Engineering seedlessness in grapevine: insights from INNER NO OUTER gene functional studies

Abstract

Seedlessness is a key quality trait in table grapes. In Vitis vinifera, seedlessness can originate from different biological processes: parthenocarpy, the fruit formation without fertilization, or steno-spermocarpy, when fertilization occurs but is followed by aborted seeddevelopment. Amajor ge-netic determinant for seedlessness from Sultanina, SdI, widely exploited in breeding, was shown to be associated to a missense mutation in the MADS-box gene VvAGL11. Alternative seedless phenotypes have been described in otherspecies, including the ‘Thai seedless’ mutant of Annona squamosa and Arabidopsis thaliana, linked to mutations at the INNER NO OUTER (INO) locus. The INO gene AT1G2340 was characterized in Arabidopsis ino-1 mutant. It encodes a YABBY-family transcriptionfactor that regulates abaxial development of the ovule outer integument. Our previous study identified VviINO as the grape YABBYortholog of AT1G2340. Functional com-plementation experiments demonstrated that VviINO fully restores the ino-1 mutant phenotype, suggesting conserved activity. Despite that a functional characterization in grapevine is still re-quired to assess its potential relevance for breeding of grape seedless cultivars.

Here, we used A. thaliana ino-1 lines complemented with VviINO CDS developed in our previous study to assessed a set of RNAguidesfor an efficient CRISPR/Cas9 knockout of the grapevine gene. Cas9 cleavage tests in vitro were first conducted. In parallel, floral dip transformation of Arabidopsis complemented lines enabled in vivo assessment of five guide RNAs constructs (three single guides and two tandem guides). Cas9-free T2 edited individuals exhibiting the rescued ino-1 phenotype and altered seed set were rescued for two of the three single guides as well as for one of the tandem guide constructs, bearing different classes of mutations. Following assessment in model system, embryogenic calli of the grape cultivars ‘Bragat Rosa’ and ‘Chardonnay’ have been transformed with constructs of the more successful guides.