Convergent evolution of plant prickles by repeated gene co-option over deep time.

An enduring question in evolutionary biology concerns the degree to which episodes of convergent trait evolution depend on the same genetic programs, particularly over long timescales. In this work, we genetically dissected repeated origins and losses of prickles-sharp epidermal projections-that convergently evolved in numerous plant lineages. Mutations in a cytokinin hormone biosynthetic gene caused at least 16 independent losses of prickles in eggplants and wild relatives in the genus Solanum. Homologs underlie prickle formation across angiosperms that collectively diverged more than 150 million years ago, including rice and roses. By developing new Solanum genetic systems, we leveraged this discovery to eliminate prickles in a wild species and an indigenously foraged berry. Our findings implicate a shared hormone activation genetic program underlying evolutionarily widespread and recurrent instances of plant morphological innovation.

Blooming balloons: Searching for mechanisms of the inflated calyx.

Studying morphological novelties offers special insights into developmental biology and evolution. The inflated calyx syndrome (ICS) is a largely unrecognized but fascinating feature of flower development, where sepals form balloon-like husks that encapsulate fruits. Despite its independent emergence in many lineages of flowering plants, the genetic and molecular mechanisms of ICS remain unknown. Early studies in the Solanaceae genus Physalis put forth key roles of MADS-box genes in ICS. However, recent work suggests these classical floral identity transcription factors were false leads. With newfound capabilities that allow rapid development of genetic systems through genomics and genome editing, Physalis has re-emerged as the most tractable model species for dissecting ICS. This review revisits current understanding of ICS and highlights how recent advancements enable a reset in the search for genetic and molecular mechanisms using unbiased, systematic approaches.

Evolutionary conservation of receptor compensation for stem cell homeostasis in Solanaceae plants.

Stem cell homeostasis is pivotal for continuous and programmed formation of organs in plants. The precise control of meristem proliferation is mediated by the evolutionarily conserved signaling that encompasses complex interactions among multiple peptide ligands and their receptor-like kinases. Here, we identified compensation mechanisms involving the CLAVATA1 (CLV1) receptor and its paralogs, BARELY ANY MERISTEMs (BAMs), for stem cell proliferation in two Solanaceae species, tomato and groundcherry. Genetic analyses of higher-order mutants deficient in multiple receptor genes, generated via CRISPR-Cas9 genome editing, reveal that tomato SlBAM1 and SlBAM2 compensate for slclv1 mutations. Unlike the compensatory responses between orthologous receptors observed in Arabidopsis, tomato slclv1 mutations do not trigger transcriptional upregulation of four SlBAM genes. The compensation mechanisms within receptors are also conserved in groundcherry, and critical amino acid residues of the receptors associated with the physical interaction with peptide ligands are highly conserved in Solanaceae plants. Our findings demonstrate that the evolutionary conservation of both compensation mechanisms and critical coding sequences between receptor-like kinases provides a strong buffering capacity during stem cell homeostasis in tomato and groundcherry.

Extreme restructuring of cis-regulatory regions controlling a deeply conserved plant stem cell regulator.

A striking paradox is that genes with conserved protein sequence, function and expression pattern over deep time often exhibit extremely divergent cis-regulatory sequences. It remains unclear how such drastic cis-regulatory evolution across species allows preservation of gene function, and to what extent these differences influence how cis-regulatory variation arising within species impacts phenotypic change. Here, we investigated these questions using a plant stem cell regulator conserved in expression pattern and function over ~125 million years. Using in-vivo genome editing in two distantly related models, Arabidopsis thaliana (Arabidopsis) and Solanum lycopersicum (tomato), we generated over 70 deletion alleles in the upstream and downstream regions of the stem cell repressor gene CLAVATA3 (CLV3) and compared their individual and combined effects on a shared phenotype, the number of carpels that make fruits. We found that sequences upstream of tomato CLV3 are highly sensitive to even small perturbations compared to its downstream region. In contrast, Arabidopsis CLV3 function is tolerant to severe disruptions both upstream and downstream of the coding sequence. Combining upstream and downstream deletions also revealed a different regulatory outcome. Whereas phenotypic enhancement from adding downstream mutations was predominantly weak and additive in tomato, mutating both regions of Arabidopsis CLV3 caused substantial and synergistic effects, demonstrating distinct distribution and redundancy of functional cis-regulatory sequences. Our results demonstrate remarkable malleability in cis-regulatory structural organization of a deeply conserved plant stem cell regulator and suggest that major reconfiguration of cis-regulatory sequence space is a common yet cryptic evolutionary force altering genotype-to-phenotype relationships from regulatory variation in conserved genes. Finally, our findings underscore the need for lineage-specific dissection of the spatial architecture of cis-regulation to effectively engineer trait variation from conserved productivity genes in crops.

Idiosyncratic and dose-dependent epistasis drives variation in tomato fruit size.

Epistasis between genes is traditionally studied with mutations that eliminate protein activity, but most natural genetic variation is in cis-regulatory DNA and influences gene expression and function quantitatively. In this study, we used natural and engineered cis-regulatory alleles in a plant stem-cell circuit to systematically evaluate epistatic relationships controlling tomato fruit size. Combining a promoter allelic series with two other loci, we collected over 30,000 phenotypic data points from 46 genotypes to quantify how allele strength transforms epistasis. We revealed a saturating dose-dependent relationship but also allele-specific idiosyncratic interactions, including between alleles driving a step change in fruit size during domestication. Our approach and findings expose an underexplored dimension of epistasis, in which cis-regulatory allelic diversity within gene regulatory networks elicits nonlinear, unpredictable interactions that shape phenotypes.

Establishing Physalis as a Solanaceae model system enables genetic reevaluation of the inflated calyx syndrome.

The highly diverse Solanaceae family contains several widely studied models and crop species. Fully exploring, appreciating, and exploiting this diversity requires additional model systems. Particularly promising are orphan fruit crops in the genus Physalis, which occupy a key evolutionary position in the Solanaceae and capture understudied variation in traits such as inflorescence complexity, fruit ripening and metabolites, disease and insect resistance, self-compatibility, and most notable, the striking inflated calyx syndrome (ICS), an evolutionary novelty found across angiosperms where sepals grow exceptionally large to encapsulate fruits in a protective husk. We recently developed transformation and genome editing in Physalis grisea (groundcherry). However, to systematically explore and unlock the potential of this and related Physalis as genetic systems, high-quality genome assemblies are needed. Here, we present chromosome-scale references for P. grisea and its close relative Physalis pruinosa and use these resources to study natural and engineered variations in floral traits. We first rapidly identified a natural structural variant in a bHLH gene that causes petal color variation. Further, and against expectations, we found that CRISPR-Cas9-targeted mutagenesis of 11 MADS-box genes, including purported essential regulators of ICS, had no effect on inflation. In a forward genetics screen, we identified huskless, which lacks ICS due to mutation of an AP2-like gene that causes sepals and petals to merge into a single whorl of mixed identity. These resources and findings elevate Physalis to a new Solanaceae model system and establish a paradigm in the search for factors driving ICS.

Extreme restructuring of cis -regulatory regions controlling a deeply conserved plant stem cell regulator.

A striking paradox is that genes with conserved protein sequence, function and expression pattern over deep time often exhibit extremely divergent cis -regulatory sequences. It remains unclear how such drastic cis -regulatory evolution across species allows preservation of gene function, and to what extent these differences influence how cis- regulatory variation arising within species impacts phenotypic change. Here, we investigated these questions using a plant stem cell regulator conserved in expression pattern and function over ∼125 million years. Using in-vivo genome editing in two distantly related models, Arabidopsis thaliana (Arabidopsis) and Solanum lycopersicum (tomato), we generated over 70 deletion alleles in the upstream and downstream regions of the stem cell repressor gene CLAVATA3 ( CLV3 ) and compared their individual and combined effects on a shared phenotype, the number of carpels that make fruits. We found that sequences upstream of tomato CLV3 are highly sensitive to even small perturbations compared to its downstream region. In contrast, Arabidopsis CLV3 function is tolerant to severe disruptions both upstream and downstream of the coding sequence. Combining upstream and downstream deletions also revealed a different regulatory outcome. Whereas phenotypic enhancement from adding downstream mutations was predominantly weak and additive in tomato, mutating both regions of Arabidopsis CLV3 caused substantial and synergistic effects, demonstrating distinct distribution and redundancy of functional cis -regulatory sequences. Our results demonstrate remarkable malleability in cis -regulatory structural organization of a deeply conserved plant stem cell regulator and suggest that major reconfiguration of cis -regulatory sequence space is a common yet cryptic evolutionary force altering genotype-to-phenotype relationships from regulatory variation in conserved genes. Finally, our findings underscore the need for lineage-specific dissection of the spatial architecture of cis -regulation to effectively engineer trait variation from conserved productivity genes in crops. Author summary: We investigated the evolution of cis -regulatory elements (CREs) and their interactions in the regulation of a plant stem cell regulator gene, CLAVATA3 (CLV3) , in Arabidopsis and tomato. Despite diverging ∼125 million years ago, the function and expression of CLV3 is conserved in these species; however, cis -regulatory sequences upstream and downstream have drastically diverged, preventing identification of conserved non-coding sequences between them. We used CRISPR-Cas9 to engineer dozens of mutations within the cis -regulatory regions of Arabidopsis and tomato CLV3. In tomato, our results show that tomato CLV3 function primarily relies on interactions among CREs in the 5' non-coding region, unlike Arabidopsis CLV3 , which depends on a more balanced distribution of functional CREs between the 5' and 3' regions. Therefore, despite a high degree of functional conservation, our study demonstrates divergent regulatory strategies between two distantly related CLV3 orthologs, with substantial alterations in regulatory sequences, their spatial arrangement, and their relative effects on CLV3 regulation. These results suggest that regulatory regions are not only extremely robust to mutagenesis, but also that the sequences underlying this robustness can be lineage-specific for conserved genes, due to the complex and often redundant interactions among CREs that ensure proper gene function amidst large-scale sequence turnover.

Automated assembly scaffolding using RagTag elevates a new tomato system for high-throughput genome editing.

Advancing crop genomics requires efficient genetic systems enabled by high-quality personalized genome assemblies. Here, we introduce RagTag, a toolset for automating assembly scaffolding and patching, and we establish chromosome-scale reference genomes for the widely used tomato genotype M82 along with Sweet-100, a new rapid-cycling genotype that we developed to accelerate functional genomics and genome editing in tomato. This work outlines strategies to rapidly expand genetic systems and genomic resources in other plant species.

Newly Discovered Alleles of the Tomato Antiflorigen Gene SELF PRUNING Provide a Range of Plant Compactness and Yield.

In tomato cultivation, a rare natural mutation in the flowering repressor antiflorigen gene SELF-PRUNING (sp-classic) induces precocious shoot termination and is the foundation in determinate tomato breeding for open field production. Heterozygous single flower truss (sft) mutants in the florigen SFT gene in the background of sp-classic provide a heterosis-like effect by delaying shoot termination, suggesting the subtle suppression of determinacy by genetic modification of the florigen-antiflorigen balance could improve yield. Here, we isolated three new sp alleles from the tomato germplasm that show modified determinate growth compared to sp-classic, including one allele that mimics the effect of sft heterozygosity. Two deletion alleles eliminated functional transcripts and showed similar shoot termination, determinate growth, and yields as sp-classic. In contrast, amino acid substitution allele sp-5732 showed semi-determinate growth with more leaves and sympodial shoots on all shoots. This translated to greater yield compared to the other stronger alleles by up to 42%. Transcriptome profiling of axillary (sympodial) shoot meristems (SYM) from sp-classic and wild type plants revealed six mis-regulated genes related to the floral transition, which were used as biomarkers to show that the maturation of SYMs in the weaker sp-5732 genotype is delayed compared to sp-classic, consistent with delayed shoot termination and semi-determinate growth. Assessing sp allele frequencies from over 500 accessions indicated that one of the strong sp alleles (sp-2798) arose in early breeding cultivars but was not selected. The newly discovered sp alleles are potentially valuable resources to quantitatively manipulate shoot growth and yield in determinate breeding programs, with sp-5732 providing an opportunity to develop semi-determinate field varieties with higher yields.

Identification of Genetic Factors Controlling the Formation of Multiple Flowers Per Node in Pepper (Capsicum spp.).

Flower production provides the foundation for crop yield and increased profits. Capsicum annuum is a pepper species with a sympodial shoot structure with solitary flowers. By contrast, C. chinense produces multiple flowers per node. C. annuum accounts for 80% of pepper production worldwide. The identification of C. chinense genes that control multiple flowers and their transfer into C. annuum may open the way to increasing fruit yield. In this study, we dissected the genetic factors were dissected controlling the multiple-flower-per-node trait in Capsicum. 85 recombinant inbred lines (RILs) between the contrasting C. annuum 'TF68' and C. chinense 'Habanero' accessions were phenotyped and genotyped. Quantitative Trait Loci (QTL) analysis identified four novel QTLs on chromosomes 1, 2, 7, and 11 that accounted for 65% of the total phenotypic variation. Genome-wide association study was also performed on a panel of 276 genotyped and phenotyped C. annuum accessions, which revealed 28 regions significantly associated with the multiple-flower trait, of which three overlapped the identified QTLs. Five candidate genes involved in the development of the shoot and flower meristems were identified and these genes could cause multiple flowers per node in pepper. These results contribute to our understanding of multiple flower formation in Capsicum and will be useful to develop high-yielding cultivars.

Dynamic evolution of small signalling peptide compensation in plant stem cell control.

Gene duplications are a hallmark of plant genome evolution and a foundation for genetic interactions that shape phenotypic diversity1-5. Compensation is a major form of paralogue interaction6-8 but how compensation relationships change as allelic variation accumulates is unknown. Here we leveraged genomics and genome editing across the Solanaceae family to capture the evolution of compensating paralogues. Mutations in the stem cell regulator CLV3 cause floral organs to overproliferate in many plants9-11. In tomato, this phenotype is partially suppressed by transcriptional upregulation of a closely related paralogue12. Tobacco lost this paralogue, resulting in no compensation and extreme clv3 phenotypes. Strikingly, the paralogues of petunia and groundcherry nearly completely suppress clv3, indicating a potent ancestral state of compensation. Cross-species transgenic complementation analyses show that this potent compensation partially degenerated in tomato due to a single amino acid change in the paralogue and cis-regulatory variation that limits its transcriptional upregulation. Our findings show how genetic interactions are remodelled following duplications and suggest that dynamic paralogue evolution is widespread over short time scales and impacts phenotypic variation from natural and engineered mutations.

Optimized sample selection for cost-efficient long-read population sequencing.

An increasingly important scenario in population genetics is when a large cohort has been genotyped using a low-resolution approach (e.g., microarrays, exome capture, short-read WGS), from which a few individuals are resequenced using a more comprehensive approach, especially long-read sequencing. The subset of individuals selected should ensure that the captured genetic diversity is fully representative and includes variants across all subpopulations. For example, human variation has historically focused on individuals with European ancestry, but this represents a small fraction of the overall diversity. Addressing this, SVCollector identifies the optimal subset of individuals for resequencing by analyzing population-level VCF files from low-resolution genotyping studies. It then computes a ranked list of samples that maximizes the total number of variants present within a subset of a given size. To solve this optimization problem, SVCollector implements a fast, greedy heuristic and an exact algorithm using integer linear programming. We apply SVCollector on simulated data, 2504 human genomes from the 1000 Genomes Project, and 3024 genomes from the 3000 Rice Genomes Project and show the rankings it computes are more representative than alternative naive strategies. When selecting an optimal subset of 100 samples in these cohorts, SVCollector identifies individuals from every subpopulation, whereas naive methods yield an unbalanced selection. Finally, we show the number of variants present in cohorts selected using this approach follows a power-law distribution that is naturally related to the population genetic concept of the allele frequency spectrum, allowing us to estimate the diversity present with increasing numbers of samples.

Dissecting cis-regulatory control of quantitative trait variation in a plant stem cell circuit.

Cis-regulatory mutations underlie important crop domestication and improvement traits1,2. However, limited allelic diversity has hindered functional dissection of the large number of cis-regulatory elements and their potential interactions, thereby precluding a deeper understanding of how cis-regulatory variation impacts traits quantitatively. Here, we engineered over 60 promoter alleles in two tomato fruit size genes3,4 to characterize cis-regulatory sequences and study their functional relationships. We found that targeted mutations in conserved promoter sequences of SlCLV3, a repressor of stem cell proliferation5,6, have a weak impact on fruit locule number. Pairwise combinations of these mutations mildly enhance this phenotype, revealing additive and synergistic relationships between conserved regions and further suggesting even higher-order cis-regulatory interactions within the SlCLV3 promoter. In contrast, SlWUS, a positive regulator of stem cell proliferation repressed by SlCLV3 (refs. 5,6), is more tolerant to promoter perturbations. Our results show that complex interplay among cis-regulatory variants can shape quantitative variation, and suggest that empirical dissections of this hidden complexity can guide promoter engineering to predictably modify crop traits.

Conserved pleiotropy of an ancient plant homeobox gene uncovered by cis-regulatory dissection.

Divergence of gene function is a hallmark of evolution, but assessing functional divergence over deep time is not trivial. The few alleles available for cross-species studies often fail to expose the entire functional spectrum of genes, potentially obscuring deeply conserved pleiotropic roles. Here, we explore the functional divergence of WUSCHEL HOMEOBOX9 (WOX9), suggested to have species-specific roles in embryo and inflorescence development. Using a cis-regulatory editing drive system, we generate a comprehensive allelic series in tomato, which revealed hidden pleiotropic roles for WOX9. Analysis of accessible chromatin and conserved cis-regulatory sequences identifies the regions responsible for this pleiotropic activity, the functions of which are conserved in groundcherry, a tomato relative. Mimicking these alleles in Arabidopsis, distantly related to tomato and groundcherry, reveals new inflorescence phenotypes, exposing a deeply conserved pleiotropy. We suggest that targeted cis-regulatory mutations can uncover conserved gene functions and reduce undesirable effects in crop improvement.

New Horizons for Dissecting Epistasis in Crop Quantitative Trait Variation.

Uncovering the genes, variants, and interactions underlying crop diversity is a frontier in plant genetics. Phenotypic variation often does not reflect the cumulative effect of individual gene mutations. This deviation is due to epistasis, in which interactions between alleles are often unpredictable and quantitative in effect. Recent advances in genomics and genome-editing technologies are elevating the study of epistasis in crops. Using the traits and developmental pathways that were major targets in domestication and breeding, we highlight how epistasis is central in guiding the behavior of the genetic variation that shapes quantitative trait variation. We outline new strategies that illuminate how quantitative epistasis from modified gene dosage defines background dependencies. Advancing our understanding of epistasis in crops can reveal new principles and approaches to engineering targeted improvements in agriculture.

Major Impacts of Widespread Structural Variation on Gene Expression and Crop Improvement in Tomato.

Structural variants (SVs) underlie important crop improvement and domestication traits. However, resolving the extent, diversity, and quantitative impact of SVs has been challenging. We used long-read nanopore sequencing to capture 238,490 SVs in 100 diverse tomato lines. This panSV genome, along with 14 new reference assemblies, revealed large-scale intermixing of diverse genotypes, as well as thousands of SVs intersecting genes and cis-regulatory regions. Hundreds of SV-gene pairs exhibit subtle and significant expression changes, which could broadly influence quantitative trait variation. By combining quantitative genetics with genome editing, we show how multiple SVs that changed gene dosage and expression levels modified fruit flavor, size, and production. In the last example, higher order epistasis among four SVs affecting three related transcription factors allowed introduction of an important harvesting trait in modern tomato. Our findings highlight the underexplored role of SVs in genotype-to-phenotype relationships and their widespread importance and utility in crop improvement.

Rapid customization of Solanaceae fruit crops for urban agriculture.

Cultivation of crops in urban environments might reduce the environmental impact of food production1-4. However, lack of available land in cities and a need for rapid crop cycling, to yield quickly and continuously, mean that so far only lettuce and related 'leafy green' vegetables are cultivated in urban farms5. New fruit varieties with architectures and yields suitable for urban farming have proven difficult to breed1,5. We identified a regulator of tomato stem length (SlER) and devised a trait-stacking strategy to combine mutations for condensed shoots, rapid flowering (SP5G) and precocious growth termination (SP). Application of our strategy using one-step CRISPR-Cas9 genome editing restructured vine-like tomato plants into compact, early yielding plants suitable for urban agriculture. Field data confirmed that yields were maintained, and we demonstrated cultivation in indoor farming systems. Targeting the same stem length regulator alone in groundcherry, another Solanaceae plant, also enabled engineering to a compact stature. Our approach can expand the repertoire of crops for urban agriculture.

RaGOO: fast and accurate reference-guided scaffolding of draft genomes.

We present RaGOO, a reference-guided contig ordering and orienting tool that leverages the speed and sensitivity of Minimap2 to accurately achieve chromosome-scale assemblies in minutes. After the pseudomolecules are constructed, RaGOO identifies structural variants, including those spanning sequencing gaps. We show that RaGOO accurately orders and orients 3 de novo tomato genome assemblies, including the widely used M82 reference cultivar. We then demonstrate the scalability and utility of RaGOO with a pan-genome analysis of 103 Arabidopsis thaliana accessions by examining the structural variants detected in the newly assembled pseudomolecules. RaGOO is available open source at https://github.com/malonge/RaGOO .

Revolutions in agriculture chart a course for targeted breeding of old and new crops.

The dominance of the major crops that feed humans and their livestock arose from agricultural revolutions that increased productivity and adapted plants to large-scale farming practices. Two hormone systems that universally control flowering and plant architecture, florigen and gibberellin, were the source of multiple revolutions that modified reproductive transitions and proportional growth among plant parts. Although step changes based on serendipitous mutations in these hormone systems laid the foundation, genetic and agronomic tuning were required for broad agricultural benefits. We propose that generating targeted genetic variation in core components of both systems would elicit a wider range of phenotypic variation. Incorporating this enhanced diversity into breeding programs of conventional and underutilized crops could help to meet the future needs of the human diet and promote sustainable agriculture.

Author Correction: Duplication of a domestication locus neutralized a cryptic variant that caused a breeding barrier in tomato.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.