Figure 1
F3H gene HRM different profiles, based on Tm, obtained for 375 bp fragment. (a) Raw Data Melt Curve. Data collected during a HRM curve experiment showing pre and post-melt regions (double bars) used to align the data, producing a clearer view of the melt curve results. The curve colours represent 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). O. europaea L. spp. was used as negative control to obtain the raw data melt curve, no amplification occurred in this DNA sample. (b) General HRM profiles, based on Tm, obtained for F3H_375bp fragment revealing four different haplotypes. (c–j) Validation of F3H_375bp different sequence variant using V. vinifera L. clones for each specific variety through Sanger sequencing. Each variety was validated using ten clones. (c,d) The same melting curve profiles was observed in clones from CS and TC, for nucleotide positions 47 and 291 bp, via melting curves normalization and sequencing HRM product. (e,f) HRM validation in FP and Vio varieties, shows the presence of variant Y [C or T] in nucleotide position 47 bp. (g,h) The presence of variant W [A or T] in nucleotide position 291 bp, in Gou and TB, was confirmed. (i,j) The evidence of SNPs in position 47 and 291 bp [C or T] was confirmed by sequencing the MF and TFi clones.
