Single Nucleotide Polymorphism Charting ofP. patensReveals Accumulation of Somatic Mutations Duringin vitroCulture on the Scale of Natural Variation by Selfing

Introduction:Physcomitrium patens(Hedw.) Mitten (previously known asPhyscomitrella patens) was collected by H.L.K. Whitehouse in Gransden Wood (Huntingdonshire, United Kingdom) in 1962 and distributed across the globe starting in 1974. Hence, the Gransden accession has been culturedin vitroin laboratories for half a century. Today, there are more than 13 different pedigrees derived from the original accession. Additionally, accessions from other sites worldwide were collected during the last decades. Methods and Results:In this study, 250 high throughput RNA sequencing (RNA-seq) samples and 25 gDNA samples were used to detect single nucleotide polymorphisms (SNPs). Analyses were performed using five differentP. patensaccessions and 13 different Gransden pedigrees. SNPs were overlaid with metadata and known phenotypic variations. Unique SNPs defining Gransden pedigrees and accessions were identified and experimentally confirmed. They can be successfully employed for PCR-based identification. Conclusion:We show independent mutations in different Gransden laboratory pedigrees, demonstrating that somatic mutations occur and accumulate duringin vitroculture. The frequency of such mutations is similar to those observed in naturally occurring populations. We present evidence that vegetative propagation leads to accumulation of deleterious mutations, and that sexual reproduction purges those. Unique SNP sets for five differentP. patensaccessions were isolated and can be used to determine individual accessions as well as Gransden pedigrees. Based on that, laboratory methods to easily determineP. patensaccessions and Gransden pedigrees are presented.