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cooling at 4° C were performed. For each primer, negative controls were included and
amplifications were repeated on 20% of samples in order to verify the repeatability of
results.

Electrophoresis and visualisation of amplification products
The PCR products were analysed by electrophoresis using a 2% agarose gel in l x

SBA buffer (Sodium borie acid Ph. 8.2) stained with l f•.tl/20 ml ethidium bromide
solution. Gels were run at 90 V (4,5 V/cm) for 2 h and ISSR banding patterns on gels
were visualized using a photo-UV transilluminator systern and recorded by digitai
photography. One hundred base pair ladder (GeneRuler lOObp Plus DNA Ladder,
Fermentas~ were run for reference with each prirner.

ISSR data analysis

ISSRs are dominant diallelic rnarkers; the presence of a given band, the dominant
phenotype, is scored asl, whereas its absence (the recessive phenotype) is scored as O.
Polymorphisrns (i.e. presence/absence of a band) are due to prirner divergence,
insertions, deletions, or chromosomal rearrangement (Wolfe & Liston 1998).

Genetic structure within the ISSR data set was assessed by means of an individuai
based approach, which rnay avoid the artefacts due to the assurnption of predefined
populations (Mank & Avise 2004). The population structure was inferred by the
Bayesian rnodel-based clustering algorithrn implernented in the software STRUCTURE
2.3.1 (Pritchard et al. 2000, Falush et al. 2007). This method assigns individuals to
clusters according to the rnultilocus genotype, without prior knowledge of their
geographical origin. The nurnber of clusters (K) is chosen in advance, and the posterior

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