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François Luro, Frédéric Laigret, Joseph-Marie Bové, and Patrick Ollitrault

We used three short repetitive nucleotide sequences [(GTG)5, (TCC)5, and (GACA)4] either as radiolabeled probes for hybridization with restricted citrus DNA or as single primers in polymerase chain reaction amplification experiments with total genomic DNA. We tested the ability of the sequences to discriminate between seedlings of zygotic or nucellar origin in the progeny of a Volkamer lemon (Citrus volkameriana Ten. & Pasq.) tree. The genetic variability within two species [Citrus sinensis (L.) Osbeck (sweet oranges) and Citrus reticulata Blanco and relatives (mandarins)] also was evaluated. DNA amplified fingerprinting with single primers was the more successful technique for discriminating between nucellar and zygotic seedlings. Although we were not able to distinguish among 10 cultivars of C. sinensis, all 10 C. reticulata cultivars tested were distinguishable. However, it still is difficult to identify the putative parents of a hybrid plant when the two parental genomes are closely related.

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Samia Lotfy, Francois Luro, Françoise Carreel, Yann Froelicher, Delphine Rist, and Patrick Ollitrault

Somatic hybridization allows the creation of new patterns of nuclear, mitochondrial and chloroplastic association. It is therefore necessary to master cytoplasmic molecular markers to determine the genetic origin of both organelles of plantlets obtained from protoplasts fusion. In the case of Citrus and related genera, only southern blot hybridization and restriction fragment-length polymorphism (RFLP) techniques were used for this task until now. Here, we describe the use in the Aurantioideae subfamily, of a simple and non labeling cleaved amplified polymorphic sequence (CAPS) technique, to determine the cytoplasmic genome origin of intergeneric somatic hybrids. Mitochondrial and chloroplastic universal primers previously selected for population genetic studies in Quercus by Demesure et al. (1995) are used with some modifications. The variability of cytoplasmic genome among somatic fusion partners is detected by coupling amplification and restriction reactions. Digested DNA fragments are analyzed by agarose gel electrophoresis (PCR-RFLP). This technique has been applied for the analysis of the cytoplasmic constitution of somatic hybrids arising from intergeneric, intersubtribal and intertribal combinations. Systematic transmission of the mitochondria from protoplasts isolated from embryogenic callus parents was confirmed.