Layer-specific genetic variation unlocks secondary metabolite diversity in long-lived clonal peppermint

Somatic mutations can generate novel clonal variants with commercially valuable traits. Periclinal chimeras, which carry genetically-differentiated meristematic layers, are common in clonally-propagated crops and can be highly stable. Given their economic and evolutionary impact, it is critical to understand the potential of layer-specific breeding in long-lived vegetatively-propagated crops. Peppermint clones are polyploid hybrids, sterile, and a source of essential oils. Using ionizing radiation, we induced large indels in an elite peppermint cultivar. We produced a haplotype-aware, high continuity assembly of this clone and used it to characterize the induced genetic variation and detected, on average, 6 large indels per individual. Importantly, the vast majority of these mutants are genetic chimeras in which the indels are confined to either the outer, L1-derived layer, responsible for mint oil production and accumulation, or derived from the inner L2/3 layers. Two mutants with very low (-)-menthol levels provided evidence that deletion of a single haplotype of the menthone-menthol reductase gene in the epidermal layer only was sufficient for altered oil composition. These results highlight new opportunities for mint oil improvement and demonstrate the potential of layer-specific variation for breeding of clonally propagated crops that suffer from genetic diversity bottlenecks.Here, we present an assembly of Black Mitcham clone obtained from the USDA ARS public repository (National Clonal Germplasm Repository, USDA ARS, Corvallis, OR), where it is identified as Mentha x piperita L. , PI 557971 (CMEN 579).