Limitations of ad hoc genotyping in detecting ash dieback tolerance in <em>Fraxinus excelsior</em>

The data presented here was generated to study common ash tolerance to Ash dieback (ADB), caused by the invasive alien fungal pathogen Hymenoscyphus fraxineus. The data was generated from a field trial aiming at the identification of ADB tolerant genotypes while encompassing the genetic diversity of common ash in Austria. Over 35,000 progenies from 611 putatively tolerant mother trees and a selection of 71 clones from two clonal seed orchards were assessed for ADB symptoms over a period of three years. One tolerant and one susceptible progeny from a subset of the mother tree range (570 trees) were genotyped using the 4TREE array, a genotyping tool developed specifically to screen common ash populations for their tolerance to ADB. Methods Please refer to this publication for a detailed description of the experimental design: Szukala, A., Unger, G.M., Trujillo-Moya C., Geburek, T., Kirisits, T., Schueler, S., and Konrad, H. (2025). Limitations of ad hoc genotyping in detecting ash dieback tolerance in Fraxinus excelsior. Tree genetics & Genomes, doi: 10.1007/s11295-025-01725-6 Summary: To establish a sufficiently large breeding base of resistant clones, we selected 611 putatively ADB tolerant and seed-bearing single trees (i.e., functional females) of F. excelsior distributed in forests across Austria. Forest stands were selected based on the rare presence of remarkably healthy or only slightly damaged trees despite these being surrounded by a high number of infected and heavily damaged or dead individuals, whereas sites in rural settings or in areas with low disease incidence were avoided because of insufficient infection pressure. Seeds were harvested in 2015 and 2017 from the selected ash trees (i.e., putatively ADB tolerant mothers, with the only exception of few mothers growing in the seed orchards). From the sown seeds 35,718 seedlings germinated and were reared in plastic multi-container blocks in the experimental nursery of the Austrian Research Centre for Forests (BFW) in Tulln an der Donau (Lower Austria). To systematically assess the susceptibility to ADB over a three-year period, the one-year-old offspring (on average about 50 seedlings per mother tree) were planted in a 1.5 ha field trial in a completely randomized block-design. To increase the level of natural infection likelihood and thus the selection pressure on the seedlings, ash leaf litter (i.e., pseudosclerotial ash leaf material collected in heavily infested ash stands) was placed between seedlings. Fraxinus excelsior is characterized by complex seed dormancy: seeds harvested in 2015 germinated during the two following years, in 2016 and 2017, while those harvested in 2017 germinated in 2018 and 2019. Thus, the field trial included four sets of seedlings germinated during four subsequent years (2016 to 2019) and planted in four separate but adjacently arranged sub-trials, named hereafter R1 (established in 2017), R2 (established in 2018), R3 (established in 2019) and R4 (established in 2020; not investigated in the present study). Progeny of sub-trials R1, R2, R3 and R4 were assessed for their susceptibility to ADB (intensity/degree of ADB) annually over a period of three years. Leaf samples were collected from all offspring seedlings during the years of trial establishment, dried on silica gel and stored at room temperature until DNA extraction. For the present study, offspring from 570 ash mother trees in the sub-trials R1, R2 and R3 were selected for genotyping. These included 503 mother trees from forests, 22 from the seed orchard in Feldkirchen (three assessed as tolerant, five as intermediate and 14 as susceptible) and 45 from the seed orchard in Bad Gleichenberg (7 assessed as tolerant, 9 as intermediate and 29 as susceptible). Where possible, one completely healthy progeny (i.e., DC = 1) and a dead one (i.e., DC = 6) from each of the 570 mother trees were sampled for genotyping in 2020, at the end of the third, second, and first assessment periods in the R1, R2 and R3 field trials, respectively. Where no healthy or dead individuals were available, individuals of intermediate damage classes were chosen instead. For the analyses the final scoring of ADB intensity (third year of documentation) was used for all genotyped samples. DNA of the selected progenies was extracted at the Austrian Institute of Technology (Tulln an der Donau, Lower Austria) using the Qiagen DNeasy 96 Plant Kit according to the manufacturer’s recommendation. DNA quality and quantity were measured using PicoGreen™ dsDNA Assay Kits (ThermoFisher). We conducted genotyping of a total of 1,107 samples, including 11 technical replicates of the same individual (ID = 32806) to control for potential plate-related technical biases. Thus, the total number of genotyped individual trees was 1,096. Genotyping was done at Thermo Fisher Scientific Inc. using the 4TREE Axiom SNP array (Guilbaud et al. 2020; Archambeau et al. 2023), which encompasses a set of 13,407 SNPs. Highest priority SNPs include 51 associated with low susceptibility to emerald ash borer (Agrilus planipennis) (Kelly et al. 2020), and 1,633 associated with tolerance to ADB (Stocks et al. 2019). Additionally, 200 SNPs are included in the array that distinguish F. angustifolia from F. excelsior based on analyses that discriminated pure individuals of F. excelsior and F. angustifolia from each other.