SummaryIntron-containing constructs encoding self-complementary 'hairpin' RNA (ihpRNA) have the potential to efficiently silence genes in a range of plant species. In this study we demonstrate the silencing of a ripeningrelated chalcone synthase (CHS) gene in strawberry fruits (Fragaria · ananassa cv. Elsanta) by a construct (ihpRNA) containing the partial sense and corresponding antisense sequences of CHS separated by an intron obtained from a F. · ananassa quinone oxidoreductase gene. An Agrobacterium strain carrying a T-DNA expressing the ihpRNA transgene was injected with a syringe into the receptacles of growing fruits still attached to the plant about 14 days after pollination. As a consequence of the reduced levels of CHS mRNA and enzymatic CHS activity, the levels of anthocyanins were downregulated and precursors of the flavonoid pathway were shunted to the phenylpropanoid pathway leading to a large increases in levels of (hydroxy) cinnamoyl glucose esters. We anticipate that this technique in combination with metabolite profiling analysis will be useful for studying the function of unknown genes during the development and ripening of strawberry fruit.

SummaryRNA interference (RNAi) has been exploited as a reverse genetic tool for functional genomics in the nonmodel species strawberry (Fragaria × ananassa) since 2006. Here, we analysed for the first time different but overlapping nucleotide sections (>200 nt) of two endogenous genes, FaCHS (chalcone synthase) and FaOMT (O‐methyltransferase), as inducer sequences and a transitive vector system to compare their gene silencing efficiencies. In total, ten vectors were assembled each containing the nucleotide sequence of one fragment in sense and corresponding antisense orientation separated by an intron (inverted hairpin construct, ihp). All sequence fragments along the full lengths of both target genes resulted in a significant down‐regulation of the respective gene expression and related metabolite levels. Quantitative PCR data and successful application of a transitive vector system coinciding with a phenotypic change suggested propagation of the silencing signal. The spreading of the signal in strawberry fruit in the 3′ direction was shown for the first time by the detection of secondary small interfering RNAs (siRNAs) outside of the primary targets by deep sequencing. Down‐regulation of endogenes by the transitive method was less effective than silencing by ihp constructs probably because the numbers of primary siRNAs exceeded the quantity of secondary siRNAs by three orders of magnitude. Besides, we observed consistent hotspots of primary and secondary siRNA formation along the target sequence which fall within a distance of less than 200 nt. Thus, ihp vectors seem to be superior over the transitive vector system for functional genomics in strawberry fruit.

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