Dissection of black rot resistance towards marker-assisted breeding in grapevine: pedigree reconstruction and phenotyping optimization coupled with high-density linkage mapping revealed a major QTL associated with bunch resistance

Viticulture is a multibillion-dollar market based on grapes, one of the most valuable fruits in agriculture, but it is also characterized by the highest uses of fungicides per hectare for disease management. In fact, despite the availability of genetic resources resistant to the most diffuse fungal pathogens, those cover less than a fifth of the worldwide vine area. The remaining 80% is represented mainly by susceptible Vitis vinifera L. varieties. Instead, re-sistance traits are known to derive from other Vitis species, due to their coevolution with the pathogens, and their exploitation in breeding is a valuable strategy to reduce pesticide treat-ments. Black rot (BR) is a destructive disease caused by the ascomycete, Phyllosticta ampeli-cida, whose telomorphic form was known as Guignardia bidwellii. In the European continental area in the last two decades its pressure increased due to the concomitant decreased usage of chemicals and the occurrence of mild-rainy summer because of the climate change. This work was established to fight the spread of BR by means of the introduction of BR resistance in susceptible genetic background. The study consisted of (i) the reconstruction of BR resistant donor pedigree based on the collection of historical phenotypic data, (ii) the eval-uation of parental lines and selections of the breeding program of the Edmund Mach Founda-tion (Italy), (iii) the microscopic inspection of disease progression, and (iv) QTL analysis of the segregating population (N=146) ‘Merzling’ (hybrid, resistant) × ‘Teroldego’ (V. vinifera, suscep-tible). The outcomes revealed a large unexplored pool of disease resistance donors of 148 va-rieties belonging to 14 different species, that permitted to evaluate and identify five new promising genotypes readily exploited for breeding. Concurrently, a new inoculation method based on spore production from fresh infected leaves was developed. Ex vivo (detached leaves) inoculation did not reveal significant differences among the assessed genotypes and highlight-ed the tendency of spores to accumulate and germinate near leaf hairs, while disease progres-sion did not occur. The screening of the segregating population, both under greenhouse and field conditions, allowed the discrimination on chromosome 14 between two distinct QTLs as-sociated with leaf/shoot and bunch resistance. The first QTL confirmed the Resistance to Gui-gnardia bidwellii (Rgb)1 locus previously identified by three studies and the high-density link-age mapping allowed to reduce it from 2.4 to 0.7 Mb along the PN40024.v4 reference genome. The region resulted enriched in genes belonging to phloem dynamics and mitochondrial pro-ton transfer. The second QTL associated with bunch resistance was designated Rgb3. Located at 9 cM (6 Mb) upstream Rgb1, it was characterized by a cluster of Germin-like protein 3 genes and lipid transfer and localization, notably known to be linked to broad spectrum disease re-sistance and stress response. No resistance (R)-genes have been annotated in the region under-lying the QTL in the PN40024 reference genome. In conclusion, this work provided new insights for grapevine breeding programs by the identification of previously unknown BR resistance donors, the development of protocols and good practices towards large-scale resistance screening as well as the discovery of a novel QTL associated with bunch resistance. Marker validation in different genetic backgrounds is ongoing for its routinary implementation in marker-assisted breeding and further studies are planned to dissect BR resistance mechanism by the sequencing of the genomic regions, the study of the Germin-like 3 gene cluster and the cell wall characterization.