Figure 3
HRM analysis of grapevine varieties for the LDOX_450bp fragment. (a) Aligned melting curves, and (b) Difference plot correspond to two representations of the same data obtained in LDOX_450bp HRM assay, using 20 varieties, and revealing three distinct genotypes. (c) Validation of LDOX_450bp SNP [T/T], [C/C] and [C or T] in nucleotide position 437 bp, by Sanger sequencing. Different grapevine varieties formed groups of melting curves, according their sequence variations. Three haplotypes are detected through HRM assay: homozygote SNP [C/C 
: DT, MF, M, Ruf, TA, TFi, TC, Vio], heterozygote [C or T 
: CS, Ch, FP, Gou, MG, Sou, TB, TR, TBr, TF, TN], and homozygote SNP [T/T 
: AB]. (d) Raw Data Melt Curve based on Tm, obtained for 201 bp fragment. Data collected during HRM curve experiment showing pre- and post-melting normalization regions (double columns) that are used to align the data, producing a clearer view of the melt curve results. O. europaea L. spp. was ussed as negative control, no amplification occurred. (e) The specificity of the HRM as technique was shown by a single melt curve (blue) using the 20 varieties (AB; CS; Ch; DT; FP; Gou; MF; M; MG; Ruf; Sou; TA; TB; TFi; TR; TC; TBr; TF; TN; Vio) for LDOX_201bp fragment amplification, and where no sequence variation was observed.
