Understanding the genetic architecture of tolerance and resistance to pathogens is important to monitor and maintain resilient tree populations. Here we investigate the genetic basis of tolerance and resistance to needle cast disease in Douglas-fir (Pseudotsuga menziesii) caused by two fungal pathogens: Swiss needle cast (SNC) caused by Nothophaeocryptopus gaeumannii, and Rhabdocline needle cast (RNC) caused by Rhabdocline pseudotsugae). We performed a case-control genome-wide association analysis (GWA) and found these traits to be polygenic and under selection. We showed that stomatal regulation as well as ethylene and jasmonic acid pathways are important for resisting SNC infection and secondary metabolite pathways play a role in tolerating SNC once the plant is infected. We identified a key upstream transcription factor of plant defence, ERF1, as the main candidate for RNC resistance. Our findings contribute to the understanding of the highly polygenic architectures underlying disease resistance and tolerance in Douglas-fir and have important implications for forestry and conservation as the climate changes.

We used a pool-sequencing approach that targeted exon regions. For probe design details, see Lind et al. (2022). Briefly, the sequence capture probes were designed using genes identified in Douglas-fir RNA-seq data from (1) daily and cyclic-induced experiments (Cronn et al., 2017) and (2) needle samples infected by the fungal pathogen causing SNC Nothophaeocryptopus gaeumannii and wasp (unpublished). Exon sequences with a length of at least 100bp were submitted to Roche NimbleGen for Custom SeqCap EZ probe design. 

DNA was extracted from Douglas-fir diploid needle tissue. DNA samples were normalised at 10ng/μl and 10 to 20 individuals were pooled per site (See File S2) by combining equimolar amounts of individual DNA samples prior to library preparation. Barcoded (Kapa, Dual-Indexed Adapter Kit) libraries were made using 100-150ng of DNA from each pooled DNA sample with an approximately 450-bp mean insert size. SeqCap library preparation was performed using custom Nimblegen SeqCap probes according to the NimbleGen SeqCap EZ HyperCap Workflow User’s Guide Ver 2 (Roche Sequencing Solutions, Inc., CA USA). Following capture, each library was sequenced in a 150bp paired-end format on an Illumina NovaSeq 6000 instrument at the Genome Quebec Innovation Centre (McGill University, Montreal, Canada).