtained that these plasmid-like DNAs crosshybridized but did not show any homology to nuclear, chloroplastic, or main mitochondrial genomes. Sequence analysis revealed that both minicircles, 1 346- and 1 160-bp long, share several stretches of homology, the most important consisting of three identical clusters of lengths 42, 47 and 38 bp. In contrast, no major homology was observed with the other higher-plant plasmid-like DNAs reported so far. Sequence analysis also revealed the presence, in the same strand of one of the minicircles, of two open reading frames potentially encoding proteins 89 and 86 amino acids in length. Interestingly, Northern analyses, using single strands of each minicircle as probes, showed the presence of two transcripts hybridizing only with the strand bearing these two open reading frames. However, computer-assisted comparison of the predicted polypeptide sequences with a protein-sequence library failed to detect any significant homology to known sequences.

The organization of the nuclear ribosomal DNA from a parental line of wheat (Triticum aestivum L., cv. ‘César’) and its anther-derived first cycle and second cycle doubled haploid lines has been analyzed by DNA-DNA molecular hybridization. Restricted DNA has been probed by three subclones of wheat nuclear ribosomal DNA covering the entire repeat unit. No significant difference was detected in the extent of methylation of ribosomal DNA of the doubled haploid lines with respect to the parental line. On the other hand, a variation has been found in the organization of the nontranscribed spacer region of ribosomal DNA of the first cycle doubled haploid line. This variation remains stable after a second cycle of in vitro androgenesis. However, one out of five second cycle doubled haploid lines so far tested showed an additional hybridization band present in the parental line but lacking in the first cycle doubled haploid line.

We have previously shown that two circular plasmid-like DNAs (the S and the R plasmids) can be found in the mitochondria of date palm (Phoenix dactylifera L.), a dioecious monocotyledonous tree. The two plasmids differ essentially by the absence, in the R plasmid, of a 109-bp DNA segment. Using 36 date palm varieties and employing a PCR-based approach, we show that the simultaneous presence of the R plasmid and absence of the S plasmid can be considered as a reliable molecular marker of resistance to a vascular wilt (Bayoud disease) caused by the fungus Fusarium oxysporum f. sp. albedinis. Conversely, the simultaneous presence of the S plasmid and absence of the R plasmid is correlated to Bayoud disease susceptibility The availibility of this diagnostic tool for plasmid characterization should subsequently allow simple, rapid and efficient selection of Bayoud-resistant individuals from the large number of date palms obtained by natural crosses which display good date quality.