Additional file 4.
Genomic organisation and transcription of two copies of F3'Hs present in the grapevine genome. In section a, exon/intron structure of F3'Hs is shown as blue boxes (exons) connected by blue lines (introns); TEs are shown as coloured boxes. In section b and c, selective amplification of exon junctions astride the terminal intron and expression of each F3'H copy are shown. Two primer pairs (orange and green triangles) were designed in the internal and terminal exons. The terminal intron varied in size between 249 bp and 96 bp in F3'Ha and -b, respectively. Each primer pair anneals perfectly to the target F3'H, but has a mismatch at the 3'-terminal nucleotide with the paralogous F3'H. Selectivity of primer pairs for either F3'Ha or F3'Hb was validated by amplifying PN40024 genomic DNA and by Sanger sequencing of the PCR amplicons. Selectivity for either F3'Ha or F3'Hb was also confirmed by assessing the size of the amplified genomic DNA (vs. the size prediction of 523 bp and 370 bp astride the second intron in F3'Ha and F3'Hb, respectively) and, for the expressed F3'Ha, by inferring intron size from the comparison between amplicons from genomic DNA and cDNA. Expression of F3'Ha was assessed by semi-quantitative PCR using cDNA from leaf, petiole, tendril, flower, shoot, and berry skin and flesh, in two grapevine cultivars ('Merlot' and 'Aglianico'). Expression of F3'Ha was also assessed in berry skin of four cultivars ('Aglianico', 'Marzemino', 'Grignolino', and 'Nebbiolo') at four stages of fruit development. cDNA was normalised using the constitutive gene VvUbiquitin. Transcripts of F3'Hb were never detected under the same experimental conditions. In section d, expression of F3'Ha was assessed by quantitative PCR in berry skin at 8 developmental stages in the cultivars 'Aglianico', 'Marzemino', 'Grignolino', and 'Nebbiolo'. Transcript levels of F3'Ha increased at full-veraison (stage of 100% coloured berries) by approximately 2-fold in all cultivars, with substantial differences among cultivars only at harvest. Transcript levels are expressed as arbitrary units, normalised using the constitutive gene coding for VvUbiquitin. Bars represent the standard deviation of three biological replicates. Letters above the histograms indicate significant differences between means, based on a Student-Newman-Keuls test (P < 0.05).
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Falginella et al. BMC Genomics 2010 11:562 doi:10.1186/1471-2164-11-562