Extreme somatic mutation variation through time and space in walnut clones

Many plants, unlike most animals, can reproduce as clones1. Cloning plants is critical for agriculture2,3 and biotechnology4, 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.