Cryptic variation fuels plant phenotypic change through hierarchical epistasis
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 is continually exposing cis-regulatory variation, along with widespread gene duplications and paralog diversification as an underappreciated source of cryptic variation within gene families and the regulatory networks in which they function. However, empirical testing of this hypothesis has been hindered by intractable genetics, limited allelic diversity, and inadequate phenotypic resolution. Here, guided by natural and engineered cis-cryptic variants in a recently evolved paralogous pair, we identified an additional  pair of redundant trans regulators, establishing a regulatory network that controls tomato inflorescence architecture. Exploiting an allelic spectrum of network components allowed a high-resolution dissection of a genotype-to-phenotype map, revealing how cryptic variants potentiate trait diversification. We combined coding mutations with a cis-regulatory allelic series in populations segregating for all four genes, systematically constructing gene dosage combinations across 216 genotypes and quantifying their effects on branching in 27,000 inflorescences. Our analysis revealed dose-dependent interactions within paralog pairs enhance branching, culminating in strong, synergistic, effects. However, modeling 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. 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. Overall design: Inflorescence meristems were collected from n = 4 plants at 8 weeks old under stereoscope magnification. Tissue was frozen, ground with beads, and RNA was extracted with TRIzol (Invitrogen) and a Direct-zol RNA Miniprep kit with on-column DNA digestion (Zymo Research). RNA was quantified with Qbit fluorimeter RNA HS assay kit (Invitrogen). Samples were treated with Ribo-Zero rRNA removal kit (Epicenter) and libraries prepared with an TruSeq V2 RNA-Seq prep kit (Illumina).