3 biological replicates of 20 berries per cultivar were collected at every deve

Syntenic regions were identified using the Genome Evolution tool. Transposable elements were annotated according to the grape genome browser information. LTRs in Copia and Gypsy retrotransposons were identified by dot plot analysis. Global DNA alignments of chromosomal segments were performed using LAGAN in a win dow of 100 bp with a minimum identity of 70%. Dot plots of segmental ARQ 197 duplications were made using Dotter. Alignments of 2 kb promoter regions were performed with DiAlign2, using a minimum HSP length of 10 bp and visualised with GEvo. DNA binding motifs were predicted by PlantCARE. Selective amplification of F35Hs and F3Hs paralogues Selective primers were designed across dissimilar exonic DNA stretches or using a 3 terminal SNP between the perfect match of the target gene copy and the mis matched annealing site of paralogous sequences.<br><br> Absence AUY922 ic50 of illegitimate cross amplifi cation of other paralogues was validated by amplification of genomic DNA, Sanger sequencing of the PCR pro ducts, and detection of variable sites inside of primer sequences that distinguished the target gene copy from other paralogues. qPCR efficiencies in amplifying the DNA of PN40024 and of the mixed haplotypes of every heterozygous cultivar used in the present study were calculated using the equation E 10 1 slope of the standard curve. The standard curve was constructed with five 10 fold serial dilutions, using cDNA from organs and developmental stages in which the specific gene copy was expressed or, if not possible, genomic DNA.<br><br> Paralogue specific Alvocidib 146426-40-6 primers with a PCR efficiency comprised between 90 and 110% in PN40024 were considered acceptable, and were used for qPCR if the standard deviation of their PCR efficiencies among the accessions under study was less than 10%. PCR pri mers that distinguished individual paleologous copies, as well as highly similar paralogues, and passed the thresh olds set for the qPCR experiment, could be developed for nine out of the sixteen F35H copies. The remaining copies were either highly identical in sequence or con tained only a few polymorphic sites within DNA seg ments unsuitable for primer design. The range of variation in average PCR efficiency of primer pairs among the accessions tested was within the bounds of 87% in Marzemino and 102% in Nebbiolo, with a similar average efficiency of 93% in Aglianico and Grignolino.<br><br> This excluded a substantial cultivar effect of the efficiency of primer annealing during qPCR on the estimation of transcript levels of the whole gene family among cultivars, caused by possible SNPs in the annealing sites across haplotypes. Experimental design and statistics in expression and metabolite analyses Variation in anthocyanin profile and in transcriptional level of duplicate genes among developmental stages and cultivars was studied using a complete randomized design, and tested for significance using ANOVA run by COSTAT statistical package. Each plot consisted of 10 in a row clonally replicated plants in north south oriented rows. Vines were grown at the germplasm repository of Vivai Cooperativi Rauscedo, northeastern Italy. Vines were trained using the Syl voz system.