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

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 2.21

SSR AND CAPILLARY ELECTROPHORESIS AS A TOOL FOR DISTINGUISHING ZYGOTIC PLANTLETS IN LEMON INTRASPECIFIC CROSSES

M.-T. SCARANO*, N. TUSA*, L. ABBATE*, D. GRAZIANO*, S. REALE**, S. FERRANTE*

*) C.N.R. IGV-sez di Palermo, Corso Calatafimi 414, 90129 Palermo, Italy

**) Dipartimento S.A.V.A, Università degli Studi del Molise, Via De Sanctis, 86100 Campobasso, Italy

Citrus, protoplast fusion, backcrosses, microsatellite

One of the most important goals in genetic improvement of lemon in the Mediterranean area is certainly the obtainment of new genotypes tolerant or resistant to mal secco disease. To improve tolerance and fruit quality, a diploid lemon cybrid with an intermediate degree of resistance to mal secco disease, spontaneously obtained by symmetric protoplast fusion between ‘Valencia’ (V) orange and ‘Femminello’ (F) lemon, was used as mother plant in backcrosses with a diploid clone of ‘Femminello’ Lemon Tolerant to the Mal Secco disease (LTMS). Backcrosses have been planned because subsequent cycles of sexual hybridization for continued advancement to eliminate negative traits will be possible. ‘Femminello’ lemon reproduces apomictically by nucellar embryony; nucellar embryos are genetically identical to the mother plant, and develop within the same embryo sac together with the zygotic embryo (polyembryony). Usually we perform flow cytometry (FCM) to distinguish zygotic embryos, but in this case, where both nucellar and zygotic plantlets are diploids, FCM is not able to distinguish zygotics from nucellars, therefore suitable methods are needed for this purpose in order to eliminate useless nucellar plants. Moreover, since the diploid lemon cybrid presents the nucleus of ‘Femminello’ lemon and the mitochondria of ‘Valencia’ orange (as previously detected) this cross is a sort of selfing, because the two parents present almost the same nucleus. We have regenerated 40 plantlets, which were subjected to microsatellite analysis with the use of capillary electrophoresis fluorescence-based technology for the analysis of the data. Genomic DNA was extracted from young leaves using the CTAB method and therefore seven microsatellite loci were analyzed (http://www.plantbiology.ucr.edu/people/faculty/rooselink.html): TAA1, TAA41, cAGG9, TAA3, CAT01, AG14, CAC39. For all the SSRs, forward primers were labeled with either 6-FAM (blue), HEX (yellow) and TET (green) fluorescent dyes and TAMRA (red)-labeled used as internal size standard (GeneScan-350). Capillary electrophoresis was carried out on an ABI PRISM 310 (Applied Biosystems) and raw data analyzed with GeneScan software (version 3.1) to estimate the variants size; each DNA band (allele) is then represented by a peak. All the seven primer pairs used in this study showed that the parental plants were heterozygous with two alleles; therefore the zygotic heterozygous plants cannot be distinguished from the nucellars, meanwhile homozygous are certainly zygotics. Among the 40 tested diploids, 3 showed locus segregation, displaying one band, confirming their zygotic origin. Combining SSR method and semi-automated analysis system like GeneScan resulted to be ideal for the molecular analysis of many genotypes and successfully utilized for a large-scale analysis. SSR coupled with fluorescence-band semi-automated allele sizing technology provided doubtless for high genetic resolution and can develop further application in Citrus genetic studies.