Genome assembly of Medicago truncatula accession SA27063 provides insight into spring black stem and leaf spot disease resistance.

Medicago truncatula, model legume and alfalfa relative, has served as an essential resource for advancing our understanding of legume physiology, functional genetics, and crop improvement traits. Necrotrophic fungus, Ascochyta medicaginicola, the causal agent of spring black stem (SBS) and leaf spot is a devasting foliar disease of alfalfa affecting stand survival, yield, and forage quality. Host resistance to SBS disease is poorly understood, and control methods rely on cultural practices. Resistance has been observed in M. truncatula accession SA27063 (HM078) with two recessively inherited quantitative-trait loci (QTL), rnpm1 and rnpm2, previously reported. To shed light on host resistance, we carried out a de novo genome assembly of HM078. The genome, referred to as MtHM078 v1.0, is comprised of 23 contigs totaling 481.19 Mbp. Notably, this assembly contains a substantial amount of novel centromere-related repeat sequences due to deep long-read sequencing. Genome annotation resulted in 98.4% of BUSCO fabales proteins being complete. The assembly enabled sequence-level analysis of rnpm1 and rnpm2 for gene content, synteny, and structural variation between SBS-resistant accession SA27063 (HM078) and SBS-susceptible accession A17 (HM101). Fourteen candidate genes were identified, and some have been implicated in resistance to necrotrophic fungi. Especially interesting candidates include loss-of-function events in HM078 because they fit the inverse gene-for-gene model, where resistance is recessively inherited. In rnpm1, these include a loss-of-function in a disease resistance gene due to a premature stop codon, and a 10.85 kbp retrotransposon-like insertion disrupting a ubiquitin conjugating E2. In rnpm2, we identified a frameshift mutation causing a loss-of-function in a glycosidase, as well as a missense and frameshift mutation altering an F-box family protein. This study generated a high-quality genome of HM078 and has identified promising candidates, that once validated, could be further studied in alfalfa to enhance disease resistance.

A view of the pan-genome of domesticated Cowpea (Vigna unguiculata [L.] Walp.).

Cowpea, Vigna unguiculata L. Walp., is a diploid warm-season legume of critical importance as both food and fodder in sub-Saharan Africa. This species is also grown in Northern Africa, Europe, Latin America, North America, and East to Southeast Asia. To capture the genomic diversity of domesticates of this important legume, de novo genome assemblies were produced for representatives of six subpopulations of cultivated cowpea identified previously from genotyping of several hundred diverse accessions. In the most complete assembly (IT97K-499-35), 26,026 core and 4963 noncore genes were identified, with 35,436 pan genes when considering all seven accessions. GO terms associated with response to stress and defense response were highly enriched among the noncore genes, while core genes were enriched in terms related to transcription factor activity, and transport and metabolic processes. Over 5 million single nucleotide polymorphisms (SNPs) relative to each assembly and over 40 structural variants >1 Mb in size were identified by comparing genomes. Vu10 was the chromosome with the highest frequency of SNPs, and Vu04 had the most structural variants. Noncore genes harbor a larger proportion of potentially disruptive variants than core genes, including missense, stop gain, and frameshift mutations; this suggests that noncore genes substantially contribute to diversity within domesticated cowpea.

Data sharing and ontology use among agricultural genetics, genomics, and breeding databases and resources of the Agbiodata Consortium.

Over the last couple of decades, there has been a rapid growth in the number and scope of agricultural genetics, genomics and breeding databases and resources. The AgBioData Consortium (https://www.agbiodata.org/) currently represents 44 databases and resources (https://www.agbiodata.org/databases) covering model or crop plant and animal GGB data, ontologies, pathways, genetic variation and breeding platforms (referred to as 'databases' throughout). One of the goals of the Consortium is to facilitate FAIR (Findable, Accessible, Interoperable, and Reusable) data management and the integration of datasets which requires data sharing, along with structured vocabularies and/or ontologies. Two AgBioData working groups, focused on Data Sharing and Ontologies, respectively, conducted a Consortium-wide survey to assess the current status and future needs of the members in those areas. A total of 33 researchers responded to the survey, representing 37 databases. Results suggest that data-sharing practices by AgBioData databases are in a fairly healthy state, but it is not clear whether this is true for all metadata and data types across all databases; and that, ontology use has not substantially changed since a similar survey was conducted in 2017. Based on our evaluation of the survey results, we recommend (i) providing training for database personnel in a specific data-sharing techniques, as well as in ontology use; (ii) further study on what metadata is shared, and how well it is shared among databases; (iii) promoting an understanding of data sharing and ontologies in the stakeholder community; (iv) improving data sharing and ontologies for specific phenotypic data types and formats; and (v) lowering specific barriers to data sharing and ontology use, by identifying sustainability solutions, and the identification, promotion, or development of data standards. Combined, these improvements are likely to help AgBioData databases increase development efforts towards improved ontology use, and data sharing via programmatic means. Database URL  https://www.agbiodata.org/databases.

Hiding in plain sight: Genome-wide recombination and a dynamic accessory genome drive diversity in Fusarium oxysporum f.sp. ciceris.

Understanding the origins of variation in agricultural pathogens is of fundamental interest and practical importance, especially for diseases that threaten food security. Fusarium oxysporum is among the most important of soil-borne pathogens, with a global distribution and an extensive host range. The pathogen is considered to be asexual, with horizontal transfer of chromosomes providing an analog of assortment by meiotic recombination. Here, we challenge those assumptions based on the results of population genomic analyses, describing the pathogen's diversity and inferring its origins and functional consequences in the context of a single, long-standing agricultural system. We identify simultaneously low nucleotide distance among strains, and unexpectedly high levels of genetic and genomic variability. We determine that these features arise from a combination of genome-scale recombination, best explained by widespread sexual reproduction, and presence-absence variation consistent with chromosomal rearrangement. Pangenome analyses document an accessory genome more than twice the size of the core genome, with contrasting evolutionary dynamics. The core genome is stable, with low diversity and high genetic differentiation across geographic space, while the accessory genome is paradoxically more diverse and unstable but with lower genetic differentiation and hallmarks of contemporary gene flow at local scales. We suggest a model in which episodic sexual reproduction generates haplotypes that are selected and then maintained through clone-like dynamics, followed by contemporary genomic rearrangements that reassort the accessory genome among sympatric strains. Taken together, these processes contribute unique genome content, including reassortment of virulence determinants that may explain observed variation in pathogenic potential.

Genome Context Viewer (GCV) version 2: enhanced visual exploration of multiple annotated genomes.

The Genome Context Viewer is a web application for identifying, aligning, and visualizing genomic regions based on their micro and macrosyntenic structures. By using functional elements such as gene annotations as the unit of search and comparison, the Genome Context Viewer can compute and display relationships between regions across many assemblies from federated data sources in real-time, enabling users to rapidly explore multiple annotated genomes and identify divergence and structural events that can help provide insight into evolutionary mechanisms associated with functional consequences. In this work, we introduce version 2 of the Genome Context Viewer and highlight new features that enhance usability, performance, and ease of deployment.

Cell-specific pathways recruited for symbiotic nodulation in the Medicago truncatula legume.

Medicago truncatula is a model legume species that has been studied for decades to understand the symbiotic relationship between legumes and soil bacteria collectively named rhizobia. This symbiosis called nodulation is initiated in roots with the infection of root hair cells by the bacteria, as well as the initiation of nodule primordia from root cortical, endodermal, and pericycle cells, leading to the development of a new root organ, the nodule, where bacteria fix and assimilate the atmospheric dinitrogen for the benefit of the plant. Here, we report the isolation and use of the nuclei from mock and rhizobia-inoculated roots for the single nuclei RNA-seq (sNucRNA-seq) profiling to gain a deeper understanding of early responses to rhizobial infection in Medicago roots. A gene expression map of the Medicago root was generated, comprising 25 clusters, which were annotated as specific cell types using 119 Medicago marker genes and orthologs to Arabidopsis cell-type marker genes. A focus on root hair, cortex, endodermis, and pericycle cell types, showing the strongest differential regulation in response to a short-term (48 h) rhizobium inoculation, revealed not only known genes and functional pathways, validating the sNucRNA-seq approach, but also numerous novel genes and pathways, allowing a comprehensive analysis of early root symbiotic responses at a cell type-specific level.

Alfalfa (Medicago sativa L.) pho2 mutant plants hyperaccumulate phosphate.

In this article, we describe a set of novel alfalfa (Medicago sativa L.) plants that hyper-accumulate Phosphate ion (Pi) at levels 3- to 6-fold higher than wild-type. This alfalfa germplasm will have practical applications reclaiming Pi from contaminated or enriched soil or be used in conservation buffer strips to protect waterways from Pi run-off. Hyper-accumulating alfalfa plants were generated by targeted mutagenesis of PHOSPHATE2 (PHO2) using newly created CRISPR/Cas9 reagents and an improved mutant screening strategy. PHO2 encodes a ubiquitin conjugating E2 enzyme (UBC24) previously characterized in Arabidopsis thaliana, Medicago truncatula, and Oryza sativa. Mutations of PHO2 disrupt Pi homeostasis resulting in Pi hyper-accumulation. Successful CRISPR/Cas9 editing of PHO2 demonstrates that this is an efficient mutagenesis tool in alfalfa despite its complex autotetraploid genome structure. Arabidopsis and M. truncatula ortholog genes were used to identify PHO2 haplotypes in outcrossing tetraploid M. sativa with the aim of generating heritable mutations in both PHO2-like genes (PHO2-B and PHO2-C). After delivery of the reagent and regeneration from transformed leaf explants, plants with mutations in all haplotypes of PHO2-B and PHO2-C were identified. These plants were evaluated for morphology, Pi accumulation, heritable transmission of targeted mutations, segregation of mutant haplotypes and removal of T-DNA(s). The Agrobacterium-mediated transformation assay and gene editing reagents reported here were also evaluated for further optimization for future alfalfa functional genomic studies.

Doing Genetic and Genomic Biology Using the Legume Information System and Associated Resources.

In this chapter, we introduce the main components of the Legume Information System ( https://legumeinfo.org ) and several associated resources. Additionally, we provide an example of their use by exploring a biological question: is there a common molecular basis, across legume species, that underlies the photoperiod-mediated transition from vegetative to reproductive development, that is, days to flowering? The Legume Information System (LIS) holds genetic and genomic data for a large number of crop and model legumes and provides a set of online bioinformatic tools designed to help biologists address questions and tasks related to legume biology. Such tasks include identifying the molecular basis of agronomic traits; identifying orthologs/syntelogs for known genes; determining gene expression patterns; accessing genomic datasets; identifying markers for breeding work; and identifying genetic similarities and differences among selected accessions. LIS integrates with other legume-focused informatics resources such as SoyBase ( https://soybase.org ), PeanutBase ( https://peanutbase.org ), and projects of the Legume Federation ( https://legumefederation.org ).

Legacy genetics of Arachis cardenasii in the peanut crop shows the profound benefits of international seed exchange.

The narrow genetics of most crops is a fundamental vulnerability to food security. This makes wild crop relatives a strategic resource of genetic diversity that can be used for crop improvement and adaptation to new agricultural challenges. Here, we uncover the contribution of one wild species accession, Arachis cardenasii GKP 10017, to the peanut crop (Arachis hypogaea) that was initiated by complex hybridizations in the 1960s and propagated by international seed exchange. However, until this study, the global scale of the dispersal of genetic contributions from this wild accession had been obscured by the multiple germplasm transfers, breeding cycles, and unrecorded genetic mixing between lineages that had occurred over the years. By genetic analysis and pedigree research, we identified A. cardenasii-enhanced, disease-resistant cultivars in Africa, Asia, Oceania, and the Americas. These cultivars provide widespread improved food security and environmental and economic benefits. This study emphasizes the importance of wild species and collaborative networks of international expertise for crop improvement. However, it also highlights the consequences of the implementation of a patchwork of restrictive national laws and sea changes in attitudes regarding germplasm that followed in the wake of the Convention on Biological Diversity. Today, the botanical collections and multiple seed exchanges which enable benefits such as those revealed by this study are drastically reduced. The research reported here underscores the vital importance of ready access to germplasm in ensuring long-term world food security.

The genome of a wild Medicago species provides insights into the tolerant mechanisms of legume forage to environmental stress.

BACKGROUND: Medicago ruthenica, a wild and perennial legume forage widely distributed in semi-arid grasslands, is distinguished by its outstanding tolerance to environmental stress. It is a close relative of commonly cultivated forage of alfalfa (Medicago sativa). The high tolerance of M. ruthenica to environmental stress makes this species a valuable genetic resource for understanding and improving traits associated with tolerance to harsh environments. RESULTS: We sequenced and assembled genome of M. ruthenica using an integrated approach, including PacBio, Illumina, 10×Genomics, and Hi-C. The assembled genome was 904.13 Mb with scaffold N50 of 99.39 Mb, and 50,162 protein-coding genes were annotated. Comparative genomics and transcriptomic analyses were used to elucidate mechanisms underlying its tolerance to environmental stress. The expanded FHY3/FAR1 family was identified to be involved in tolerance of M. ruthenica to drought stress. Many genes involved in tolerance to abiotic stress were retained in M. ruthenica compared to other cultivated Medicago species. Hundreds of candidate genes associated with drought tolerance were identified by analyzing variations in single nucleotide polymorphism using accessions of M. ruthenica with varying tolerance to drought. Transcriptomic data demonstrated the involvements of genes related to transcriptional regulation, stress response, and metabolic regulation in tolerance of M. ruthenica. CONCLUSIONS: We present a high-quality genome assembly and identification of drought-related genes in the wild species of M. ruthenica, providing a valuable resource for genomic studies on perennial legume forages.

Expression Partitioning of Duplicate Genes at Single Cell Resolution in Arabidopsis Roots.

Gene duplication is a key evolutionary phenomenon, prevalent in all organisms but particularly so in plants, where whole genome duplication (WGD; polyploidy) is a major force in genome evolution. Much effort has been expended in attempting to understand the evolution of duplicate genes, addressing such questions as why some paralog pairs rapidly return to single copy status whereas, in other pairs, both paralogs are retained and may diverge in expression pattern or function. The effect of a gene - its site of expression and thus the initial locus of its function - occurs at the level of a cell comprising a single cell type at a given state of the cell's development. Using Arabidopsis thaliana single cell transcriptomic data we categorized patterns of expression for 11,470 duplicate gene pairs across 36 cell clusters comprising nine cell types and their developmental states. Among these 11,470 pairs, 10,187 (88.8%) had at least one copy expressed in at least one of the 36 cell clusters. Pairs produced by WGD more often had both paralogs expressed in root cells than did pairs produced by small scale duplications. Three quarters of gene pairs expressed in the 36 cell clusters (7,608/10,187) showed extreme expression bias in at least one cluster, including 352 cases of reciprocal bias, a pattern consistent with expression subfunctionalization. More than twice as many pairs showed reciprocal expression bias between cell states than between cell types or between roots and leaves. A group of 33 gene pairs with reciprocal expression bias showed evidence of concerted divergence of gene networks in stele vs. epidermis. Pairs with both paralogs expressed without bias were less likely to have paralogs with divergent mutant phenotypes; such bias-free pairs showed evidence of preservation by maintenance of dosage balance. Overall, we found considerable evidence of shifts in gene expression following duplication, including in >80% of pairs encoding 7,653 genes expressed ubiquitously in all root cell types and states for which we inferred the polarity of change.

A genome-wide association and meta-analysis reveal regions associated with seed size in cowpea [Vigna unguiculata (L.) Walp].

KEY MESSAGE: This paper combined GWAS, meta-analysis and sequence homology comparison with common bean to identify regions associated with seed size variation in domesticated cowpea. Seed size is an important trait for yield and commercial value in dry-grain cowpea. Seed size varies widely among different cowpea accessions, and the genetic basis of such variation is not yet well understood. To better decipher the genetic basis of seed size, a genome-wide association study (GWAS) and meta-analysis were conducted on a panel of 368 cowpea diverse accessions from 51 countries. Four traits, including seed weight, length, width and density were evaluated across three locations. Using 51,128 single nucleotide polymorphisms covering the cowpea genome, 17 loci were identified for these traits. One locus was common to weight, width and length, suggesting pleiotropy. By integrating synteny-based analysis with common bean, six candidate genes (Vigun05g036000, Vigun05g039600, Vigun05g204200, Vigun08g217000, Vigun11g187000, and Vigun11g191300) which are implicated in multiple functional categories related to seed size such as endosperm development, embryo development, and cell elongation were identified. These results suggest that a combination of GWAS meta-analysis with synteny comparison in a related plant is an efficient approach to identify candidate gene (s) for complex traits in cowpea. The identified loci and candidate genes provide useful information for improving cowpea varieties and for molecular investigation of seed size.

The genome sequence of segmental allotetraploid peanut Arachis hypogaea.

Like many other crops, the cultivated peanut (Arachis hypogaea L.) is of hybrid origin and has a polyploid genome that contains essentially complete sets of chromosomes from two ancestral species. Here we report the genome sequence of peanut and show that after its polyploid origin, the genome has evolved through mobile-element activity, deletions and by the flow of genetic information between corresponding ancestral chromosomes (that is, homeologous recombination). Uniformity of patterns of homeologous recombination at the ends of chromosomes favors a single origin for cultivated peanut and its wild counterpart A. monticola. However, through much of the genome, homeologous recombination has created diversity. Using new polyploid hybrids made from the ancestral species, we show how this can generate phenotypic changes such as spontaneous changes in the color of the flowers. We suggest that diversity generated by these genetic mechanisms helped to favor the domestication of the polyploid A. hypogaea over other diploid Arachis species cultivated by humans.

The genome of cowpea (Vigna unguiculata [L.] Walp.).

Cowpea (Vigna unguiculata [L.] Walp.) is a major crop for worldwide food and nutritional security, especially in sub-Saharan Africa, that is resilient to hot and drought-prone environments. An assembly of the single-haplotype inbred genome of cowpea IT97K-499-35 was developed by exploiting the synergies between single-molecule real-time sequencing, optical and genetic mapping, and an assembly reconciliation algorithm. A total of 519 Mb is included in the assembled sequences. Nearly half of the assembled sequence is composed of repetitive elements, which are enriched within recombination-poor pericentromeric regions. A comparative analysis of these elements suggests that genome size differences between Vigna species are mainly attributable to changes in the amount of Gypsy retrotransposons. Conversely, genes are more abundant in more distal, high-recombination regions of the chromosomes; there appears to be more duplication of genes within the NBS-LRR and the SAUR-like auxin superfamilies compared with other warm-season legumes that have been sequenced. A surprising outcome is the identification of an inversion of 4.2 Mb among landraces and cultivars, which includes a gene that has been associated in other plants with interactions with the parasitic weed Striga gesnerioides. The genome sequence facilitated the identification of a putative syntelog for multiple organ gigantism in legumes. A revised numbering system has been adopted for cowpea chromosomes based on synteny with common bean (Phaseolus vulgaris). An estimate of nuclear genome size of 640.6 Mbp based on cytometry is presented.

Uneven recombination rate and linkage disequilibrium across a reference SNP map for common bean (Phaseolus vulgaris L.).

Recombination (R) rate and linkage disequilibrium (LD) analyses are the basis for plant breeding. These vary by breeding system, by generation of inbreeding or outcrossing and by region in the chromosome. Common bean (Phaseolus vulgaris L.) is a favored food legume with a small sequenced genome (514 Mb) and n = 11 chromosomes. The goal of this study was to describe R and LD in the common bean genome using a 768-marker array of single nucleotide polymorphisms (SNP) based on Trans-legume Orthologous Group (TOG) genes along with an advanced-generation Recombinant Inbred Line reference mapping population (BAT93 x Jalo EEP558) and an internationally available diversity panel. A whole genome genetic map was created that covered all eleven linkage groups (LG). The LGs were linked to the physical map by sequence data of the TOGs compared to each chromosome sequence of common bean. The genetic map length in total was smaller than for previous maps reflecting the precision of allele calling and mapping with SNP technology as well as the use of gene-based markers. A total of 91.4% of TOG markers had singleton hits with annotated Pv genes and all mapped outside of regions of resistance gene clusters. LD levels were found to be stronger within the Mesoamerican genepool and decay more rapidly within the Andean genepool. The recombination rate across the genome was 2.13 cM / Mb but R was found to be highly repressed around centromeres and frequent outside peri-centromeric regions. These results have important implications for association and genetic mapping or crop improvement in common bean.

Correction to: The transcriptome landscape of early maize meiosis.

CORRECTION: Following publication of the original article [1], the authors reported that the number of genes overlaying the bar graph in Fig. 3A were incorrectly counted and inserted (i.e. including a title tile, and in inverse order). The corrected numbers are below and match with the listed genes supplied in Additional File: Table S2.

A transcriptome-SNP-derived linkage map of Apios americana (potato bean) provides insights about genome re-organization and synteny conservation in the phaseoloid legumes.

KEY MESSAGE: We report a linkage map for Apios americana and describe synteny with selected warm-season legumes. A translocation event in common bean and soybean is confirmed against Apios and Vigna species. Apios (Apios americana; "apios"), a tuberous perennial legume in the Phaseoleae tribe, was widely used as a food by Native Americans. Work in the last 40 years has led to several improved breeding lines. Aspects of the pollination biology (complex floral structure and tripping mechanism) have made controlled crosses difficult, and the previous reports indicated that the plant is likely primarily an outcrosser. We used a pseudo-testcross strategy to construct a genetic map specific to the maternal parent. The map was built using single-nucleotide polymorphism markers identified by comparing the expressed sequences of individuals in the mapping population against a de novo maternal reference transcriptome assembly. The apios map consists of 11 linkage groups and 1121 recombinationally distinct loci, covering ~ 938.6 cM. By sequencing the transcriptomes of all potential pollen parents, we were able to identify the probable pollen donors and to discover new aspects of the pollination biology in apios. No selfing was observed, but multiple pollen parents were seen within individual pods. Comparisons with genome sequences in other species in the Phaseoleae showed extended synteny for most apios linkage groups. This synteny supports the robustness of the map, and also sheds light on the history of the Phaseoleae, as apios is relatively early diverging in this tribe. We detected a translocation event that separates apios and two Vigna species from Phaseolus vulgaris and Glycine max. This apios mapping work provides a general protocol for sequencing-based construction of high-density linkage maps in outcrossing species with heterogeneous pollen parents.

Anilinopyrimidine Resistance in Botrytis cinerea Is Linked to Mitochondrial Function.

Crop protection anilinopyrimidine (AP) fungicides were introduced more than 20 years ago for the control of a range of diseases caused by ascomycete plant pathogens, and in particular for the control of gray mold caused by Botrytis cinerea. Although early mode of action studies suggested an inhibition of methionine biosynthesis, the molecular target of this class of fungicides was never fully clarified. Despite AP-specific resistance having been described in B. cinerea field isolates and in multiple other targeted species, the underlying resistance mechanisms were unknown. It was therefore expected that the genetic characterization of resistance mechanisms would permit the identification of the molecular target of these fungicides. In order to explore the widest range of possible resistance mechanisms, AP-resistant B. cinerea UV laboratory mutants were generated and the mutations conferring resistance were determined by combining whole-genome sequencing and reverse genetics. Genetic mapping from a cross between a resistant field isolate and a sensitive reference isolate was used in parallel and led to the identification of an additional molecular determinant not found from the characterized UV mutant collection. Together, these two approaches enabled the characterization of an unrivaled diversity of resistance mechanisms. In total, we report the elucidation of resistance-conferring mutations within nine individual genes, two of which are responsible for almost all instances of AP resistance in the field. All identified resistance-conferring genes encode proteins that are involved in mitochondrial processes, suggesting that APs primarily target the mitochondria. The functions of these genes and their possible interactions are discussed in the context of the potential mode of action for this important class of fungicides.