The whole-genome sequence of carnation (Dianthus caryophyllus L.) cv. ‘Francesco’ was determined using a combination of different new-generation multiplex sequencing platforms. The total length of the non-redundant sequences was 568 887 315 bp, consisting of 45 088 scaffolds, which covered 91% of the 622 Mb carnation genome estimated by k-mer analysis. The N50 values of contigs and scaffolds were 16 644 bp and 60 737 bp, respectively, and the longest scaffold was 1 287 144 bp. The average GC content of the contig sequences was 36%. A total of 1050, 13, 92 and 143 genes for tRNAs, rRNAs, snoRNA and miRNA, respectively, were identified in the assembled genomic sequences. For protein-encoding genes, 43 266 complete and partial gene structures excluding those in transposable elements were deduced. Gene coverage was ∼98%, as deduced from the coverage of the core eukaryotic genes. Intensive characterization of the assigned carnation genes and comparison with those of other plant species revealed characteristic features of the carnation genome. The results of this study will serve as a valuable resource for fundamental and applied research of carnation, especially for breeding new carnation varieties. Further information on the genomic sequences is available at .

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Abstract:Higher plants can produce a wide variety of anthocyanin molecules through modification of the six common anthocyanin aglycons that they present. Thus, hydrophilic anthocyanin molecules can be formed and stabilized by glycosylation and acylation. Two types of glycosyltransferase (GT) and acyltransferase (AT) have been identified, namely cytoplasmic GT and AT and vacuolar GT and AT. Cytoplasmic GT and AT utilize UDP-sugar and acyl-CoA as donor molecules, respectively, whereas both vacuolar GT and AT use acyl-glucoses as donor molecules. In carnation plants, vacuolar GT uses aromatic acyl-glucoses as the glucose donor in vivo; independently, vacuolar AT uses malylglucose, an aliphatic acyl-glucose, as the acyl-donor. In delphinium and Arabidopsis, p-hydroxybenzoylglucose and sinapoylglucose are used in vivo as bi-functional donor molecules by vacuolar GT and AT, respectively. The evolution of these enzymes has allowed delphinium and Arabidopsis to utilize unique donor molecules for production of highly modified anthocyanins.

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Flower color intensity is largely determined by the amount of accumulated anthocyanins. Delphinium flowers show a wide range of colors from pale pink to deep orange to red to dark blue. Here, we demonstrated that the level of anthocyanin accumulation in dark blue, orange and red varieties was higher than in pale blue and pale pink varieties. Since dihydroflavonol 4-reductase (DFR) is a key enzyme in anthocyanin biosynthesis and accumulation in plants, we investigated the relationship between flower color intensity and the level of DFR gene expression. Six delphinium varieties with different flower colors were analyzed. Varieties that accumulated relatively high levels of anthocyanin also had high levels of DFR expression and enzyme activity in crude protein extracts. By contrast, DFR expression and activity was low in varieties with low anthocyanin accumulation. Alignment of DFR amino acid sequences in the six varieties showed the presence of two types, termed DgDFR and DnDFR. Recombinant DgDFR and DnDFR proteins had similar substrate specificities, but the kinetic turnover rate of the DnDFR enzyme was higher than that of DgDFR. We conclude that DFR expression level is closely correlated with flower color intensity and that DFR is an important factor that determines anthocyanin accumulation and delphinium flower color intensity.

Grafting of non-transgenic scion onto genetically modified (GM) rootstocks provides superior agronomic traits in the GM rootstock, and excellent fruits can be produced for consumption. In such grafted plants, the scion does not contain any foreign genes, but the fruit itself is likely to be influenced directly or indirectly by the foreign genes in the rootstock. Before market release of such fruit products, the effects of grafting onto GM rootstocks should be determined from the perspective of safety use. Here, we evaluated the effects of a transgene encoding β-glucuronidase (GUS) on the grafted tomato fruits as a model case. An edible tomato cultivar, Stella Mini Tomato, was grafted onto GM Micro-Tom tomato plants that had been transformed with the GUS gene. The grafted plants showed no difference in their fruit development rate and fresh weight regardless of the presence or absence of the GUS gene in the rootstock. The fruit samples were subjected to transcriptome (NGS-illumina), proteome (shotgun LC-MS/MS), metabolome (LC-ESI-MS and GC-EI-MS), and general food ingredient analyses. In addition, differentially detected items were identified between the grafted plants onto rootstocks with or without transgenes (more than two-fold). The transcriptome analysis detected approximately 18,500 expressed genes on average, and only 6 genes were identified as differentially expressed. Principal component analysis of 2,442 peaks for peptides in proteome profiles showed no significant differences. In the LC-ESI-MS and GC-EI-MS analyses, a total of 93 peak groups and 114 peak groups were identified, respectively, and only 2 peak groups showed more than two-fold differences. The general food ingredient analysis showed no significant differences in the fruits of Stella scions between GM and non-GM

Carnations carrying a recessive I gene show accumulation of the yellow pigment chalcononaringenin 2′-glucoside (Ch2′G) in their flowers, whereas those with a dominant I gene do accumulation the red pigment, anthocyanin. Although this metabolic alternative at the I gene could explain yellow and red flower phenotypes, it does not explain the development of orange flower phenotypes which result from the simultaneous accumulation of both Ch2′G and anthocyanin. The carnation whole genome sequencing project recently revealed that two chalcone isomerase genes are present, one that is consistent with the I gene (Dca60979) and another (Dca60978) that had not been characterized. Here, we demonstrate that Dca60979 shows a high level of gene expression and strong enzyme activity in plants with a red flower phenotype; however, functional Dca60979 transcripts are not detected in plants with an orange flower phenotype because of a dTdic1 insertion event. Dca60978 was expressed at a low level and showed a low level of enzyme activity in plants, which could catalyze a part of chalcone to naringenin to advance anthocyanin synthesis but the other part remained to be catalyzed chalcone to Ch2′G by chalcone 2′-glucosyltransferase, resulting in accumulation of anthocyanin and Ch2′G simultaneously to give orange color.