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Key message We identified marker-trait associations for key faba bean agronomic traits and genomic signatures of selection within a global germplasm collection. Abstract Faba bean (Vicia faba L.) is a high-protein grain legume crop with great potential for sustainable protein production. However, little is known about the genetics underlying trait diversity. In this study, we used 21,345 high-quality SNP markers to genetically characterize 2678 faba bean genotypes. We performed genome-wide association studies of key agronomic traits using a seven-parent-MAGIC population and detected 238 significant marker-trait associations linked to 12 traits of agronomic importance. Sixty-five of these were stable across multiple environments. Using a non-redundant diversity panel of 685 accessions from 52 countries, we identified three subpopulations differentiated by geographical origin and 33 genomic regions subjected to strong diversifying selection between subpopulations. We found that SNP markers associated with the differentiation of northern and southern accessions explained a significant proportion of agronomic trait variance in the seven-parent-MAGIC population, suggesting that some of these traits were targets of selection during breeding. Our findings point to genomic regions associated with important agronomic traits and selection, facilitating faba bean genomics-based breeding.

Increasing the proportion of locally produced plant protein in currently meat-rich diets could substantially reduce greenhouse gas emissions and loss of biodiversity1. However, plant protein production is hampered by the lack of a cool-season legume equivalent to soybean in agronomic value2. Faba bean (Vicia faba L.) has a high yield potential and is well suited for cultivation in temperate regions, but genomic resources are scarce. Here, we report a high-quality chromosome-scale assembly of the faba bean genome and show that it has expanded to a massive 13 Gb in size through an imbalance between the rates of amplification and elimination of retrotransposons and satellite repeats. Genes and recombination events are evenly dispersed across chromosomes and the gene space is remarkably compact considering the genome size, although with substantial copy number variation driven by tandem duplication. Demonstrating practical application of the genome sequence, we develop a targeted genotyping assay and use high-resolution genome-wide association analysis to dissect the genetic basis of seed size and hilum colour. The resources presented constitute a genomics-based breeding platform for faba bean, enabling breeders and geneticists to accelerate the improvement of sustainable protein production across the Mediterranean, subtropical and northern temperate agroecological zones.

Many plant species have succeeded in colonizing a wide range of diverse climates through local adaptation, but the underlying molecular genetics remain obscure. We previously found that winter survival was a direct target of selection during colonization of Japan by the perennial legume Lotus japonicus and identified associated candidate genes. Here, we show that two of these, FERONIA-receptor like kinase (LjFER) and a S-receptor-like kinase gene (LjLecRK), are required for non-acclimated freezing tolerance and show haplotype-dependent cold-responsive expression. Our work suggests that recruiting a conserved growth regulator gene, FER, and a receptor-like kinase gene, LecRK, into the set of cold-responsive genes has contributed to freezing tolerance and local climate adaptation in L. japonicus, offering functional genetic insight into perennial herb evolution.

Higher plants encode four DICER-LIKE (DCL) enzymes responsible for the production of small non-coding RNAs which function in RNA interference (RNAi). Different RNAi pathways in plants effect transposon silencing, antiviral defense and endogenous gene regulation.DCL2acts genetically redundantly withDCL4to confer basal antiviral defense, but in other settings,DCL2has the opposite function ofDCL4, at least in formal genetic terms. For example, knockout ofDCL4causes growth defects that are suppressed by inactivation ofDCL2. Current models maintain that the biochemical basis of both of these effects is RNAi via DCL2-dependent small interfering RNAs (siRNAs). Here, we report that neither DCL2-mediated antiviral resistance nor growth defects can be explained by silencing effects of DCL2-dependent siRNAs. Both functions are defective in genetic backgrounds that maintain high levels of DCL2-dependent siRNAs, either through specific point mutations in DCL2 or simply by reducing DCL2 dosage in plants heterozygous fordcl2knockout alleles. Intriguingly, however, all functions of DCL2 depend on it having some level of catalytic activity. We discuss this requirement for catalytic activity, but not for the resulting siRNAs, in the light of recent findings that reveal a function of DCL2 in activation of innate immunity in response to cytoplasmic double-stranded RNA.

Legume-rhizobium signaling during establishment of symbiotic nitrogen fixation restricts rhizobium colonization to specific cells. A limited number of root hair cells allow infection threads to form, and only a fraction of the epidermal infection threads progress to cortical layers to establish functional nodules. Here we use single-cell analysis to define the epidermal and cortical cell populations that respond to and facilitate rhizobium infection. We then identify high-confidence nodulation gene candidates based on their specific expression in these populations, pinpointing genes stably associated with infection across genotypes and time points. We show that one of these, which we nameSYMRKL1, encodes a protein with an ectodomain predicted to be nearly identical to that of SYMRK and is required for normal infection thread formation. Our work disentangles cellular processes and transcriptional modules that were previously confounded due to lack of cellular resolution, providing a more detailed understanding of symbiotic interactions.

Increasing the proportion of locally produced plant protein in currently meat-rich diets could substantially reduce greenhouse gas emission and loss of biodiversity. However, plant protein production is hampered by the lack of a cool-season legume equivalent to soybean in agronomic value. Faba bean (Vicia faba L.) has a high yield potential and is well-suited for cultivation in temperate regions, but genomic resources are scarce. Here, we report a high-quality chromosome-scale assembly of the faba bean genome and show that it has grown to a massive 13 Gb in size through an imbalance between the rates of amplification and elimination of retrotransposons and satellite repeats. Genes and recombination events are evenly dispersed across chromosomes and the gene space is remarkably compact considering the genome size, though with significant copy number variation driven by tandem duplication. Demonstrating practical application of the genome sequence, we develop a targeted genotyping assay and use high-resolution genome-wide association (GWA) analysis to dissect the genetic basis of hilum colour. The resources presented constitute a genomics-based breeding platform for faba bean, enabling breeders and geneticists to accelerate improvement of sustainable protein production across Mediterranean, subtropical, and northern temperate agro-ecological zones.

Transposable elements (TEs) constitute a large proportion of genomes of multicellular eukaryotes including flowering plants. TEs are normally maintained in a silenced state and their transpositions rarely occur. Hybridization between distant species has been regarded as a "shock" that stimulates genome re-organization, including TE mobilization. However, whether crosses between genetically close parents that result in viable and fertile offspring can induce TE transpositions has remained obscure. Here, we investigated the activation of long terminal repeat (LTR) retrotransposons in three Lotus japonicus recombinant inbred line (RIL) populations. We found that at least six LTR retrotransposon families were activated and transposed in 78% of the investigated RILs. LORE1a, one of the transposed LTR retrotransposons, showed transgenerational epigenetic activation, indicating the long-term effects of epigenetic instability induced by hybridization. Our study highlights TE activation as unexpectedly common events in plant reproduction.

Faba bean (Vicia faba L.) is a high-protein grain legume crop with great potential for further cultivation. However, little is known about the genetics underlying trait diversity. In this study, we use 21,345 high-quality SNP markers to genetically characterise 2,678 faba bean genotypes. We perform genome-wide association studies of key agronomic traits using a Seven-parent-MAGIC population and detect 238 significant marker-trait associations linked to 12 traits of agronomic importance, with 65 of these being stable across multiple environments. Using a non-redundant diversity panel of 685 accessions from 52 countries, we identify 3 subpopulations differentiated by geographical origin and 33 genomic regions subject to strong diversifying selection between subpopulations. We find that SNP markers associated with the differentiation of northern and southern accessions were able to explain a significant proportion of agronomic trait variance in the Seven-parent-MAGIC population, suggesting that some of these traits have played an important role in breeding. Altogether, our findings point to genomic regions associated with important agronomic traits and selection in faba bean, which can be used for breeding purposes.Key MessageWe identified marker-trait associations for key faba bean agronomic traits and genomic signatures of selection within a global germplasm collection.

Many plant species have succeeded in colonizing a wide range of diverse climates through local adaptation, but the underlying molecular genetics remain obscure. We previously found that winter survival was a direct target of selection during colonization of Japan by the perennial legume Lotus japonicus and identified associated candidate genes. Here, we show that two of these, the FERONIA-receptor like kinase (LjFER) and a novel S-receptor-like kinase (LjSRK) are required for non-acclimated freezing tolerance and show haplotype-dependent cold-responsive expression. Our work demonstrates that recruiting a conserved growth regulator, FER, and a novel receptor-like kinase, SRK, into the set of cold-responsive genes has contributed to freezing tolerance and local climate adaptation in L. japonicus, offering new functional genetic insight into perennial herb evolution.

Biological interactions between plants and their root microbiomes are pivotal for plant growth. Even though the plant genotype [G], soil microbiome [C], and growth conditions (environment) [E] are core factors shaping the root microbiome, their relationships remain unclear. We disentangled the effects of G, C, E, and their interactions on the Lotus root microbiome and plant growth using a cross-inoculation approach that reconstructed the interactions between nine Lotus accessions and four soil microbiomes under two different environmental conditions. We found that a large proportion of the root microbiome composition was determined by C and E and that G-related (G, G × C, and G × E) effects were significant but small. In contrast, the interactions between G and C had a more pronounced effect on plant shoot growth than C alone. Our findings indicate that most microbiome variations controlled by C have little effect on the plant phenotype, whereas G × C interactions have more significant effects. Plant genotype-dependent interactions with soil microbes warrant more attention in efforts to optimize crop yield and resilience.

The Lotus Retrotransposon 1 (LORE1) is used for genome-wide mutagenesis of the model legume Lotus japonicus. Characterization of the LORE1 insertion sites in individual mutant lines is critical for development and use of the resource. Here we present guidelines for use of the LORE1 reverse genetics resource and provide detailed protocols for insertion site identification and validation. For high-throughput identification of insertions in up to 9,216 pooled lines, the FSTpoolit protocol takes advantage of Splinkerette adapters, molecular barcoding, 2D pooling, Illumina sequencing, and automated data analysis using the freely available FSTpoolit software. Complementing the high-throughput approach, we describe a simplified sequence-specific amplification polymorphism (SSAP) protocol well suited for quick identification of insertion sites in a limited number of lines. Both the FSTpoolit and simplified SSAP protocols are generally applicable to insertion site identification in any insertional mutagenesis setup.

Use of insertion mutants facilitates functional analysis of genes, but it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics in most plant species. The main challenge is developing efficient high-throughput procedures for both mutagenesis and identification of insertion sites. To date, only floral-dip T-DNA transformation of Arabidopsis has produced independent germinal insertions, thereby allowing generation of mutant populations from seeds of single plants. In addition, advances in insertion detection have been hampered by a lack of protocols, including software for automated data analysis, that take full advantage of high-throughput next-generation sequencing. We have addressed these challenges by developing the FSTpoolit protocol and software package, and here we demonstrate its efficacy by detecting 8935 LORE1 insertions in 3744 Lotus japonicus plants. The identified insertions show that the endogenous LORE1 retrotransposon is well suited for insertion mutagenesis due to homogenous gene targeting and exonic insertion preference. As LORE1 transposition occurs in the germline, harvesting seeds from a single founder line and cultivating progeny generates a complete mutant population. This ease of LORE1 mutagenesis, combined with the efficient FSTpoolit protocol, which exploits 2D pooling, Illumina sequencing and automated data analysis, allows highly cost-efficient development of a comprehensive reverse genetic resource.