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Abstract Cryptic genetic variants exert minimal phenotypic effects alone but are hypothesized to form a vast reservoir of genetic diversity driving trait evolvability through epistatic interactions^1–3. This classical theory has been reinvigorated by pan-genomics, which is revealing pervasive variation within gene families,cis-regulatory regions and regulatory networks^4–6. Testing the ability of cryptic variation to fuel phenotypic diversification has been hindered by intractable genetics, limited allelic diversity and inadequate phenotypic resolution. Here, guided by natural and engineeredcis-regulatory cryptic variants in a paralogous gene pair, we identified additional redundanttransregulators, establishing a regulatory network controlling tomato inflorescence architecture. By combining coding mutations withcis-regulatory alleles in populations segregating for all four network genes, we generated 216 genotypes spanning a wide spectrum of inflorescence complexity and quantified branching in over 35,000 inflorescences. Analysis of this high-resolution genotype–phenotype map using a hierarchical model of epistasis revealed a layer of dose-dependent interactions within paralogue pairs enhancing branching, culminating in strong, synergistic effects. However, we also identified a layer of antagonism between paralogue pairs, whereby accumulating mutations in one pair progressively diminished the effects of mutations in the other. Our results demonstrate how gene regulatory network architecture and complex dosage effects from paralogue diversification converge to shape phenotypic space, producing the potential for both strongly buffered phenotypes and sudden bursts of phenotypic change. 

Developmental transitions require precise temporal and spatial control of gene expression. In plants, such regulation is critical for flower formation, which involves the progressive maturation of stem cell populations within shoot meristems to floral meristems, followed by rapid sequential differentiation into floral organs. Across plant taxa, these transitions are orchestrated by the F-box transcriptional cofactor geneUNUSUAL FLORAL ORGANS(UFO). The conserved and pleiotropic functions ofUFOoffer a useful framework for investigating how evolutionary processes have shaped the intricatecis-regulation of key developmental genes. By pinpointing a conserved promoter sequence in an accessible chromatin region of the tomato ortholog ofUFO, we engineered in vivo a series ofcis-regulatory alleles that caused both loss- and gain-of-function floral defects. These mutant phenotypes were linked to disruptions in predicted transcription factor binding sites for known transcriptional activators and repressors. Allelic combinations revealed dosage-dependent interactions between opposing alleles, influencing the penetrance and expressivity of gain-of-function phenotypes. These phenotypic differences support that robustness in tomato flower development requires precise temporal control ofUFOexpression dosage. Bridging our analysis toArabidopsis, we found that although homologous sequences to the tomato regulatory region are dispersed within theUFOpromoter, they maintain similar control over floral development. However, phenotypes from disrupting these sequences differ due to the differing expression patterns ofUFO. Our study underscores the complexcis-regulatory control of dynamic developmental genes and demonstrates that critical short stretches of regulatory sequences that recruit both activating and repressing machinery are conserved to maintain developmental robustness. 

ABSTRACT Cryptic genetic variants exert minimal or no phenotypic effects alone but have long been hypothesized to form a vast, hidden reservoir of genetic diversity that drives trait evolvability through epistatic interactions. This classical theory has been reinvigorated by pan-genome sequencing, which has revealed pervasive variation within gene families and regulatory networks, including extensive cis-regulatory changes, gene duplication, and divergence between paralogs. Nevertheless, empirical testing of cryptic variation’s capacity to fuel phenotypic diversification has been hindered by intractable genetics, limited allelic diversity, and inadequate phenotypic resolution. Here, guided by natural and engineered cis-regulatory cryptic variants in a recently evolved paralogous gene pair, we identified an additional pair of redundant trans regulators, establishing a regulatory network that controls tomato inflorescence architecture. By combining coding mutations with a cis-regulatory allelic series in populations segregating for all four network genes, we systematically constructed a collection of 216 genotypes spanning the full spectrum of inflorescence complexity and quantified branching in over 27,000 inflorescences. Analysis of the resulting high-resolution genotype-phenotype map revealed a layer of dose-dependent interactions within paralog pairs that enhances branching, culminating in strong, synergistic effects. However, we also uncovered an unexpected layer of antagonism between paralog pairs, where accumulating mutations in one pair progressively diminished the effects of mutations in the other. Our results demonstrate how gene regulatory network architecture and complex dosage effects from paralog diversification converge to shape phenotypic space under a hierarchical model of epistatic interactions. Given the prevalence of paralog evolution in genomes, we propose that paralogous cryptic variation within regulatory networks elicits hierarchies of epistatic interactions, catalyzing bursts of phenotypic change. Keyword:cryptic mutations, paralogs, redundancy, cis-regulatory, tomato, inflorescence, gene regulatory network, modeling, epistasis 

Abstract Pan-genomics and genome-editing technologies are revolutionizing breeding of global crops^1,2. A transformative opportunity lies in exchanging genotype-to-phenotype knowledge between major crops (that is, those cultivated globally) and indigenous crops (that is, those locally cultivated within a circumscribed area)^3–5to enhance our food system. However, species-specific genetic variants and their interactions with desirable natural or engineered mutations pose barriers to achieving predictable phenotypic effects, even between related crops^6,7. Here, by establishing a pan-genome of the crop-rich genusSolanum⁸and integrating functional genomics and pan-genetics, we show that gene duplication and subsequent paralogue diversification are major obstacles to genotype-to-phenotype predictability. Despite broad conservation of gene macrosynteny among chromosome-scale references for 22 species, including 13 indigenous crops, thousands of gene duplications, particularly within key domestication gene families, exhibited dynamic trajectories in sequence, expression and function. By augmenting our pan-genome with African eggplant cultivars⁹and applying quantitative genetics and genome editing, we dissected an intricate history of paralogue evolution affecting fruit size. The loss of a redundant paralogue of the classical fruit size regulatorCLAVATA3(CLV3)^10,11was compensated by a lineage-specific tandem duplication. Subsequent pseudogenization of the derived copy, followed by a large cultivar-specific deletion, created a single fusedCLV3allele that modulates fruit organ number alongside an enzymatic gene controlling the same trait. Our findings demonstrate that paralogue diversifications over short timescales are underexplored contingencies in trait evolvability. Exposing and navigating these contingencies is crucial for translating genotype-to-phenotype relationships across species. 

Abstract 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. 

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. 

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 genusSolanum. Homologs underlie prickle formation across angiosperms that collectively diverged more than 150 million years ago, including rice and roses. By developing newSolanumgenetic 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. 

Abstract 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. 

Abstract 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. Here 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 genusSolanum. Strikingly, homologs promote prickle formation across angiosperms that collectively diverged over 150 million years ago. By developing newSolanumgenetic 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.