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Protoplasts isolated from an embryogenic callus line of `Femminello siracusano' lemon [Citrus limon (L.) Burm. f.] were incubated with 0.5 μm toxin of Phoma tracheiphila (Petri) Kanc. et Ghik., the pathogen of the mal secco disease, which seriously damages most commercial lemon cultivars. Two toxin-tolerant cell lines were obtained, and plants were regenerated from each line. The selected protoclones were tested for their tolerance by exposing callus and protoplasts to the toxin and detecting chitinase (a pathogenesis-related protein) among the intra- and extracellular proteins extracted from leaves of regenerated plants and suspension culture, respectively. The tolerance of the protoclones in these tests was equivalent to the tolerant lemon cultivar Monachello, and they were substantially more tolerant than their mother cultivar Femminello siracusano.
Specific primers were designed for 61 cloned RAPD fragments and from 10 Citrus EST sequences for the production of SCAR, CAPS, and STS markers for a Citrus grandis `DPI 6-4' × Poncirus trifoliata `Rubideaux' F1 pseudo-testcross population. Fifteen SCAR, three CAPS, and one EST/STS markers were developed. An additional 17 SCAR and CAPS primer pairs developed at the Citrus Research and Education Center for a Citrus grandis `Thong Dee' × (Citrus grandis `Thong Dee' × Poncirus trifoliata `Pomeroy') BC1 population were screened in the pseudo-testcross population. A total of 27 markers were identified and scored in the pseudo-testcross population in which 24 were mapped; 13 in the Citrus parental linkage map on seven linkage groups and 11 in the Poncirus parental map on five linkage groups. In the BC1 population, 20 of 27 markers tested were found to be polymorphic and 13 mapped to seven of nine linkage groups. Of these, 11 were mapped in both populations and could be used for aligning presumed homologous regions on the three linkage maps.