Intra-specific NLR allelic diversity and genomic landscape for plant resistance association studies

The identification of nucleotide-binding domain leucine-rich repeat receptor genes (NLRs) across multiple accessions is essential to capture their genetic diversity within a plant species. Using Nanopore adaptive sampling and de novo assembly, we accurately reconstructed the NLR regions across 143 Cucumis melo accessions representing diverse botanical groups and geographical origins. NLR annotation evidenced diverse cluster architectures and unexpected variation in NLR content across accessions, leading to unsaturated allelic diversity curves. Using this diversity, we further proposed pan-NLRome graph- and k-mer-based genome-wide association studies (GWAS), which, using Fusarium wilt races 1 and 2 severity data, accurately identified Fom-1, Fom-2, and novel non-NLR candidates. Furthermore, we extended these approaches for the identification of a candidate gene for flaccid necrosis caused by zucchini yellow mosaic virus. Our study offers a comprehensive view of NLR diversity in melon, overcoming the limitations of single-reference analyses, and supporting future efforts in NLR-focused GWAS and resistance breeding.