François Belzile

Significant yield losses in soybean production are imputable to Phytophthora root and stem rot (PRR) caused by Phytophthora sojae . Resistance to PRR relies on resistance genes ( Rps ) that recognize key effectors from the pathogen, which are encoded by avirulence genes ( Avr ). Key Avr haplotypes can be monitored in natural P. sojae populations and exploited to predict which resistance gene can be deployed to provide protection to the soybean crop. In this study, we reassessed the identity of the avirulence gene whose protein product is recognized by Rps6 . Following extensive soil sampling and single‐spore isolation of a large population of P. sojae , we found two salient isolates carrying a rare recombination between the closely linked effectors Avr3c and Avr4/6 . Using a PCR assay and molecular markers, we showed that only allelic variations at the Avr3c locus were in perfect association with the phenotypes of the isolates on Rps3c , Rps4 and Rps6 resistance. Consistent with these findings, a soybean protoplast cell‐death assay further showed that only Avr3c , and not Avr4/6 , triggered Rps6 ‐dependent recognition. Furthermore, whole‐genome resequencing and de novo assembly of the two isolates revealed the full extent of this genomic rearrangement. Together, these results establish Avr3c as the bona fide avirulence gene corresponding to Rps6 and offer a more reliable target for the pathotyping of P. sojae , which ultimately leads to a better deployment of resistant soybean cultivars.

Effective management of Phytophthora root rot in soybean is compromised by the rapid loss of efficacy of the most widely deployed resistance genes to Phyto

Nitrogen fixation in soybeans, facilitated by symbiotic interactions with rhizobia, is a cornerstone of sustainable agriculture, reducing reliance on synthetic fertilizers. However, the efficiency of symbiotic nitrogen fixation (SNF) varies due to natural genetic variation in SNF‐related genes. Our study underscores the pivotal role of the GmNFR5α gene not only in nodulation but also in root hair development, which is crucial for effective nutrient uptake and plant yield. Through detailed genetic analyses and clustered regularly interspaced short palindromic repeats (CRISPR)‐based manipulations, we identified and characterized multiple knockout mutants, notably GmNFR5α ‐KO and combined GmNFR5α + GmROP6 ‐KO, which exhibited significant reductions in root hair density and nodulation. These phenotypic changes correspond with the downregulation of key root hair development genes such as TTG , RHD1 , RHD2 , and KJK , establishing a clear link between GmNFR5α function and root hair formation. The potential of leveraging these genetic insights to improve nitrogen fixation in legumes and introduce SNF capabilities into cereal crops could revolutionize crop fertilization strategies, offering a sustainable solution to global agricultural challenges.

Significant yield losses in soybean production are imputable to Phytophthora root and stem rot (PRR) caused by Phytophthora sojae . Soybean resistance to the pathogen depends on the presence of resistance genes ( Rps ) that recognize key effectors from P. sojae , which are encoded by avirulence genes ( Avr ). A unique molecular signature associated with these genes allows the prediction of the outcome of infection with great accuracy making the interaction Rps-Avr central to reduce disease incidence. In this study, we reassessed the identity of the avirulence gene whose protein product is recognized by Rps6 . Following extensive soil sampling and single-spore isolation of a large population of P. sojae , we found two salient isolates carrying a rare recombination between two effectors, Avr3c and Avr4/6 . Using a PCR assay and molecular markers, we showed that only alleles at the Avr3c locus were in perfect association with the phenotypes of the isolates. Furthermore, whole-genome resequencing and de novo assembly of the two isolates revealed the full extent of this genomic rearrangement. These results bring to light an unsuspected connection between Avr3c and Rps6 and offer a more reliable target for the pathotyping of P. sojae , which ultimately leads to a better use of resistant soybean material.

To exploit allelic variation in Hordeum vulgare subsp. spontaneum, the Wild Barley Diversity Collection was subjected to paired-end Illumina sequencing at ∼9 × depth and evaluated for several agronomic traits. We discovered 240.2 million single nucleotide polymorphisms (SNPs) after alignment to the Morex V3 assembly and 24.4 million short (1 to 50 bp) insertions and deletions. A genome-wide association study of lemma color identified one marker-trait association (MTA) on chromosome 1H close to HvBlp, the cloned gene controlling black lemma. Four MTAs were identified for seedling stem rust resistance, including 2 novel loci on chromosomes 1H and 6H and one co-locating to the complex RMRL1-RMRL2 locus on 5H. The whole-genome sequence data described herein will facilitate the identification and utilization of new alleles for barley improvement.

Efficient and consistent DNA extraction is crucial for genotyping but often hindered by the limitations of traditional manual processes, which are labour-intensive, error-prone, and costly. We introduce a semi-automated, robotic-assisted DNA extraction (RoboCTAB) tailored for large-scale plant genotyping, leveraging advanced yet affordable liquid-handling robotic systems. The protocol/workflow integrates a CTAB extraction protocol specifically adapted for a robotic liquid-handling system, making it compatible with high-throughput genotyping techniques such as SNP genotyping and sequencing. Various plant parts (leaves, roots, manual seed chip) were explored as the source material for DNA extractions, with the aim of identifying the tissue best suited for collection on a large scale. Young roots (radicle) proved the easiest to harvest at scale, while the harvest of leaves and seed chips were more laborious and error-prone. DNA yield and quality from both leaves and roots (but not seed chips) were similar and sufficient for downstream analysis. Interestingly, root tissue could still be extracted from imbibed seeds, even if the seeds failed to germinate, thus proving useful for DNA extraction. Cost analysis indicates significant savings in labour costs, highlighting the approach’s suitability for large-scale projects. Quality assessments demonstrate that the robotic process yields high-quality DNA, maintaining integrity for downstream applications. This semi-automated DNA extraction system represents a scalable, reliable solution for large-scale genotyping that is accessible to many users who cannot implement highly sophisticated and costly systems as are known to exist in large multinational seed companies. RoboCTAB, a low-cost, optimized method for high-throughput DNA extraction, minimizes the risk of cross-contamination. RoboCTAB is capable of processing up to four 96-well plates (384 samples) simultaneously in a single run, improving cost-efficiency and providing seamless integration with laboratory workflows, potentially setting new standards for efficiency and quality in DNA processing and sequencing at scale.

Exploitation of disease resistance genes in soybean ( Glycine max (L.) Merr.), as an effective method for management of Phytophthora sojae (Kauf. & Gerd.), is on the verge of an impasse. Few of the known resistance genes are commercially exploited, and even fewer have been precisely identified. Therefore, little is known about the identities or relationships between those genes, a hindrance preventing optimal introgression of new sources of resistance into elite soybean lines. In this study, we have applied state‐of‐the‐art nucleotide‐binding and leucine‐rich repeat gene capture (RenSeq) using a set of approximately 80,000 unique baits on near‐isogenic lines, whole‐genome resequencing, and bulked segregant analysis to uncover a resistance gene that has remained elusive for 40 years. This work highlights the reassessment of the Rps3b locus from Chr13 to Chr7 and the description of two alleles, from Turkish and Chinese landraces, of a sole candidate gene. We have identified Rps3b in four, fully resequenced, genetic backgrounds, including the original PI from 1985, in which the resistance gene was originally described. Specificity of the resistant alleles was achieved through phenotypic characterization with field isolates carrying virulent and avirulent forms of the corresponding effector, Avr3b . Surprisingly, these alleles showed extremely high synteny and sequence identity with Rps11 consistent with allelism, and conferred a resistance phenotype indistinguishable from that of the recently cloned Rps11 . These results offer new sources of resistance for breeders that are effective against the current P. sojae pathotypes in the field.

Typically, root system architecture (RSA) is not visible, and realistically, high-throughput methods for RSA trait phenotyping should capture key features of developing root systems in solid substrates in 3D. In a published 2-D study using thin rhizoboxes, vermiculite as a growing medium, and photography for imaging, triplicates of 137 soybean cultivars were phenotyped for their RSA. In the transition to 3-D work using X-ray computed tomography (CT) scanning and mineral soil, two research questions are addressed: (1) how different is the soybean RSA characterization between the two phenotyping systems; and (2) is a direct comparison of the results reliable? Prior to a full-scale study in 3D, we grew, in pots filled with sand, triplicates of the Casino and OAC Woodstock cultivars that had shown the most contrasting RSAs in the 2-D study, and CT scanned them at the V1 vegetative stage of development of the shoots. Differences between soybean cultivars in RSA traits, such as total root length and fractal dimension (FD), observed in 2D, can change in 3D. In particular, in 2D, the mean FD values are 1.48 ± 0.16 (OAC Woodstock) vs. 1.31 ± 0.16 (Casino), whereas in 3D, they are 1.52 ± 0.14 (OAC Woodstock) vs. 1.24 ± 0.13 (Casino), indicating variations in RSA complexity.

To exploit allelic variation in Hordeum vulgare subsp. spontaneum , the Wild Barley Diversity Collection was evaluated for several agronomic traits and subjected to paired-end Illumina sequencing at ∼9X depth, generating 109.5 million single nucleotide polymorphisms after alignment to the Morex V3 assembly. A genome-wide association study of lemma color identified one marker-trait association (MTA) on chromosome 1HL close to HvBlp , the cloned gene controlling black lemma. Four MTAs were identified for stem rust resistance: one co-locating to the complex RMRL1-RMRL2 locus on 5HL, and three novel loci on 1HS, 1HL, and 5HL. Six MTAs for days to heading (DTH) on vernalized plants were identified on all chromosomes except 1H and 6H. Two MTAs for DTH on non-vernalized plants were identified on chromosomes 1HL and 2HS. All MTAs for DTH were novel. The whole genome sequence data described herein will facilitate the identification and utilization of new alleles for barley improvement.

Soybean is a crucial crop for the Brazilian economy, but it faces challenges from the biotrophic fungus Phakopsora pachyrhizi, which causes Asian Soybean Rust (ASR). In this study, we aimed to identify SNPs associated with resistance within the Rpp1 locus, which is effective against Brazilian ASR populations. We employed GWAS and re-sequencing analyzes to pinpoint SNP markers capable of differentiating between soybean accessions harboring the Rpp1, Rpp1-b and other alternative alleles in the Rpp1 locus and from susceptible soybean cultivars. Seven SNP markers were found to be associated with ASR resistance through GWAS, with three of them defining haplotypes that efficiently distinguished the accessions based on their ASR resistance and source of the Rpp gene. These haplotypes were subsequently validated using a bi-parental population and a diverse set of Rpp sources, demonstrating that the GWAS markers co-segregate with ASR resistance. We then examined the presence of these haplotypes in a diverse set of soybean genomes worldwide, finding a few new potential sources of Rpp1/Rpp1-b. Further genomic sequence analysis revealed nucleotide differences within the genes present in the Rpp1 locus, including the ULP1-NBS-LRR genes, which are potential R gene candidates. These results provide valuable insights into ASR resistance in soybean, thus helping the development of resistant soybean varieties through genetic breeding programs.