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Diapositiva 1 CNR - Istituto di Bioscienze e Biorisorse, Perugia Luciana Baldoni Composizione varietale degli oli di oliva mediante analisi del DNA I Metodi di Controllo – Il Controllo dei Metodi Torino, 9-10 Novembre 2017 Gli oli extra vergine di oliva L’ampia gamma di oli extra vergine di oliva è conseguenza: del grandissimo numero di varietà ancora in coltivazione, dell’area geografica di produzione e delle condizioni pedo-climatiche locali delle tecnologie di produzione, estrazione e stoccaggio L’olio di oliva è il prodotto alimentare sottoposto al maggior numero di contraffazioni in Europa Le più diffuse contraffazioni degli oli extra vergine di oliva si basano sulla diversa reputazione, notorietà e prezzo di certi oli rispetto ad altri (es. Olio Italiano 100%, DOP e IGP) Il principale bersaglio di questo tipo di contraffazioni sono le varietà impiegate per costruire l’olio Le analisi chimiche e fisiche classiche non sono in grado di rilevare le miscelazioni improprie degli oli Il patrimonio varietale dell’olivo Italia 538 42% del patrimonio mondiale Spagna 183 14% Francia 88 7% Grecia 52 4% Turchia 45 3,5% Tunisia 44 3,5% Algeria 41 3,2% Portogallo 24 1,9% Altri paesi 260 20% TOTALE 1.275 Perchè la caratterizzazione molecolare è necessaria? Confusione sull’identità varietale dovuta a diverse ragioni Varietà locali Varietà cosmopolite Varietà migranti Sinonimia (Frantoio-Raggiola) Omonimia (Rosciola) Toponimia (Nostrale di Rigali, Moraiolo dei Monti Martani) Morfonimia (Pendolino, Limona) Cultivar, Cloni? Ecotipi locali, Popolazioni varietali? Genotipi rari, Selvatici Presenza di virus o altri microrganismi non patogeni Principali varietà del Mediterraneo Ad ogni paese / regione / territorio di coltivazione corrispondono varietà locali specifiche e ben diverse da quelle coltivate altrove Taggiasca, Moraiolo, Olivastra, Bianchera, Rasara, Casaliva, Gargna’, Nostrana Brisighella, Frantoio, Leccino, Dritta, Canino, RajaSabina, Gentile Chieti , Raggiola, Piantone Mogliano, Nera di Collotorto, Cima Melfi, Carolea, Cassanese, Ottobratica, OgliarolaLeccese, Coratina, Peranzana, Cima Bitonto, Cellina Nardò, Rotondella, Pisciottana, Gentile Larino, Biancolilla, Cerasuola, Tonda Iblea, NocellaraBelice, Nera Di Gonnos, Bosana Aglandau, Oblica, Bouteillan, Levantinka, Grossane, Lastovka, Lucques, Bianchera, Picholine, Zutica, Buga Tanche Galega, Azeiteira, Arbequina, Kalinjot, Bardi Cordovil, Cornicabra, Tirana Cobrancosa Blanqueta, Empeltre, Koroneiki, Adramitini, Ayvalik, Domat, Villalonga, Farga Amigdalolia, Gemlik, Izmir Sofralik, Chalkidiki, Kalamon, Memecik, Picual, Hojiblanca, Picudo, Sigoise, Chemlal Konservolia, Lechinde Sevilla, Manzanilla, Mastoidis Zaity, Sorani, Kaissy Gordal, ChemlaliSfax, Chetoui, Oueslati Soury Picholine Nabali, Barnea, Marocaine, Kadesh, Haouzia, Menara Merhavia, Toffahi, Hamed Strutturazione geografica delle varietà di olivo in Italia Diffusione geografica di alcune varietà di olivo Solo poche varietà hanno raggiunto una diffusione molto ampia, interessando diversi paesi e continenti (es. Arbequina, Arbosana, Koroneiki) Marker molecolari ‘regionali’ Le varietà locali sono fortemente imparentate tra loro e quindi si possono distinguere come gruppi separati Ciascun gruppo è discriminato da marcatori regione-specifici Disponibilità di una banca dati dei profili DNA delle principali varietà di olivo Progetto Europeo BEFORE Coordinamento scientifico: CNR-ISAFOM-IBBR Perugia, Italia Obiettivi Stabilire protocolli comuni di identificazione molecolare e morfologica delle varietà di olivo Collection Mondiale de l’Oliviere - Marrakech, Marocco Olive Germplasm Bank - Cordova, Spagna Collezione Germoplasma di Olivo CNR – Regione Umbria - Lugnano in Teverina (TR), Italia Altre collezioni di Grecia, Libano, Giordania, Argentina L’analisi del DNA applicata agli oli di oliva extra vergine Solo l’analisi del DNA è in grado di identificare le varietà e/o specie che hanno contribuito alla preparazione di un olio Il DNA caratterizza in maniera inequivocabile ciascun individuo, varietà o specie diversa Consente di stabilire a posteriori quali sono la/le varietà da cui l’olio è stato estratto E’ utile per verificare se sono state operate miscelazioni o sostituzioni con oli di altre specie Potenzialità applicative Identificare le cultivar usate per la preparazione dell’olio Dimostrare l’assenza di varietà diverse da quelle attese Verificare l’assenza di oli di specie oleaginose diverse da olivo (nocciolo, girasole, colza, mais, o soia) Rilevare l’assenza di olive danneggiate dalla mosca (Bactrocera oleae) o da altri patogeni Testare l’assenza di OGM Presupposti della rintracciabilità molecolare Il DNA si conserva in tracce nell’olio per lungo tempo (1-3 anni) Il DNA si degrada progressivamente, rimanendo in frammenti sempre più brevi ma rilevabili Le tracce di DNA si possono estrarre dall’olio, purificare e rendere analizzabile (Budker et al., 2002) Il DNA è l’unica molecola che può essere moltiplicata in vitro tramite PCR Da 1 frammento di DNA Si ottengono Frammento di DNA originale milioni di Nuovo frammento copie per PCR (Polymerase Dopo 1 ciclo PCR: Chain 2 copie Reaction) Dopo 2 cicli PCR: 4 copie Dopo 3 cicli PCR: 8 copie Dopo 4 cicli PCR: 16 copie Sviluppo di nuovi marcatori varietà-specifici basati su Single Nucleotide Polymorphisms (SNP) A Sequenza comune a più varietà A G Mutazione su varietà target A Altre applicazioni NGS, GBS Applicazioni RNA-seq Mappatura .15.000 SNP ottenuti da Genotyping genetica approccio Genotyping-By- Sequencing Breeding, .3.000 SNP ottenuti dal Analisi del Selezione sequenziamento dei geni DNA degli oli assistita espressi SNP nella sequenza del gene SUT1 Cultivar 1 Heterozygous AG Cultivar 2 Homozygous AA Cultivar 3 Homozygous GG I marcatori SSR usati per l’analisi del DNA degli oli 1 2 3 156 145 160 145 148 154 156 210 183 210 190 210 190 198 4 5 6 156 156 148 154 145 160 190 198 183 190 210 210 Profilo SSR di 6 varietà A quali varietà corrispondono quegli alleli nella bottiglia? I marcatori SNP varietà-specifici 1 2 3 A G T 4 5 6 G C T 7 8 9 T A C Profilo SNP di 8 varietà L'olio è stato fatto con le cv. 1, 4, 7 Confronto tra profili di DNA estratto da foglia e da olio ACP1 alleli 1-3C cv. MORAIOLO DNA da foglia 304 316 bp ACP1 alleli 1-3C cv. MORAIOLO DNA da olio 304 316 bp Olio delle cv. Moraiolo e Coratina cv. MORAIOLO alleli 350-378 bp 350 378 bp cv. CORATINA alleli 378-426 bp 378 426 bp DNA da miscela di olio MORAIOLO/CORATINA alleli 350-378-426 bp 350 378 bp 426 bp Marcatori plastidiali specie-specifici (bar-coding) LD 100 bp LD 100 bp ccSSR-11 ccSSR-12 CCMP1 NOCCIOLO MAIS OLIVO NOCCIOLO OLIVO NOCCIOLO MAIS MAIS NOCCIOLO MAIS OLIVO OLIVO SSR plastidiali polimorfici tra specie oleaginose diverse (nocciolo, mais, olivo) Quantificazione del contributo di ciascuna varietà in un blend di oli Mediante Real Time PCR è possibile quantificare il contributo percentuale di ciascuna varietà in una miscela di oli Ma va tenuta in considerazione la possibile diversa storia di ciascun olio componente il blend Delta Rn Delta PCR Cycles PCR Cycles Nuovi sistemi di amplificazione-detection ad alta sensibilità, rapidità, portabilità, quantificabilità LAMP: Loop-mediated isothermal AMPlification Digital PCR Microfluidics NALF (Nucleic Acid Lateral Flow) Point-of-Care methods CONSIGLIO NAZIONALE DELLE RICERCHE Istituto di Bioscienze e Biorisorse, Perugia Luciana Baldoni Nicolò Cultrera Roberto Mariotti Saverio Pandolfi www.cnr.ibbr.it [email protected] Tel. (+39) 075 5014866 Mob. (+39) 328 3760912.
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