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138 Figure 1. Map of distribution of 16 Brassica populations investi-
gated.
tives could thus be very interesting for the breeding of
agronomical varieties. According to the enzyme system, the compositions
of the gels and the buffers were as follows:
Gladis and Hammer (2001) propose to unite in one
species (Brassica oleracea) all these wild and culti- 1) aconitase (ACO), leucine aminopeptidase
vated forms and to treat the Atlantic and the wild (LAP), phosphoglucoisomerase (PGI), phospho-
Brassicas at subspecies and variety level. glucomutase (PGM)
• gel buffer: histidine pH 7 (DL-histidine, 5 mM
Sicily represents one of the two centres of differen-
tiation of this section, since the other one is in the titrated with NaOH);
eastern Mediterranaean area (Raimondo 1997a). In • electrode buffer: Tris pH 7, 0.13 M titrated with
fact, three species, B. rupestris, B. villosa and B.
macrocarpa, are endemic to the Sicilian area and two citric acid;
other species, B. incana and B. insularis, also occur
there. 2) 6-phosphogluconate dehydrogenase (6-PGD)
• gel buffer: morpholine-citrate pH 6.1; 2 mM
Their habitats consist of limestone cliffs at sea level
until 1000–1200 m. The populations are often re- (1 / 20 dilution of electrode buffer)
stricted in size and distribution, basically because of • electrode buffer: citric acid 40 mM with pH ad-
the limited areas of cliffs, the competition with other
species and the human disturbance i.e. grazing, fire, justed to 6.1 with N-3-aminopropylmorpholine.
quarries, etc. Consequently, some of these popula-
tions are endangered or threatened and need to be The enzymes migrated towards the anode. 35 mA
preserved by genetic resources conservation measures were applied in pH 6.1 gels and 100 V in pH 7 gels
(Raimondo et al. 1994). during the first ten minutes. After the wicks were
removed, gels were run at 40 mA and 150 V respec-
This paper evaluates the isozyme diversity of 16 tively.
wild populations from Sicily and Calabria in order to
obtain genetic resource information for meaningful Gel slices were incubated in a staining substrate
implementation of the conservation programs. solution, specific to each enzyme tested. Staining
solutions for ACO and LAP were described by Wen-
Materials and methods del and Stuber (1984) and Arus and Orton (1983),
respectively. Solutions for PGI, PGM and 6-PGD
A total of 16 populations was collected from natural were as reported by Vallejos (1983).
habitats of Sicily, Egadi islands and Calabria in 1997
(Table 1, Figure 1). Seeds were collected from seven Zymograms and alleles nomenclature referred to
to ten different individuals and pooled to obtain a Che`vre et al. (1995) (Figure 2).
good representative sample of each population. Seeds
were stored under appropriate conditions (15 8C, low
humidity).

Thirtyfive plants per population, grown in indi-
vidual pots in a heated greenhouse (25 8C), were
studied.

Isozyme analysis

Fresh young leaves were crushed in 100 mL of
extraction buffer containing tris HCl pH 7.5 and 1%
reduced glutathion. Soaked wicks of each sample
were inserted into a slit made across the gel (Che`vre
et al. 1995). The technique used for horizontal elec-
trophoresis in 11% starch gels is described by Kephart
(1990).