Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
Identification OF zygotic AND NUCELLAR seedlings in CITRUS interploid crosses by means of isozymes, flow cytometry and ISSR-PCR
Tusa N.*, Abbate L.*, Ferrante S.*, Lucretti S.**, Scarano M.-T.*
*) Istituto di Ricerca per la Genetica degli Agrumi - C.N.R. Viale delle Scienze 11, 90128 Palermo, Italy
**) ENEA C.R. Casaccia, Sezione Genetica e Genomica Vegetale (026) Via Anguillarese 301, 00060 S.M. di Galeria (Roma), Italy
Citrus, protoplast fusion, cybrids, ISSR-PCR
Malsecco is a systemic fungal disease of Citrus caused by Phoma tracheiphila (Petri) Kantsch & Gik. and occurs widely in the Mediterranean and Black Sea areas. The fungus penetrates through wounds and colonizes the xylem, inducing withering and dieback of twigs and branches. Development of improved lemon cultivars with tolerance or resistance to mal secco disease is an important breeding objective in these area. Our approach has been to develop somatic hybrids by protoplast fusion of lemon and other species or varieties tolerant to malsecco in attempt to introgress genes for tolerance to the disease into a new genotype. The resulting hybrids were then used as pollen parents in backcrosses with lemon cultivars. Here we report on the investigations effected on the offspring derived from the backcrosses between the allotetraploid somatic hybrid ‘Milam’ lemon (purported hybrid of Citrus jambhiri Lush) + ‘Femminello’ lemon (Citrus limon L.Burm. f.) and diploid ‘Femminello’ lemon. Our goal was to generate malsecco tolerance in different populations of seedlees triploid lemon types with good fruit quality. Many Citrus species reproduce apomictically by seed, through nucellar embryony: these embryos are genetically identical to the mother plant, and develop within the same embryo-sac together with zygotic embryo. Therefore, in order to distinguish zygotic seedlings from the nucellars specific methods are needed. We have tested three different techniques, isozymes analysis, flow cytometry and ISSR-PCR to analyze a total of 137 seedlings. Pgi (phosphoglucose isomerase) and Pgm (phosphoglucomutase) isozymes were used for early screening, but they were not always able to reveal locus segregation and consequently they were not useful to detect the two classes of embryos. Flow cytometry was able to actually distinguish very easily and accurately triploid genotypes (zygotic seedlings) from diploids (nucellars), but the main drawback of this technique is that it is only able to detect true hybrids in interploid crosses. ISSR-PCR technique resulted to be very effective in detecting differences between zygotic and nucellar seedlings, independently from the ploidy of the two parents. Among 8 triploid hybrids tested, 2 genotypes showed a banding pattern equal to that of ‘Femminello’ lemon, while 6 genotypes displayed locus segregation. It is known that this technique is not very informative because of its dominant nature (i.e. heterozygous and homozygous genotypes cannot be distinguished), which requires to score a high number of loci and therefore a great number of primers must be tested. This may explain why ISSR-PCR procedure was unable to reveal the hybrid nature of 2 tested triploid hybrids. This procedure requires an easy PCR amplification of genomic DNA using a single primer composed of a microsatellite sequence anchored at the 5’ or 3’ end by two to three arbitrary nucleotides. In our hands, ISSR-PCR showed to be speedy, highly reproducible and requiring low quantities of DNA, making it very useful for the analysis of Citrus hybrids.