A haplotype-resolved chromosome-level assembly and annotation of European hazelnut (C. avellana cv. Jefferson) provides insight into mechanisms of eastern filbert blight resistance.

European hazelnut (Corylus avellana L.) is an important tree nut crop. Hazelnut production in North America is currently limited in scalability due to Anisogramma anomala, a fungal pathogen that causes Eastern Filbert Blight (EFB) disease in hazelnut. Successful deployment of EFB resistant cultivars has been limited to the state of Oregon, where the breeding program at Oregon State University (OSU) has released cultivars with a dominant allele at a single resistance locus identified by classical breeding, linkage mapping, and molecular markers. C. avellana cultivar "Jefferson" is resistant to the predominant EFB biotype in Oregon and has been selected by the OSU breeding program as a model for hazelnut genetic and genomic research. Here, we present a near complete, haplotype-resolved chromosome-level hazelnut genome assembly for "Jefferson". This new assembly is a significant improvement over a previously published genome draft. Analysis of genomic regions linked to EFB resistance and self-incompatibility confirmed haplotype splitting and identified new gene candidates that are essential for downstream molecular marker development, thereby facilitating breeding efforts.

Rare but diverse off-target and somatic mutations found in field and greenhouse grown trees expressing CRISPR/Cas9.

Introduction: CRISPR gene editing, while highly efficient in creating desired mutations, also has the potential to cause off-target mutations. This risk is especially high in clonally propagated plants, where editing reagents may remain in the genome for long periods of time or in perpetuity. We studied a diverse population of Populus and Eucalyptus trees that had CRISPR/Cas9-containing transgenes that targeted one or two types of floral development genes, homologs of LEAFY and AGAMOUS. Methods: Using a targeted sequence approach, we studied approximately 20,000 genomic sites with degenerate sequence homology of up to five base pairs relative to guide RNA (gRNA) target sites. We analyzed those sites in 96 individual tree samples that represented 37 independent insertion events containing one or multiples of six unique gRNAs. Results: We found low rates of off-target mutations, with rates of 1.2 × 10-9 in poplar and 3.1 × 10-10 in eucalypts, respectively, comparable to that expected due to sexual reproduction. The rates of mutation were highly idiosyncratic among sites and not predicted by sequence similarity to the target sites; a subset of two gRNAs showed off-target editing of four unique genomic sites with up to five mismatches relative to the true target sites, reaching fixation in some gene insertion events and clonal ramets. The location of off-target mutations relative to the PAM site were essentially identical to that seen with on-target CRISPR mutations. Discussion: The low rates observed support many other studies in plants that suggest that the rates of off-target mutagenesis from CRISPR/Cas9 transgenes are negligible; our study extends this conclusion to trees and other long-lived plants where CRISPR/Cas9 transgenes were present in the genome for approximately four years.