Biology subjects

Steffenson, B. J.

Publications and source records attributed to Steffenson, B. J..

15 recordsLinked to original sources

bioRxiv2026

Haplotype-phased genomes of the barley leaf rust pathogen reveal evidence of repeat element expansion and somatic hybridization

Barley leaf rust disease, caused by Puccinia hordei, leads to substantial yield losses and diminished malting quality of barley across temperate growing regions worldwide. To address the paucity of high-resolution genomic resources for this pathogen, we generated haplotype-phased, chromosome-scale assemblies for ten globally distributed isolates using PacBio HiFi and Hi-C sequencing. Phylogenomic analysis revealed seven distinct lineages of P. hordei, including evidence of nuclear exchange, with a shared nuclear haplotype detected between two US lineages. Nuclear genome sizes ranged from [~]140-147Mbp, with the exception of isolate 90ISR03 from Israel ([~]163Mbp), which also harbored a 6.2Mbp supernumerary scaffold in one nucleus exhibiting chromosomal characteristics. Consistent with its larger genome, P. hordei had a higher repeat content ([~]70%) than related cereal rust fungi, driven primarily by the proliferation of LTR retroelements and DNA transposons. Across the global pan-genome of 13 unique nuclear haplotypes, approximately one-third of all protein orthogroups were conserved across all isolates. Only 18% of predicted effector orthogroups were shared between all haplotypes, reflecting the highly dynamic and variable nature of the effector repertoire. The long-term propagation of clonal P. hordei lineages is apparent both within the US and globally, and nuclear exchange is important for generating novel diversity and virulence profiles. The high degree of genome plasticity is evident in extensive structural variation, including large-scale translocations and inversions as well as a putative accessory chromosome. These chromosome-level, haplotype-resolved genomes provide a foundational resource for exploring the evolution, diversity, and avirulence gene repertoire of P. hordei.

genomics↗

bioRxiv2025

Novel clonal lineages of wheat stem rust in the Southern Cone of South America

Puccinia graminis f. sp. tritici (Pgt) causes wheat stem rust, a devastating disease of cereals. Recent approaches to examine populations at a genomic level have provided valuable information on the genotypic diversity of Pgt populations in Africa, Europe and North America and evolutionary mechanisms underlying the emergence of new races. However, an in-depth characterisation of Pgt populations in South America has been lacking. To bridge this knowledge gap, 91 Pgt isolates were collected from Argentina and Uruguay in 2020 and 2021 and used to generate transcriptome and whole genome sequence data and pathotype information. Phylogenetic analyses revealed that this South American Pgt population includes three clonal lineages, two of which have not been detected elsewhere. The third lineage is globally dispersed, including isolates from Africa and the Middle East. The predominant lineage, unique to South America, encompassed 90% of the samples and showed related pathotypes differing by virulence on single resistance genes consistent with evolution by stepwise mutation within the clonal lineage. There was no evidence of sexual recombination giving rise to new genetic diversity in this population. These observations urge to routinely incorporate and compare genotypic data from South American Pgt isolates to surveillance data from other geographic regions.

evolutionary biology↗

bioRxiv2025

Haplotype-resolved genomes of diverse oat crown rust isolates reveal both global dispersion of long-lived clonal haplotypes and limited recombination between haplotypes.

Genetic diversity of pathogen populations plays an important role in host adaptation. While single high quality genome references are a valuable resource, a compilation of genome references from individuals increases the breadth of genetic variation within a species that is captured. Puccinia coronata f. sp. avenae (Pca), a fungal pathogen causing crown rust of oat, demonstrates rapid virulence evolution and adaptation to newly released cultivars. To broaden the geographic and temporal distribution of available Pca genomes, we generated nuclear haplotype-resolved genome references for ten isolates from Europe, Africa and the Middle East and compared these with existing references for USA and Australian isolates. Of the full collection of 52 haplotypes, 40 were unique. Importantly, the presence of a nearly identical haplotype in a UK isolate collected in 1984 and in USA isolates from 1990 and 2017 supports the existence of long-lived clonal haplotypes in the global population that have been exchanged between lineages. Taken together with the identification of infrequent recombination between haplotypes from geographically dispersed isolates, this evidence reflects a globally mobile population of Pca that is mostly comprised of persistent clonal lineages with some influence from rare recombination events. Analysis of the core and non-core proteome suggests that while the core proteome is enriched for predicted secreted and effector proteins, sequence and expression variation are most prevalent in non-core orthogroups. We anticipate that this expanded collection of haplotypes will facilitate the development of new surveillance technologies and identification of virulence loci.

genomics↗

bioRxiv2024

A haplotype-based evolutionary history of barley domestication

Barley is an old crop with a complex history. Its evolution and the molecular basis of domestication have been intensely studied. This research has ruled out a single origin of the crop and motivated a model of mosaic genomics ancestry. As more and better genome sequences have become available, this concept can be refined: where do the building blocks of the mosaic, that is, the haplotypes come from? Were all cultivated barleys created equal or did some wild barley populations contribute more to some domesticated lineages? To answer these and other questions, we pursue a haplotype-based approach to connect diversity and population structure in wild and domesticated barley. We sequence the genomes of 628 genebank accessions and 23 archaeological specimens. Using these data, we infer the spatiotemporal origins of haplotypes and map the contributions of different wild barley populations either during the initial phase of domestication or through later gene flow. Ancient DNA sequences corroborate our genome-wide analysis in present-day samples. Our results indicate that an early domesticated founder population formed in the Fertile Crescent during an extended period of pre-domestication cultivation. A practical implication of our findings is that the high haplotype differentiation between barley populations, which arose possibly without, and most likely on top of, selective forces, complicates the mapping of adaptive loci.

plant biology↗

bioRxiv2024

A wheat tandem kinase sensor activates an NLR helper to trigger immunity

Most plant resistance genes encode membrane-anchored receptor-like proteins or intracellular nucleotide-binding and leucine-rich repeat (NLR) receptors. In wheat and barley, tandem kinases (TKs) have emerged as a new class of resistance determinants. To understand the modus operandi of the wheat stem rust resistance protein Sr62TK, we identified two genetic interactors-- a host gene required for Sr62TK function and the corresponding fungal AvrSr62 effector. We discovered that the SR62 locus consists of a digenic module encoding Sr62TK and an NLR (Sr62NLR). AvrSr62 binds to the N-terminal kinase of Sr62TK. This triggers displacement of the C-terminal kinase allowing it to recruit Sr62NLR for activation of immune responses. Understanding the mechanism of this two-component resistance complex will help engineering and breeding for durable resistance.

plant biology↗

bioRxiv2024

Phenotypically wild barley shows evidence of introgression from cultivated barley

Plant conservation hinges on preserving biodiversity, which is crucial for long-term adaptation. Multiple studies have reported genetic evidence of crop-to-wild introgression in phenotypically wild accessions of wild barley (Hordeum vulgare ssp. spontaneum). We examined 318 Wild Barley Diversity Collection (WBDC) accessions for evidence of introgression from cultivated barley. Using SNP genotype and exome capture data, we performed local ancestry inference between the 318 WBDC accessions and cultivated barley to identify genomic regions with evidence of introgression. Using the genomic intervals for well-characterized genes involved in domestication and improvement, we examined the evidence for introgression at genomic regions potentially important for maintaining a wild phenotype. Our analysis revealed that nearly 16% (48 of 318) of WBDC accessions showed evidence of introgression from cultivated barley, and up to 16.6% of the genome has been introgressed. All accessions identified as introgressed based on domestication-related phenotypes show clear genetic evidence of introgression. The size of runs of identity by state and local ancestry inference suggests that most introgression did not occur recently. This study suggests a long history of genetic exchange between wild and cultivated barley, highlighting the potential for introgression to influence the genetic makeup and future adaptation of wild populations, with implications for plant conservation strategies.

evolutionary biology↗

bioRxiv2024

A high-resolution haplotype pangenome uncovers somatic hybridization, recombination and intercontinental migration in oat crown rust

Basidiomycetes like rust fungi have complex reproductive cycles and dikaryotic life stages which influence their population structure and evolution. Puccinia coronata f. sp. avenae (Pca), the causal agent of oat crown rust, is a pathogen of global economic importance. To investigate the genetic diversity of the species, as well as the role of mating type system and nuclear exchange (somatic hybridization) in host adaptation of Pca we acquired whole genome sequencing data of Taiwanese and Australian isolates, adding to existing data for USA and South African populations. An atlas of 30 chromosome-level, fully-phased nuclear haplotypes from six USA isolates and nine Australian isolates was generated to capture the genomic composition of key oat crown rust lineages. This study provides evidence of nuclear exchange and recombination of haplotypes in both the USA and Australian Pca populations as mechanisms for the introduction of genetic diversity. The limitations of assuming clonal evolutionary history from virulence phenotyping is demonstrated by the detection of either sexual or cryptic genetic recombination in the Pca Australian population. Overall, the characterization of intercontinental migration of Pca at the haplotype level provides further impetus for molecular monitoring of rust pathogen populations on a global scale.

genomics↗

bioRxiv2023

A barley MLA receptor is targeted by a non-ribosomal peptide effector of the necrotrophic spot blotch fungus for disease susceptibility

The evolutionary history of plant interactions with necrotrophic pathogens that feed on dying host cells and their virulence mechanisms remains fragmentary. We have isolated the barley gene Scs6, which is required for the necrotrophic fungus Bipolaris sorokiniana isolate ND90Pr to cause spot blotch disease. Scs6 is located at the disease resistance gene locus Mildew locus a (Mla) and encodes an intracellular nucleotide-binding leucine-rich repeat receptor (NLR). In transgenic barley, Scs6 is sufficient to confer susceptibility to ND90Pr in accessions naturally lacking the receptor, resulting in infection-associated host cell death. Expression of Scs6 in evolutionarily distant Nicotiana benthamiana reconstitutes a cell death response to an uncharacterized non-ribosomal peptide effector produced by ND90Pr-specific non-ribosomal peptide synthetases (NRPSs) encoded at the VHv1 virulence locus. Our data suggest that the heat-resistant effector directly activates the SCS6 receptor. Scs6 is an allelic variant of functionally diversified Mla resistance genes each conferring strain-specific immunity to barley powdery mildew isolates with a matching proteinaceous pathogen effector. Domain swaps between MLA and SCS6 NLRs and expression of the resulting hybrid proteins in N. benthamiana reveal that the SCS6 leucine-rich repeat domain is a specificity determinant for the NRPS-derived effector to activate the receptor. Scs6 evolved after the divergence of barley from wheat and is maintained in several wild barley populations with an incidence of 8%, suggesting a beneficial function for the host. Evolution of the bona fide immune receptor SCS6 targeted by the NRPS-derived effector was key for the emergence of strain-specific spot blotch disease in domesticated barley.

plant biology↗

bioRxiv2023

An RGB based deep neural network for high fidelity Fusarium head blight phenotyping in wheat

Fusarium head blight (FHB) in wheat is an economically important disease, which can cause yield losses exceeding 50% and the causal pathogen that infects spikes produces harmful mycotoxins. Breeding for host resistance remains the most effective disease control method; but time, labor, and human subjectivity during disease scoring limits selection advancements. In this study we describe an innovative, high-throughput phenotyping rover for capturing in-field RGB images and a deep neural network pipeline for wheat spike detection and FHB disease quantification. The image analysis pipeline successfully detects wheat spikes from images under variable field conditions, segments spikes and diseased tissue in the spikes, and quantifies disease severity as the region of intersection between spike and disease masks. Model inferences on an individual spike and plot basis were compared to human visual disease scoring in the field and on imagery for model evaluation. The precision and throughput of the model surpassed traditional field rating methods. The accuracy of FHB severity assessments of the model was equivalent to human disease annotations of images, however individual spike disease assessment was influenced by field location. The model was able to quantify FHB in images taken with different camera orientations in an unseen year, which demonstrates strong generalizability. This innovative pipeline represents a breakthrough in FHB phenotyping, offering precise and efficient assessment of FHB on both individual spikes and plot aggregates. The model is robust to different conditions and the potential to standardize disease evaluation methods across the community make it a valuable tool for studying and managing this economically significant fungal disease.

plant biology↗

bioRxiv2022

A chromosome-level, fully phased genome assembly of the oat crown rust fungus Puccinia coronata f. sp. avenae: a resource to enable comparative genomics in the cereal rusts

Advances in sequencing technologies as well as development of algorithms and workflows have made it possible to generate fully phased genome references for organisms with non-haploid genomes such as dikaryotic rust fungi. To enable discovery of pathogen effectors and further our understanding of virulence evolution, we generated a chromosome-scale assembly for each of the two nuclear genomes of the oat crown rust pathogen, Puccinia coronata f. sp. avenae (Pca). This resource complements two previous released partially phased genome references of Pca, which display virulence traits absent in the isolate of historic race 203 (isolate Pca203) which was selected for this genome project. A fully phased, chromosome-level reference for Pca203 was generated using PacBio reads and Hi-C data and a recently developed pipeline named NuclearPhaser for phase assignment of contigs and phase switch correction. With 18 chromosomes in each haplotype and a total size of 208.10 Mbp, Pca203 has the same number of chromosomes as other cereal rust fungi such as Puccinia graminis f. sp. tritici and Puccinia triticina, the causal agents of wheat stem rust and wheat leaf rust, respectively. The Pca203 reference marks the third fully-phased chromosome-level assembly of a cereal rust to date. Here, we demonstrate that the chromosomes of these three Puccinia species are syntenous and that chromosomal size variations are primarily due to differences in repeat element content.

genomics↗

bioRxiv2021

Identification of candidate susceptibility genes to Puccinia graminis f. sp. tritici in wheat

Wheat stem rust disease caused by Puccinia graminis f. sp. tritici (Pgt) is a global threat to wheat production. Fast evolving populations of Pgt limit the efficacy of plant genetic resistance and constrain disease management strategies. Understanding molecular mechanisms that lead to rust infection and disease susceptibility could deliver novel strategies to deploy crop resistance through genetic loss of disease susceptibility. We used comparative transcriptome-based and orthology-guided approaches to characterize gene expression changes associated with Pgt infection in susceptible and resistant Triticum aestivum genotypes as well as the non-host Brachypodium distachyon. We targeted our analysis to genes with differential expression in T. aestivum and genes suppressed or not affected in B. distachyon and report several processes potentially linked to susceptibility to Pgt, such as cell death suppression and impairment of photosynthesis. We complemented our approach with a gene co-expression network analysis to identify wheat targets to deliver resistance to Pgt through removal or modification of putative susceptibility genes.

plant biology↗

bioRxiv2020

Ancient BED-domain-containing immune receptor from wild barley confers widely effective resistance to leaf rust

Leaf rust, caused by Puccinia hordei is a devastating fungal disease affecting barley (Hordeum vulgare subsp. vulgare) production globally. Race-specific resistance (R) genes have been deployed widely; however, their durability is often compromised due to the rapid emergence of virulent P. hordei races, prompting the search for new sources of broad-spectrum resistance. Here we report on the cloning of Rph15, a broadly effective resistance gene derived from the wild progenitor Hordeum vulgare subsp. spontaneum. We demonstrate using introgression mapping, mutation and complementation that Rph15 encodes a coiled-coil nucleotide-binding leucine-rich repeat (NLR) protein with an integrated Zinc-finger BED (ZF-BED) domain. The allelic variation at the Rph15 locus was assessed using barley exome capture data that traced its origin to the western region of the Fertile Crescent bordering Jordan and Israel. To unravel the genetic relationship of two other leaf rust resistance genes (Rph14 and Rph16) mapped at similar locus on chromosome 2H, we re-sequenced the Rph15 gene from the near-isogenic line for Rph15 (Bowman+Rph15) and the two donor accessions of Rph14 (PI 584760) and Rph16, (PI 405292, Hs 680). Both whole genome and Sanger sequencing confirmed that Hs 680 carried Rph15, while Rph14 in PI 584760 was an independent locus. A perfect diagnostic KASP marker was developed and validated to permit efficient introduction of Rph15 into cultivated barley.

plant biology↗

bioRxiv2018

RNA-seq reveals few differences in resistant and susceptible responses of barley to infection by the spot blotch pathogen Bipolaris sorokiniana

Spot blotch, caused by Bipolaris sorokiniana (Sacc.) Shoem., is an economically important disease affecting barley (Hordeum vulgare L.). The disease has largely been controlled in the Upper Midwest region of the USA through a suite of quantitative trait loci (QTL) termed the Midwest Six-rowed Durable Resistance Haplotype (MSDRH). These QTL have been bred into all six-rowed Midwest barley cultivars, including the widely used cultivar Morex. We identified a gamma ray- induced Morex mutant (MUT) that exhibits spot blotch susceptibility at the seedling stage. This mutant also spontaneously develops extremely large necrotic lesions in the absence of the pathogen at the adult plant stage. Spot blotch susceptibility at the seedling stage and necrotic lesion formation at the adult plant stage are highly correlated. To start dissecting the molecular responses underlying the observed symptoms at the seedling stage, we conducted a time course RNA-seq experiment comparing the wild type (WT) and the mutant (MUT) Morex at 12, 24 and 36 h after B. sorokiniana inoculation. Mock-inoculated controls were also included. A total of 10,772 and 11,530 genes were differentially expressed between treatments for WT and MUT genotypes, respectively, while 277 and 195 genes were differentially expressed between fungal and mock-inoculated genotypes, respectively. The transcript expression profiles of WT and MUT Morex samples were similar for most treatments. Two genes whose expression was putatively knocked out in the MUT were identified: HORVU3Hr1G019920 (glycine-rich protein) and HORVU5Hr1G120850 (Long- chain-fatty-acid--CoA ligase 1). The latter appears to be genetically intact, but not expressed. Collectively, these data suggest that MUT susceptibility to B. sorokiniana is a result of minor, rather than major, differences in the defense responses.

genetics↗

bioRxiv2018

The wheat Sr22, Sr33, Sr35 and Sr45 genes confer resistance against stem rust in barley

In the last 20 years, stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), has re-emerged as a major threat to wheat and barley cultivation in Africa and Europe. In contrast to wheat with 82 designated stem rust (Sr) resistance genes, barleys genetic variation for stem rust resistance is very narrow with only seven resistance genes genetically identified. Of these, only one locus consisting of two genes is effective against Ug99, a strain of Pgt which emerged in Uganda in 1999 and has since spread to much of East Africa and parts of the Middle East. The objective of this study was to assess the functionality, in barley, of cloned wheat Sr genes effective against Ug99. Sr22, Sr33, Sr35 and Sr45 were transformed into barley cv. Golden Promise using Agrobacterium-mediated transformation. All four genes were found to confer effective stem rust resistance. The barley transgenics remained susceptible to the barley leaf rust pathogen Puccinia hordei, indicating that the resistance conferred by these wheat Sr genes was specific for Pgt. Cloned Sr genes from wheat are therefore a potential source of resistance against wheat stem rust in barley.

plant biology↗

bioRxiv2018

Resistance gene discovery and cloning by sequence capture and association genetics

Genetic resistance is the most economic and environmentally sustainable approach for crop disease protection. Disease resistance (R) genes from wild relatives are a valuable resource for breeding resistant crops. However, introgression of R genes into crops is a lengthy process often associated with co-integration of deleterious linked genes1, 2 and pathogens can rapidly evolve to overcome R genes when deployed singly3. Introducing multiple cloned R genes into crops as a stack would avoid linkage drag and delay emergence of resistance-breaking pathogen races4. However, current R gene cloning methods require segregating or mutant progenies5-10, which are difficult to generate for many wild relatives due to poor agronomic traits. We exploited natural pan-genome variation in a wild diploid wheat by combining association genetics with R gene enrichment sequencing (AgRenSeq) to clone four stem rust resistance genes in <6 months. RenSeq combined with diversity panels is therefore a major advance in isolating R genes for engineering broad-spectrum resistance in crops.

genomics↗