Nanopore Adaptive sampling: A powerful approach to decipher the genetic diversity of the NLRome/resistome in melon - INRAE - Institut national de recherche pour l’agriculture, l’alimentation et l’environnement Access content directly
Conference Papers Year : 2023

Nanopore Adaptive sampling: A powerful approach to decipher the genetic diversity of the NLRome/resistome in melon

Abstract

Nucleotide-binding-site-leucine-rich-repeat (NLR) disease resistance genes encode the most important family of plant resistance proteins. Moreover, due to the dynamic evolutionary interactions between plants and pathogens, NLR genes are also one of the most variable gene families in plants. The characterization of the complete NLR-like resistance genes of a species (or NLRome) represents a major challenge for sustainable agriculture, as it would facilitate the creation of varieties with a very broad spectrum of resistance by maximizing the use of the NLRome. However, although many efforts have been made, the specific role of each NLR gene remains largely unknown in melon (Cucumis melo L.), especially in relation to quantitative resistances. This lack of information is mainly due to the complex structure and organization of these genes. They are frequently arranged in clusters that include a large number of transposable and repetitive elements. This fact, together with the repetitive intra-structure of many NLR genes (domain leucine-rich-repeat) makes them prone to major evolutionary structural changes like duplication or transposition. For these reasons, NLR clusters commonly present a high level of presence-absence (PAV) polymorphisms. This implies that reference genomes likely include only a fraction of the NLR gene collection of a species, making necessary the sequencing of a large number of genotypes and the construction of an NLRome for a complete characterization of the NLR genes of a species. These structural singularities of NLR genes make sequencing methods using short reads often ineffective to decipher them. To solve this problem, long reads sequencing methods such as Oxford Nanopore Technology may provide a valuable information. Concretely, Nanopore Adaptive Sampling (NAS) is presumed to be a good approach here, since the sequencing of the entire genome is not necessary. This approach reduces the quantity of information to manage, especially when working with a large number of genotypes, while increasing the coverage of the target regions compared to a standard sequencing. In addition, it is a cost and labor-effective solution compared to other targeted sequencing methods that require somewhat difficult and time-consuming probe design as well as highly stringent hybridization conditions. Our project pursues the evaluation and implementation of NAS for the sequencing and characterization of the NLRome of melon, a eudicot diploid plant species of high economic value, using ≈140 melon varieties. For that purpose, our final goal is to perform genetic association studies using the NLRome sequence variability of the ≈140 lines and the phenotypic variability against a group of selected pests and pathogens. In a first step, we demonstrated the performance (= increase of enrichment) of the NAS approach against WGS (whole genome sequencing) for the sequencing and assembly of the previously defined NLR clusters in a well-studied melon variety. Using the recently released kit 14/R10.4.1 flowcell, a great increase of enrichment of the target regions was observed in the NAS experiment compared to WGS, allowing better assemblies of the NLR clusters. We also assessed the transferability of our design to other melon varieties, in simplex- and multiplex-sequencing experiments. In the next steps of the project, we plan to increase the multiplexing of genotypes per run, as well as continue exploring the possibilities of this promising targeted sequencing approach.
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hal-04037782 , version 1 (20-03-2023)

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Attribution - NonCommercial - NoDerivatives

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  • HAL Id : hal-04037782 , version 1

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Javier Belinchon-Moreno. Nanopore Adaptive sampling: A powerful approach to decipher the genetic diversity of the NLRome/resistome in melon. Conference, talk, Oxford Nanopore Technologies, Mar 2023, Paris institut du cerveau (ICM), France. ⟨hal-04037782⟩
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