A deep-time landscape of plant cis-regulatory sequence evolution

Developmental gene function is conserved over deep time, but similar cis-regulatory sequence conservation is rarely found. However, rapid sequence turnover, paleopolyploidy, structural variation, and limited phylogenomic sampling have impeded conserved non-coding sequence (CNS) discovery. Using Conservatory, an algorithm that leverages microsynteny and iterative alignments to map CNS-gene associations over evolution, we uncovered ~2.3M CNSs, including over 3,000 predating angiosperms, from 284 plant species spanning 400 million years of diversification. Ancient CNSs were enriched near developmental regulators, and mutagenizing those near HOMEOBOX genes produced strong phenotypes. Tracing CNS evolution uncovered key principles: CNS spacing varies, but order is conserved; genomic rearrangements form new CNS-gene associations; and ancient CNSs are preferentially retained among paralogs but often are lost as cohorts or evolve into lineage-specific CNSs. Overall design: Six replicates of ATAC-seq data from young leaves of Solanum lycopersicum cv. M82 were generated on an Illumina MiSeq SY-410-1003 instrument. Two replicates of Hi-C data from Solanum lycopersicum cv. M82 anantha inflorescences (meristematic enriched tissue) were generated on an Illumina NovaSeq 6000 with SP flow cell configuration.