Extreme somatic mutation variation through time and space in walnut clones

Many plants, unlike most animals, can reproduce as clones 1 . Cloning plants is critical for agriculture 2,3 and biotechnology 4 , but the extent of somatic mutation arising during different propagation methods remains an important question for both fundamental research and agriculture. Here, we discover a surprisingly complex mutational history in a multi-decade natural experiment of isogenic walnut clones derived and maintained through three alternative methods: budwood propagation of field-grown trees, in vitro shoot culturing, and in vitro somatic embryogenesis. We generated a haplotype-phased reference genome assembly and revealed a >3500% increase in somatic embryo mutation rates compared to field-grown trees, along with a distinct mutation spectrum. The assembly also helped reveal extreme genomic instability in the somatic embryos, including multiple chromosomal duplications, megabase-scale deletions, telomere expansions, somatic recombination events, and ongoing transposable element activation. Our survey of somatic mutation also provides high-resolution insight into clonal stem cell dynamics, confirming the canonical meristem layers of flowering plants in the tree and shoot clones, while uncovering clear evidence of frequent single-cell bottlenecks in the somatic embryos. These discoveries inform practical questions about mutagenesis through plant tissue culture and serve as a benchmark to complement emerging paradigms of somatic mutation research in humans and other organisms.