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tives could thus be very interesting for the breeding of
agronomical varieties.
Gladis and Hammer (2001) propose to unite in one
species (Brassica oleracea) all these wild and culti-
vated forms and to treat the Atlantic and the wild
Brassicas at subspecies and variety level.
Sicily represents one of the two centres of differen-
tiation of this section, since the other one is in the
eastern Mediterranaean area (Raimondo 1997a). In
fact, three species, B. rupestris, B. villosa and B.
macrocarpa, are endemic to the Sicilian area and two
other species, B. incana and B. insularis, also occur
there.
Their habitats consist of limestone cliffs at sea level
until 1000–1200 m. The populations are often re-
stricted in size and distribution, basically because of
the limited areas of cliffs, the competition with other
species and the human disturbance i.e. grazing, fire, Figure 1. Map of distribution of 16 Brassica populations investi-
gated.
quarries, etc. Consequently, some of these popula-
tions are endangered or threatened and need to be
preserved by genetic resources conservation measures
(Raimondo et al. 1994). According to the enzyme system, the compositions
This paper evaluates the isozyme diversity of 16 of the gels and the buffers were as follows:
wild populations from Sicily and Calabria in order to 1) aconitase (ACO), leucine aminopeptidase
obtain genetic resource information for meaningful (LAP), phosphoglucoisomerase (PGI), phospho-
implementation of the conservation programs. glucomutase (PGM)
• gel buffer: histidine pH 7 (DL-histidine, 5 mM
titrated with NaOH);
• electrode buffer: Tris pH 7, 0.13 M titrated with
Materials and methods
citric acid;
A total of 16 populations was collected from natural
habitats of Sicily, Egadi islands and Calabria in 1997 2) 6-phosphogluconate dehydrogenase (6-PGD)
(Table 1, Figure 1). Seeds were collected from seven • gel buffer: morpholine-citrate pH 6.1; 2 mM
to ten different individuals and pooled to obtain a (1/20 dilution of electrode buffer)
good representative sample of each population. Seeds • electrode buffer: citric acid 40 mM with pH ad-
were stored under appropriate conditions (15 8C, low justed to 6.1 with N-3-aminopropylmorpholine.
humidity).
Thirtyfive plants per population, grown in indi-
The enzymes migrated towards the anode. 35 mA
vidual pots in a heated greenhouse (25 8C), were
were applied in pH 6.1 gels and 100 V in pH 7 gels
studied.
during the first ten minutes. After the wicks were
removed, gels were run at 40 mA and 150 V respec-
Isozyme analysis tively.
Gel slices were incubated in a staining substrate
Fresh young leaves were crushed in 100 mL of solution, specific to each enzyme tested. Staining
extraction buffer containing tris HCl pH 7.5 and 1% solutions for ACO and LAP were described by Wen-
reduced glutathion. Soaked wicks of each sample del and Stuber (1984) and Arus and Orton (1983),
were inserted into a slit made across the gel (Chevre respectively. Solutions for PGI, PGM and 6-PGD
`
et al. 1995). The technique used for horizontal elec- were as reported by Vallejos (1983).
trophoresis in 11% starch gels is described by Kephart Zymograms and alleles nomenclature referred to
`
(1990). Chevre et al. (1995) (Figure 2).