Marcatori molecolari per l iden3ficazione varietale. Tracciabilità molecolare

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Lamberto Berardino
8 anni fa
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1 Marcatori molecolari per l iden3ficazione varietale Tracciabilità molecolare

2 Regolamento Europeo 178/2002 Nella tracciabilità rientrano le a.vità volte a iden1ficare le sostanze che cos1tuiscono il cibo o che si suppone saranno introdo8e negli alimen1 durante tu8e le fasi di produzione, processamento e distribuzione

il cibo o che si suppone saranno introdo8e negli alimen1

3 Tracciabilità basata sul DNA

4 Tracciabilità basata sul DNA

5 Tracciabilità basata sul DNA

6 Tracciabilità basata sul DNA

7 Real- Time qpcr vs.tradi3onal PCR Starting Template PCR Round 1 End-point analysis Agarose gel for product detection 2X Template PCR Round 2 4X Template Fluorescence

8 Sequence Detec3on System

9 TaqMan Fluorogenic Probe Reporter! (Fluoresceine)! Quencher! (Rhodamine)! Energy Transfer! 5! Fam! Vic! Tet! 3! Phosphate! Group! Laser Excitation!

10 Taqman PCR Chemistry Denaturation Annealing Polymerization R = Reporter Q = Quencher forward primer R Q 5 3 5

11 Taqman PCR Chemistry. Cleavage R = Reporter Q = Quencher R Q forward primer 5 3 5

12 Molecular beacons

13 SYBR Double- Stranded DNA Binding Dye Minor Groove Binding

14 Real- Time qpcr Nucleo1de sequences screening for iden1fica1on of a species- specific DNA marker Real- 1me PCR assay development Tes1ng assay for specificity on a panel of genotypes Tes1ng assay for efficiency and sensi1vity Valida1on on food vs official analy1cal methods

assay for specificity on a panel of genotypes Tes1ng assay for

15 Tracciabilità molecolare di specie cerealicole

16 Marcatori AFLP ed SSR per il genotyping

17 Costruzione di una matrice 1/0 primer ## inallettabile1-farina inallettabile1-pane inallettabile2-farina inallettabile2-pane mentana-farina mentana-pane gentil rosso-farina gentil rosso-pane canove-farina canove-pane terminillo-farina terminillo-pane apulia precoce mix-farina apulia precoce mix-pane marzuolo mix-farina marzuolo mix-pane risciola-farina risciola-pane bolero-farina bolero-pane autonomiab-farina autonomiab-pane sasson-farina sasson-pane

0 0 0 inallettabile2-farina 0 0 1 0 0 1 1 0 0 0 1 0 0 1 0 0 0 0 0 0 1 0 0 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 0 inallettabile2-pane 0 0 1 0 1 0 1 0 0 0 1 0 0 1 0 0 0 0 0 0 1 0 0 1 0 0 0 0 1 0 0 0 1 0 0

18 Tracciabilità molecolare di specie cerealicole AFLP analysis Distance es1ma1on

19 Modified RAPD markers 42 tomato varie1es from different geographical origins and seed merchants

22 All of the varie1es were clearly separated and individually iden1fied by reproducible fingerprints of only 6 RAPD primers.

23 Tomato cul1vars for industrial processing are very different, not only in fruit characteris1cs (size, shape), but also in lycopene and an1oxidant contents. The aim of this study was the chemotyping and genotyping of the tomato varie1es Heinz 3402, Leader and Perfectpeel, (1) to evaluate the gene1c traceability of these varie1es, and (2) to determine whether their func1onal an1oxidants compounds are useful markers of traceability.

25 Fig. 1. Typical RAPD complex fingerprint generated with A9 primer, separated in agarose gel, 2% (w/v) (A) and virtual gel obtained with Bioanalyzer 2100 (B). M: molecular marker; 1 2: Heinz 3402; 3 4: Leader; 5 6: Perfectpeel; 7: Ciliegino tomato; 8: Grappolo tomato.

28 Also a more consolidated (and less expensive) technique like RAPD coupled with PCA, as well as some chemotype parameters (firstly mineral content and then an1oxidant func1onal compounds) allows the univocal iden1fica1on/tracking of tomato cul1vars at varietal level.

34 The process of RADSeq. (A) Genomic DNA is sheared with a restric1on enzyme of choice (SbfI in this example). (B) P1 adapter is ligated to SbfI- cut fragments. The P1 adapter is adapted from the Illumina sequencing adapter (full sequence not shown here), with a molecular iden1fier (MID; CGATA in this example) and a cut site overhang at the end (TGCA in this example). (C) Samples from mul1ple individuals are pooled together and all fragments are randomly sheared. Only a subset of the resul1ng fragments contains restric1on sites and P1 adapters. (D) P2 adapter is ligated to all fragments. The P2 adapter has a divergent end. (E) PCR amplifica1on with P1 and P2 primers. The P2 adapter will be completed only in the fragments ligated with P1 adapter, and so only these fragments will be fully amplified. (F) Pooled samples with different MIDs are separated bioinforma1cally and SNPs called (C/G SNP underlined). (G) As fragments are sheared randomly, paired end sequences from each sequenced fragment will cover a bp region downstream of the restric1on site.

37 RAD tags were generated from the genomic DNA of a pair of eggplant mapping parents, and sequenced to produce ~17.5 Mb of sequences arrangeable into ~78,000 con1gs. The resul1ng non- redundant genomic sequence dataset consisted of ~45,000 sequences, of which ~29% were puta1ve coding sequences and ~70% were in common between the mapping parents. The shared sequences allowed the discovery of ~10,000 SNPs and nearly 1,000 indels, equivalent to a SNP frequency of 0.8 per Kb and an indel frequency of 0.07 per Kb.

38 214 tomato accessions (which include cul1vated landraces, commercial varie1es and wild rela1ves) genotyped with a custom- made Illumina SNP- panel. Most of the 175 successfully scored SNP loci were found to be polymorphic.

42 In a previous study, by using 19 SSRs on a subset of the De la Pera and Muchamiel Spanish cul1vars here analysed, we were not able to discriminate all the accessions evaluated, although they had different phenotypes as assessed by morphological characteriza1on. It is therefore remarkable that the use of the (GATA)4 oligonucleo1de probe, although labour intensive and not amenable to high- throughput genotyping methods, detected clear and reproducible differences between all the accessions inves1gated.

43 (GATA)4 oligonucleo3de probe

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