Modern breeding has created significant evolutionary bottlenecks in most major crops, but many specialty crops retain considerable genetic diversity. This is particularly true for long-lived perennial crops, which are often unsuitable for accelerated breeding. Pecan (*Carya illinoinensis*) is a specialty crop cultivated worldwide. It co-evolved with various pests and pathogens, including *Phylloxera* species and the scab-causing fungus (*Venturia effusa*), which significantly impact yield. Although resistant cultivars exist, modern pecan breeding has yielded only modest improvements, partly due to the long generation times involved. Contemporary pecan breeding primarily uses stocks from early 20th-century crosses of wild trees. While progress has been made in selecting for larger nut size, improved quality, and stress tolerance, molecular markers for early trait assaying would significantly accelerate this process. The outbred and highly diverse nature of pecan complicates molecular breeding because crucial genes for selection might be absent in many genotypes. This necessitates a shift towards using multiple outbred genomes rather than a single inbred reference.

The authors cite previous research highlighting the genetic bottlenecks in major crops and the challenges of breeding long-lived perennial crops. They reference studies on pecan pests and pathogens, including *Phylloxera* and *Venturia effusa*. Existing literature on pecan breeding programs and the limited success in improving traits through traditional methods is also reviewed. The complexities of breeding outbred and diverse species like pecan, and the need for a multi-genome approach, are discussed based on existing literature.

This study generated de novo genome assemblies and annotations for four outbred pecan genotypes: 'Pawnee', 'Lakota', 'Elliott', and 'Oaxaca'. 'Pawnee' was assembled using PacBio HiFi reads, achieving a chromosome-scale assembly of both haplotypes. The other three were assembled using a combination of PacBio and Illumina data. Synteny and Hi-C data were used to order and orient contigs. Each genome was annotated using homology-based and RNA-seq supported methods. A pan-genome annotation was constructed to identify presence-absence variation (PAV) among the four genomes. The synonymous and non-synonymous substitution rates were calculated for single-copy orthologs to assess evolutionary conservation. Admixture analysis was performed to identify genomic introgressions from related *Carya* species. RNA-seq was used to analyze the gene expression response of the 'Desirable' cultivar to *V. effusa* inoculation. Finally, a quantitative trait locus (QTL) mapping approach was used on a ‘Lakota’ x ‘Oaxaca’ F1 population to identify loci associated with phylloxera resistance.

The study generated four high-quality pecan genome assemblies, with 'Pawnee' exhibiting the most contiguous assembly. The pan-genome analysis revealed substantial PAV and interspecific genomic introgressions, including a large block from bitternut hickory in the 'Lakota' pedigree. Allergen protein sequences were highly conserved across the genomes. The analysis of introgressions identified candidate genes for disease resistance in regions with non-pecan ancestry. Differential gene expression analysis in 'Desirable' upon *V. effusa* inoculation revealed significant changes in genes related to wounding and chitin responses. QTL mapping in the ‘Lakota’ x ‘Oaxaca’ F1 population identified a significant QTL on chromosome 16 associated with phylloxera resistance, with a focus on presence-absence variation in LRR genes in 'Lakota'.

The findings highlight the importance of using multiple outbred genomes for functional genomics in diverse crop species. The identified introgressions and PAVs provide valuable resources for pecan breeding, particularly for disease resistance. The QTL analysis demonstrates the utility of this approach in identifying candidate genes for pest resistance. This multi-genome approach is particularly relevant for outbred species with complex evolutionary histories and significant gene content variation. The results suggest that introgressions have played a significant role in pecan adaptation and provide potential targets for marker-assisted selection.

This research provides high-quality genome assemblies and annotations for four diverse pecan genotypes, creating a significant resource for pecan breeding. The pan-genome approach identified introgressions and PAVs as sources of adaptive variation. The study successfully applied QTL mapping to identify a major QTL related to phylloxera resistance. Future research could focus on functional validation of candidate genes and the development of molecular markers for marker-assisted selection.

The study focused on a limited number of genotypes, potentially not fully capturing the overall pecan genetic diversity. The QTL analysis was performed on a single population, limiting the generalizability of results. The RNA-seq analysis was conducted only at a single time point, providing a snapshot of the early response to pathogen inoculation rather than a more comprehensive understanding of the dynamic interactions.