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Consequently, the airn ofthe present study was to analyse the rnitochondrial COI sequence
of P. ulyssiponensis s.l., P. caerulea, P. vulgata and P. candei, in order to identify a
species-specific restriction site. This site can be used as diagnostic RFLP rnarkers to
facilitate unequivocal distinction of P. ulyssiponensis s.l. frorn the other three species,
whose correct taxonornic attribution on rnorphological bases alone rnay raise concems.

2. Materials and Methods
2.1. Designing ofspecific COI primers and RFLPs
A new pair of COI primers was designed, since universal primers for this gene (Folmer et
al. 1994) did not always give satisfactory results for P. ulyssiponensis s.l. frorn
Mediterranean. Specific primers (L: 5'-TTAATTCGAGCTGAACTTGG-3'; H: 5'-
ATTCGTTCAAGTTGCATAGC-3') were designed after the analysis of COI sequences -
aligned by rneans ofthe software Clustal W Multiple alignrnent irnplernented in BioEdit
7.0.5.2 (Hall 1999)- on a total of 131 individuals of P. ulyssiponensis s.I. (51
Mediterranean and Atlantic specimens), P. caerulea (24), P. vulgata (11) and P. candei
(45) (GenBank Accession No. are given in Table 1), choosing two conserved internai
regions suitable for the annealing (see Sanna et al. 2009 for the rnethodology). These
primers allowed amplifications of an internai portion of the "Folmer region" (Folmer et al.
1994) which was 437 base pair (bp) long.
Forty COI sequences ofthe following congenerics: Patella depressa Pennant, 1777
(GenBank Accession Nos.: DQ089613-15; EF462972-75; EU073890-95), Patella lugubris
Gmelin, 1791 (DQ089576-77; EU073889),Patellaferruginea Gmelin, 1791 (AY996038-
43; GQ469870-76), Patella rustica Linnaeus, 1758 (AB238579; EF462954-56;
GQ469878-82), and Patella pellucida Linnaeus, 1758 (DQ089620-21) were also added to
the above-rnentioned 131 sequences in order to find a restriction site able to unequivocally

103

Gìan Luca Dedola: ANALISI DELLA VARIABILITÀ GENETICA DI PATELLA FéRRUGINEA, PATEL-LA ULl'SSIPONENSIS (MOLLUSCA: GASTROPODA) E PINNA
NOBILIS (MOLLUSCA: BIVALVIA): lLCONTRJBUTO DEl DATI MOLECOLARI 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