Page 159 - Dedola2011

2. MATERlALS AND METHODS
A total of 195 specimens were collected from tidal flats at six different sites within the
Gulf of Olbia at an average depth of 5-6 m. The sediment structure at the six sites was
mostly characterized by coarse sand (0.5-1.0 mm grain size). Furthermore, 99
specimens were sampled within a farming area in the Lagoon of Sacca di Goro (SGR).
For each individuai, the totallength, width, and height were measured (Table l , Fig. l).
Genomic DNA was isolated either from the muscle or from the mantle tissue using a
modified alkaline/CTAB/NaCl lysis method and subsequent silicon dioxide treatment
(Neudecker and Grimm, 2000). Approximately 50 mg of frozen tissue was incubated
for 30 min at 65°C in 200 111 of extraction buffer (NaOH 0.02 M, NaCl 2.5 M, CTAB
1%). The extract was then neutralized with the addition of0.02 M Tris-HCl buffer (0.01
M Tris pH 8.0, 0.02 M HCl).
Six microsatellite loci (Asaril 6, Asari24, Asari43, Asari55, Asari62, and Asari64)
(Yasuda et al. , 2007) were used in this study. PCR was performed in a 25 111 solution
containing 20-40 ng of genomic DNA, l x reaction buffer, 2 mM MgC12, 0.2 mM of
each dNTP, lU ofEuroTaq (Euroclone), multiplexed fluorescent dye-labeled primers at
different concentrations, and deionized water. PCR was performed in a MJ DNA
Engine PTC-1 00 thermal cycler under the following conditions: an initial cycle at 95°C
for 5 min, followed by 30 cycles of94°C for 30 s, 50°C for 30 s, and 72°C for 30 s.
Microsatellite loci were visualized on an ABI PRISM 3130xl Genetic Analyzer
(Applied Biosystems) and analyzed using GENEMAPPER 4.0 software (Applied
Biosystems).
Genetic relationships among the sampling sites were visualized by means of a principal
coordinate analysis (PCoA). The ordination plot was built using GENALAEX 6.3
software (Peakall and Smouse, 2006) on a matrix of genotypic distances.

159

Gìan Luca Dedola: ANALISI DELLA VARIABILITÀ GENETICA DI PA TELLA FéRRUGINEA, PATEL.LA UL l'SSIPONENSIS (MOLLUSCA: GASTROPODA) E PINNA
NOBILIS (MOLLUSCA: BIVALVIA): lLCONTRJBUTO DEl DATI MOLECOLARI ALLA CONSERVAZIONE DI SPECIE MINACCIATE - Tesi d i douorato in Scienze
della natura e delle sue risorse • indiri7.zo BK>logi.a Ambientale, Università degli Studi di Sassarl