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 - 1.06
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.