[Thesis defence] 17/12/2025 – Javier Belinchon Moreno: «Genetic and functional diversity of the NLRome/resistome in melons» (UR GAFL)

Mr Javier BELINCHON MORENO will publicly defend his thesis entitled: «Genetic and functional diversity of the NLRome/resistome in melons», supervised by Ms Nathalie BOISSOT and Ms Patricia FAIVRE RAMPANT, on Wednesday 17 December 2025.

Laboratory

UR GAFL

Composition of the jury

Ms Nathalie BOISSOT	INRAE	Thesis supervisor
Ms Camille RUSTENHOLZ	INRAE	Rapporteur
Mr François SABOT	IRD	Rapporteur
Mrs Faivre-Rampant PATRICIA	INRAE	Thesis co-director
Mr Marc OLIVER	Syngenta	Examiner
Mr Michel PITRAT	INRAE	Examiner

Summary

Nucleotide-binding leucine-rich repeat (NLR) genes are one of the main families of resistance genes in plants and play a central role in Effector-Triggered Immunity. These NLRs are frequently organised into gene clusters with numerous variations in copy number and presence-absence polymorphisms. This organisation influences the modes of NLR evolution: it promotes their diversification via unequal recombination, gene conversion or other sources of variation, while maintaining functional stability when necessary. In melon (Cucumis melo) and more broadly within Cucurbitaceae, the number of NLRs is relatively low compared to other species of similar genomic size, raising the question of how these crops cope with high pathogen pressure with such a small repertoire. However, the intraspecific diversity of NLRs remains poorly characterised in melon, mainly due to the difficulties in analysing these complex loci using short-read sequencing. We applied the Nanopore Adaptive Sampling (NAS) targeted sequencing method to selectively enrich NLR regions («NLRome») in 143 melon accessions representing a wide botanical and geographical diversity. This selective sequencing allowed for good enrichment and accurate reconstruction of the targeted regions, making it possible to construct a pan-NLRome. Annotation of NLRs revealed significant variation in cluster architecture, presence-absence profiles, and NLR allelic content, with unsaturated allelic diversity curves highlighting the limitations of single-reference-based approaches. Indeed, diversity estimates suggested that nearly half of the allelic diversity of NLRs remains to be discovered after the assembly of 143 NLRomes. Using this dataset, we implemented NLRome-targeting GWAS based on k-mers as well as GWAS using a pan-NLRome graph. These approaches provided higher resolution than conventional SNP-based GWAS, allowing us to locate with high precision experimentally validated resistance genes, such as Fom-1 and Fom-2 against fusarium wilt. They also identified new NLR candidates as well as non-NLR genes located within NLR clusters. The availability of multiple NLRome assemblies also made it possible to discriminate between resistant and susceptible haplotypes within the diversity panel. We extended these approaches to dissect the genetic architecture of resistance to the CUC1 clone of Aphis gossypii, which is emerging in Europe. Genome-wide SNP-based GWAS combined with genetic mapping identified loci of quantitative traits acting at different stages of the plant-aphid interaction. GWAS targeting the NLRome provided allelic resolution of a QTL for resistance to colonisation on chromosome 5, corresponding to the well-known Vat locus. Functional characterisation of 20 Vat homologues with four R65aa motifs, likely involved in aphid resistance, revealed distinct response profiles to five A. gossypii clones, effectively limiting viral transmission in a clone-specific manner. Overall, our results provide a high-resolution view of NLR diversity in melon, revealing multiple quantitative resistance determinants that would remain undetectable in a single-reference paradigm. These resources and analytical frameworks pave the way for precise dissection of the genetic architecture of resistance to multiple biotic stresses, while supporting the strategic deployment of identified resistances in breeding programmes.

Keywords : Cucumis melo, NLR, targeted assembly, pan-genomic, quantitative genetics, biotic resistance