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).

隐蔽遗传变异（cryptic genetic variants）单独存在时仅对表型产生极微弱或无影响，但长期以来学界假设其构成了一个庞大的隐蔽遗传多样性库，可通过上位性互作（epistatic interactions）推动性状的可进化性。这一经典理论因泛基因组测序（pan-genome sequencing）的发展重新获得关注，该技术不断揭示出顺式调控变异（cis-regulatory variation），以及广泛存在的基因重复与旁系同源基因分化（paralog diversification）——这一此前未被充分重视的变异来源，广泛存在于基因家族及其所属的调控网络中。

然而，该假说的实证检验长期受限于难以解析的遗传机制、有限的等位基因多样性，以及不足的表型分辨率。本研究依托新近演化得到的一对旁系同源基因的天然与人工改造的顺式隐蔽变异（cis-cryptic variants），鉴定出另一对冗余的反式调控因子（trans regulators），构建了调控番茄花序结构（tomato inflorescence architecture）的调控网络。

我们利用网络组分的等位基因谱（allelic spectrum），实现了基因型-表型映射（genotype-to-phenotype map）的高分辨率解析，揭示了隐蔽遗传变异如何促进性状多样化。我们在同时分离四个基因的群体中，结合编码区突变（coding mutations）与顺式调控等位基因系列（cis-regulatory allelic series），系统构建了涵盖216种基因型的基因剂量组合（gene dosage combinations），并对27000个花序的分枝情况进行量化分析。

分析结果显示，旁系同源基因对内部的剂量依赖性互作（dose-dependent interactions）可增强分枝，最终产生强烈的协同效应（synergistic effects）。但建模分析还揭示了旁系同源基因对之间未曾预料的拮抗作用（antagonism）：其中一对基因的突变积累会逐步削弱另一对基因突变的效应。

本研究结果阐明了基因调控网络的架构与旁系同源基因分化带来的复杂剂量效应，二者共同塑造了表型空间（phenotypic space）。鉴于基因组中旁系同源基因演化的普遍性，我们提出：调控网络内的旁系同源隐蔽遗传变异，可引发层级化的上位性互作，进而推动表型突变式涌现。

整体实验设计：
在体视显微镜下，于植株生长8周时从n=4株植株中采集花序分生组织。将组织速冻后用磁珠研磨，采用TRIzol（Invitrogen）与带柱上DNA消化步骤的Direct-zol RNA Miniprep试剂盒（Zymo Research）提取RNA。使用Qbit荧光分光光度计RNA HS检测试剂盒（Invitrogen）对RNA进行定量。样品经Ribo-Zero rRNA去除试剂盒（Epicenter）处理后，采用TruSeq V2 RNA测序建库试剂盒（Illumina）制备测序文库。