Single Nucleotide Polymorphism Charting of P. patens Reveals Accumulation of Somatic Mutations During in vitro Culture on the Scale of Natural Variation by Selfing

Abstract

Introduction: Physcomitrium patens (Hedw.) Mitten (previously known as Physcomitrella 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 cultured in vitro in 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 different P. patens accessions 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 during in vitro culture. 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 different P. patens accessions were isolated and can be used to determine individual accessions as well as Gransden pedigrees. Based on that, laboratory methods to easily determine P. patens accessions and Gransden pedigrees are presented.

A vertically transmitted amalgavirus is present in certain accessions of the bryophyte Physcomitrium patens

“…Indeed, we detected three different base pairs in the sequence obtained from Kaskaskia and one different base pair in the sequence obtained from Lviv with respect to the sequence of the virus present in Reute. RNA‐Seq data from P. patens Kaskaskia and Villersexel have been recently reported (Haas et al., 2020; Kamisugi et al., 2016). We did not detect reads corresponding to PHPAV1 in the Villersexel libraries, in line with the qRT‐PCR results discussed above, which suggests that PHPAV1 is not present in this accession.…”

“…We used wild‐type P. patens accessions of different origins: Gransden (Gd) pedigree Gd_JP_St Louis (Haas et al., 2020) and Reute‐K1 (Re) (Hiss et al., 2017), which are routinely used in laboratory settings, and Villersexel‐K3 (Vx), Kaskaskia (Ka), Lviv (Lv), Trondheim‐K2 (Td), and Uppsala‐K1 (Up), which were obtained from the International Moss Stock Center (https://www.moss-stock-center.org/). In addition, we used the Wisconsin (Wi) accession, which has been recently isolated from the wild (Haas et al., 2020). In order to facilitate the isolation of crossed plants, we used transgenic P. patens expressing a fluorescent protein.…”

“…We checked for the presence of PHPAV1 in Gransden and Reute accessions using the RNA‐Seq data available in the first release of the P. patens gene atlas (Lang et al., 2018) and in Kaskaskia and Villersexel accessions using RNA‐Seq data deposited in NCBI SRA in BioProjects PRJNA601618 and PRJNA602303 (Haas et al., 2020) and libraries SRX031156 and SRX031155 (Kamisugi et al., 2016).…”

“…Moreover, although no virus has been described to infect P. patens in the wild, it has been shown that P. patens can be infected in the laboratory with viruses such as the tomato spotted wilt virus (H€ uhns et al, 2003). Here we used the NGS-based approach described in (Gilbert et al, 2019) to look for viral sequences in available P. patens RNA sequencing (RNA-Seq) libraries (Haas et al, 2020;Kamisugi et al, 2016;Perroud et al, 2018) and we describe the first P. patens-associated virus, P. patens amalgavirus 1 (PHPAV1).…”

The nuclear GUCT domain-containing DEAD-box RNA helicases govern gametophytic and sporophytic development in Physcomitrium patens

“…Physcomitrium patens (previously known as Physcomitrella patens ) (Medina et al 2019 ) Gransden wild-type (WT), pedigree Gd_JP_St Louis (Haas et al 2020 ) was used as starting plant material for this study. Maintenance and amplification tissue culture for the WT and mutant strains were performed either directly on BCDA medium (Cove et al 2009 ) for spot inoculum or on cellophane overlaying the medium for blended tissue used for protoplasting.…”