François Belzile

Genome‐wide association studies (GWAS) are powerful statistical methods that detect associations between genotype and phenotype at genome scale. Despite their power, GWAS frequently fail to pinpoint the causal variant or the gene controlling a given trait in crop species. Assessing genetic variants other than single‐nucleotide polymorphisms (SNPs) could alleviate this problem. In this study, we tested the potential of structural variant (SV)‐ and k ‐mer‐based GWAS in soybean by applying these methods as well as conventional SNP/indel‐based GWAS to 13 traits. We assessed the performance of each GWAS approach based on loci for which the causal genes or variants were known from previous genetic studies. We found that k ‐mer‐based GWAS was the most versatile approach and the best at pinpointing causal variants or candidate genes. Moreover, k ‐mer‐based analyses identified promising candidate genes for loci related to pod color, pubescence form, and resistance to Phytophthora sojae . In our dataset, SV‐based GWAS did not add value compared to k ‐mer‐based GWAS and may not be worth the time and computational resources invested. Despite promising results, significant challenges remain regarding the downstream analysis of k ‐mer‐based GWAS. Notably, better methods are needed to associate significant k ‐mers with sequence variation. Our results suggest that coupling k ‐mer‐ and SNP/indel‐based GWAS is a powerful approach for discovering candidate genes in crop species.

The use of resistance genes in elite soybean cultivars is one of the most widely used methods to manage Phytophthora sojae . This method relies on effector‐triggered immunity, where a Resistant to P. sojae ( Rps ) gene product from the plant recognizes a specific effector from the pathogen, encoded by an avirulence ( Avr ) gene. Many Avr genes from P . sojae have been identified in the last decade, allowing a better exploitation of this type of resistance. The objective of the present study was to identify the Avr gene triggering immunity derived from the soybean resistance gene Rps8 . The analysis of a segregating F 2 progeny coupled with a genotyping‐by‐sequencing approach led to the identification of a putative Avr8 locus. The investigation of this locus using whole‐genome sequencing data from 31 isolates of P . sojae identified Avr3a as the likely candidate for Avr8 . Long‐read sequencing also revealed that P . sojae isolates can carry up to five copies of the Avr3a gene, compared to the four previously reported. Haplotype and transcriptional analyses showed that amino acid changes and absence of Avr3a transcripts from P. sojae isolates caused changes in virulence towards Rps8 . Functional analyses using CRISPR/Cas9 knockout and constitutive expression demonstrated that Rps8 interacted with Avr3a . We also showed that a specific allele of Avr3a is recognized by Rps3a but not Rps8 . While Rps3a and Rps8 have been previously described as closely linked, this is the first report of a clear distinction hitherto undefined between these two resistance genes.