An apple amiRNA efficiently silences the phytoene desaturase gene in apple.

Abstract

National audienceArtificial miRNA (amiRNA) is a powerful technology to silence genes of interest with a high specificity, either to validate their function or to create new traits. To set up such a gene regulation tool for apple, we designed two amiRNA constructs based on an apple endogenous miRNA backbone that was previously characterized (Md-miRNA156h), and we checked their efficiency on an easily scorable marker gene: the phytoene desaturase gene (PDS). Two pairs of miRNA:miRNA* regions were designed according to the recommendations published by Whartman et al. (2008). The monocistronic Md-miRNA156h with these PDS targets was placed under the CaMV 35S promoter and cloned using the Gateway recombination method in the destination plasmid pK7WG2D, generating the two plasmids pAmiPDS-h and pAmiPDS-w. Two Agrobacterium-mediated transformation experiments were performed on the cultivar ‘Gala’, with a rate of transformation of 2 % for pAmiPDS-w and 3.4 % for pAmiPDS-h. In total, 5 and 10 independent transgenic clones were recovered, respectively. Most transgenic lines had a typical albino and dwarf phenotype. However, three clones had a wild type green phenotype. Molecular analyses are underway to correlate the phenotype with the degree of expression of the amiRNA gene and of the PDS gene. This study is the first demonstration in apple of the functionality of an amiRNA based on an endogenous miRNA backbone. It provides important opportunities for apple genetic functional studies as well as apple genetic improvement projects