Figure 2
High-resolution DNA melting curve analysis for a 532 bp amplicon with seven haplotypes in F3H gene. (a) Raw Data Melt Curve. Data collected during a HRM curve experiment showing pre and post-melt regions (vertical bars) that are used to align the data, producing a clearer view of the melt curve results. A melting curve generated by a DNA negative control (O. europaea L.) was used, no amplification was observed. The curve colours represent the sequence variations among 20 samples (AB - Alicante Bouschet; CS - Cabernet Sauvignon; Ch - Chardonnay; DT - Donzelinho Tinto; FP - Fernão Pires; Gou - Gouveio; MF - Malvasia Fina; M - Merlot; MG - Moscatel Galego; Ruf - Rufete; Sou - Sousão; TA - Tinta Amarela; TB - Tinta Barroca; TFi - Tinta Francisca; TR - Tinta Roriz; TC - Tinto Cão; TBr - Touriga Brasileira; TF - Touriga Franca; TN - Touriga Nacional; Vio - Viosinho). (b) HRM profiles, based on F3H_532bp fragment Tm, revealing seven haplotypes. (c–j) Validation of F3H_532bp sequence variants by Sanger sequencing using V. vinifera L. clones. Ten clones of each variety were used to validate HRM assay with the coincident melting curve profiles. (c,d) HRM product validation in CS, TFi and TN varieties, display the presence of variant in position 1039/40 [A or T], 1065 [A or C], and 1318 bp [C or T] for TFi variety. The sequence validation for TN was confirmed by SNPs presence in positions 1157 [C or G], 1381 [A or G], and 1464 bp [A or T]. (e,f) The evidence of SNPs in position 1039/40 [A or T], and 1318 bp [C or T] was confirmed by sequencing Vio and Ruf clones. (g,h) FP clones validate the variations of HRM assay for nucleotide position 1039/40 [A or T], 1157 [C or G], 1318 [C or T], 1381 [A or G] and 1464 bp [A or T]; and TR clones for position 1065 bp [A]. (i,j) The evidence of SNPs [A or C] in position 1065 bp was confirmed by sequencing Gou and TB clones.
