Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 1.05

MOLECULAR CHARACTERIZATION OF CITRUS SYMMETRIC AND ASYMMETRIC SOMATIC HYBRIDS BY MEANS OF ISSR-PCR AND PCR-RFLP

Scarano M.-T.*, Abbate L.*, Ferrante S.*, Lucretti S.**, Tusa N.*

*) Istituto di Ricerca per la Genetica degli Agrumi-C.N.R., Viale delle Scienze 11, 90128 Palermo, Italy

**) ENEA CR Casaccia, Sezione Genetica e Genomica Vegetale (026), Via Anguillarese 301, 00060 S.M. di Galeria (Roma), Italy

Citrus, protoplast fusion, ISSR-PCR, RFLP-PCR

Several protoplast fusion experiments have been carried out in order to obtain new somatic hybrids for genetic improvement of mandarin. Four different fusion were planned: ‘Redblush’ grapefruit (Citrus paradisi Macfadyen) + ‘Avana’ mandarin (Citrus deliciosa Tenore); ‘Duncan’ grapefruit + ‘Tardivo di Ciaculli’ mandarin; ‘Duncan’ grapefruit + ‘Fortune’ mandarin (Citrus reticulata Blanco) and ‘Murcott’ tangor (Citrus sinensis L. Osb x Citrus unshiu Marc.) + ‘Duncan’ grapefruit. Flow cytometry and isozyme banding pattern analysis were used for early screening. DNA of the hybrids was then extracted and subjected to ISSR-PCR using different primers anchored at 5’ and 3’ ends. PCR products were resolved on 1.5% agarose gel and stained with ethidium bromide. The great number of amplicons generated consist of the region between neighboring and inverted SSRs. From flow cytometry, the following genotypes: ‘Redblush’ grapefruit + ‘Avana’ mandarin; ‘Duncan’ grapefruit + ‘Tardivo di Ciaculli’ mandarin and ‘Duncan’ grapefruit + ‘Fortune’ mandarin resulted to be all tetraploids; meanwhile ‘Murcott’ tangor +’Duncan’ genotypes were all diploids, with morphological traits very similar to ‘Duncan’ (leaf parent). Among the tested loci, Pgi (phosphoglucose isomerase) and Pgm (phosphoglucomutase) showed the clearest evidence of the hybridity of the tested plantlets. ‘Duncan+Fortune’ and ‘Murcott+Duncan’ hybrids showed a banding pattern equal to that of ‘Fortune’ and ‘Duncan’ respectively (leaf parent). The hybrids obtained from the other two combinations displayed the isozyme banding pattern as expected in somatic hybrids. As for ISSR-PCR results, all the tetraploid plantlets showed the banding pattern of both parents, confirming their somatic hybridity. ‘Murcott+Duncan’ genotypes (2n) displayed only the banding pattern of ‘Duncan‘ grapefruit (leaf parent). Since, in Citrus, leaf protoplasts cannot regenerate, we could assume that we obtained, spontaneously, asymmetric hybrids (cybrids), in which the nucleus is inherited from the leaf parent and mitocondria from callus parent. In order to confirm this hypothesis, these hybrids were analyzed with PCR-RFLP using universal mtDNA primers to analyze mitochondria inheritance; PCR products were digested with four different restriction enzymes and resolved on 3% Metaphore agarose gel. All the hybrids presented the banding pattern of the ‘Murcott‘ tangor (callus parent) proving that these new genotypes are nevertheless cybrids. The speed, high reproducibility, technical simplicity and the requirement for low quantities of DNA make ISSR-PCR and PCR-RFLP more than suitable for the analysis of somatic hybrids of Citrus and, surely, of a wide range of other plant species. Therefore, these techniques could be proposed as a reliable screening procedure for the identification of citrus symmetric and asymmetric hybrids, provided that a sufficient number of primers is tested.