The mosaic oat genome gives insights into a uniquely healthy cereal crop

Oat (Avena sativa) is a member of the grass family Poaceae and is grown worldwide, ranking seventh in cereal production. Avena species exist as diploids, tetraploids and hexaploids, with considerable genetic diversity around the Mediterranean, Middle East, Canary Islands and Himalayas. Compared with other cereals, oat cultivation generally requires fewer insecticide, fungicide or fertilizer inputs. Whole-grain oats are a healthy source of antioxidants, polyunsaturated fatty acids, proteins and dietary fibre, particularly β-glucan, which contributes to reduced post-prandial glycaemic responses and cardiovascular risk. Unlike wheat, barley and rye that store high levels of gluten proteins in grain, oat and rice predominantly store globular proteins. Despite oat’s agronomic and nutritional value, the absence of a fully annotated reference genome has limited the resolution of its complex evolutionary history, genome architecture and functional gene dynamics. The aim of this study is to generate and analyze a chromosome-scale, fully annotated hexaploid oat reference genome, place it in the context of its progenitor species, dissect the mosaic chromosome architecture and subgenome interactions, and demonstrate its utility for trait mapping and for understanding storage protein biology relevant to human health.

Previous large-genome cereal projects (for example, wheat, barley and rye) established strategies for chromosome-scale assemblies and annotation, enabling advances in Triticeae research. Prior molecular marker studies in oat mapping and breeding populations provided evidence for frequent inter-subgenomic translocations and pseudo-linkage phenomena, implicating breeding barriers. Health-related literature has established β-glucan’s benefits for blood cholesterol and glycaemic control, and clinical assessments support the safety of oats in gluten-free diets. Work on cellulose synthase-like genes (such as CslF6) in barley and wheat has linked these genes to β-glucan biosynthesis. Comparative studies of cereal prolamins highlighted the high immunogenicity and extensive expansion of wheat α-gliadins relative to other cereals. Collectively, these studies motivated the need for a high-quality oat reference to resolve genome structure, validate and reinterpret prior mapping data, and to analyze gene families impacting human nutrition and allergenicity.

- Assembly and validation: Generated a chromosome-scale reference sequence for the spring oat cultivar Sang, comprising 21 pseudochromosomes (1A–7D) using a short-read strategy similar to those for wheat, barley and rye. Assembly integrity was assessed via Hi-C contact matrices and a consensus genetic map, and by comparison to an independent long-read assembly of hexaploid oat OT3098 (version 2). BUSCO v5.1.2 scores quantified completeness (genome: 98.7%). - Progenitor assemblies and subgenome assignment: Assembled pseudochromosomes for diploid Avena longiglumis (AA) and tetraploid Avena insularis (CCDD), presumed progenitors of the A and CD subgenomes, respectively. Phylogenomic analyses and synteny with barley, A. eriantha, A. longiglumis and A. insularis were used to assign and orient oat chromosomes to A, C and D subgenomes while preserving core region orientation. - Annotation: Used an automated pipeline assisted by RNA-seq, Iso-Seq, protein homology and ab initio prediction to identify protein-coding loci. Identified 80,608 high-confidence protein-coding genes (98.5% BUSCO) and 71,727 low-confidence loci. Evaluated transposable element content and composition across subgenomes; quantified tandem repeats and rDNA representation. - Genome architecture and rearrangements: Employed whole-genome alignments, subgenome-specific k-mers, and clustering of orthologous and homoeologous genes into syntenic blocks across four Avena species to detect and trace large-scale rearrangements and inter-subgenomic translocations. Reanalyzed historical mapping data to interpret pseudo-linkage and breeding implications. - Expression analyses: Defined 7,726 homoeologous gene triads (1:1:1 across A, C, D) and profiled expression across multiple tissues/stages to assess homoeologue expression bias and co-expression network module assignments. Assessed expression in triads located within translocated regions. - Gene family analyses: Catalogued cellulose synthase (GT2) and callose synthase (GT48) gene families; constructed phylogenies and evaluated expression dynamics during seed development. Compared copy number and expansions relative to other grasses. - Storage protein and proteogenomics: Identified genes encoding avenins, HMW-glutenin-like proteins and α-amylase/trypsin inhibitors (ATIs) and assessed genomic distribution. Analyzed Pfam domains, cysteine patterns, protein length/composition, and mapped known coeliac disease T cell epitopes. Performed LC–MS/MS discovery proteomics to detect storage proteins and corroborate expression timing. - Trait mapping (wax mutant): Conducted mapping-by-sequencing of the epicuticular wax mutant glossy1 using pooled sequencing and sliding-window allele frequency analysis (windows of 100 variants; total allelic depth ≥30). Anchored candidate contig via Hi-C to chromosome 1C, identified a candidate α/β-hydrolase gene (AVESA.00010b.r2.UnG1403470) orthologous to barley Cer-q, and validated with an independent mutant (glossy2). Assessed metabolite profiles (β-diketone) and scanning electron microscopy of cuticle. Analyzed expression of homoeologous gene clusters on 1C, 3A and 2C.

- Reference genome: Produced a high-quality, chromosome-scale hexaploid oat (cv. Sang) reference with 21 pseudochromosomes and 98.7% BUSCO completeness. Annotated 80,608 high-confidence protein-coding genes (98.5% BUSCO), with 83.5% supported by transcription. - Repeats and subgenomes: Transposable elements comprise 64% of the genome. The C subgenome is ~1 Gb larger than the A or D subgenomes, consistent with higher historical transposon activity (1.3× more full-length LTR-RTs, enriched TE-related Pfam domains, more tandem repeats and TE/low-confidence genes). Short-read assembly underrepresents tandem repeats and rDNA loci, with reduced gene density in peri/centromeric regions. - Mosaic architecture and translocations: Identified seven major rearrangements traced to eight inter-subgenomic translocation events among A, C and D, spanning 4.3% of the genome and ~7.9% of high-confidence genes. Oat subgenomes exhibit unbalanced gene counts; the C subgenome has ~12% fewer genes than A or D. Ancestral reconstruction indicates ≥226 Mb of gene-rich regions moved from C to A/D, accounting for the lower gene count without requiring post-hexaploidization gene loss. Reanalysis of mapping data revealed pseudo-linkage consistent with translocations, explaining breeding barriers and trait associations. - Meiosis gene context: The apparent absence of a TaZIP4-B2 orthologue (wheat Ph1-associated) may relate to oat’s mosaic architecture and difficulties with interploidy crosses/alien introgressions. - Expression balance: Among 7,726 ancestral triads, expression is largely balanced (84.1% balanced; 3.4% single-homoeologue dominant; 12.6% suppressed). Average subgenome contributions were similar (A 33.76%, D 33.53%, C 32.32%; Kruskal–Wallis P=0.054). C-subgenome homoeologues more frequently resided in divergent co-expression modules (χ2 P=2.085×10−6). Triads within translocated regions showed broadly similar patterns, with subtle shifts in suppression categories (χ2 P=0.019). - Cellulose synthase superfamily: Identified 134 GT2 cellulose synthase-related genes (CesA and Csl subfamilies) and 28 GT48 callose synthases. CesA and CslF genes showed highest expression in seed development; CslE and CslF (including the C-subgenome copy of CslF6) were upregulated late in seed development. No major family expansions versus other grasses, aside from some duplications (CesA, CslC, CslE, CslI), suggesting β-glucan traits are driven by allelic/transcriptional regulation rather than copy number. - Storage proteins and immunogenicity: Discovered 25 avenin genes, 6 HMW-glutenin-like genes and 61 ATI/prolamin-related genes; 135 globulin genes mapped mainly to A and D subgenomes with no storage protein loci on C. Oat lacks α- and ω-gliadins; avenins co-cluster with γ-gliadins, LMW glutenins and B-hordeins. Oat HMW-GS and avenins are shorter with fewer glutamine/proline-rich repeats; cysteine patterns and Pfam domains are conserved. Proteomics detected numerous globulins and corroborated expression timing: avenins increase from mid seed development; 11S globulins initiate earlier and are more abundant. Few oat avenins contain coeliac disease-associated T cell epitopes compared with wheat/barley. Together with low copy number of immunogenic genes and lower avenin proportion, findings support oat inclusion in gluten-free diets. - Trait mapping (epicuticular wax): Mapping-by-sequencing localized glossy1 to chromosome 1C and implicated AVESA.00010b.r2.UnG1403470 (α/β-hydrolase; lipase/carboxyltransferase) orthologous to barley Cer-q. The glossy1 mutation (P243S) lies near a known deleterious site (F219L) in barley CER-Q; glossy mutants lacked the β-diketone hentriacontane-14,16-dione and wax tubules. Homologous gene clusters to barley Cer-cqu were identified on oat 1C, 3A and 2C; genes in the 1C cluster (except SDR) showed 3–6× higher expression than 3A, with low expression on 2C. Results establish the oat Cer-q gene and provide a basis for manipulating epicuticular wax for stress adaptation.

The assembled and annotated hexaploid oat reference resolves long-standing uncertainties about oat genome structure, revealing a pronounced mosaic architecture shaped by inter-subgenomic translocations. This structural context explains pseudo-linkage and segregation anomalies observed in historical mapping studies and suggests mechanistic barriers to alien introgression and interploidy crosses, potentially linked to the absence of a ZIP4-B2 orthologue implicated in meiotic stabilization in wheat. Despite genome rearrangements, homoeologue expression remains largely balanced across subgenomes, indicating functional buffering and coordinated regulation, with nuanced divergence of C-subgenome expression modules and modest effects of translocated context. Functional analyses demonstrate that high β-glucan content is not due to expanded cellulose synthase gene families but likely to allelic and regulatory differences. The proteogenomic characterization of storage proteins underscores oat’s distinct profile relative to gluten-rich cereals, with fewer immunogenic sequences and different nitrogen storage strategies, supporting clinical observations of oat safety in gluten-free diets. The reference enabled efficient mapping-by-sequencing of a wax biosynthetic mutant, identifying the oat Cer-q orthologue and associated gene clusters, illustrating the resource’s power for trait dissection and targeted breeding.

This work delivers the first fully annotated, chromosome-scale reference genome for hexaploid oat alongside assemblies of close diploid and tetraploid relatives. It defines a mosaic genome architecture with multiple inter-subgenomic translocations, clarifies subgenome gene content differences, and shows largely balanced homoeologue expression. Comprehensive analyses of cellulose synthase-related genes and storage protein families provide mechanistic insight into β-glucan biosynthesis and the lower immunogenic potential of oats compared with other cereals, supporting their inclusion in gluten-free diets. A case study mapping the glossy wax mutant demonstrates the utility of the reference for rapid gene discovery and for manipulating adaptive traits. Looking forward, anchoring known QTL to this reference, leveraging the transcriptome atlas and co-expression networks, and applying genome editing and gene pyramiding will accelerate oat improvement. The resource also sets the stage for an Avena pan-genome and reanalysis of quantitative trait studies across diverse germplasm.

- Assembly constraints: The short-read-based assembly exhibits lower contiguity in highly repetitive regions, leading to under-representation of tandem repeats and ribosomal DNA loci, reduced gene density in centromeric/pericentromeric regions, and a set of unplaced genes. - Structural complexity: Extensive translocations and rearrangements complicate comparative analyses and breeding, contributing to pseudo-linkage and potential barriers to introgression. - Expression analyses: Homoeologue expression assessments rely on defined triads and available tissue/stage transcriptomes; context-specific biases or rare transcripts may be under-sampled. - Functional validation: While gene family assignments, epitope mapping and candidate loci (e.g., Cer-q) are strongly supported by comparative genomics, expression and proteomics, broader functional validation across diverse oat germplasm and environments remains to be completed.