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.
We investigated gravitropic bending of sporangiophores of the zygomycete fungus Phycomyces blakesleeanus in response to centrifugal accelerations to test the so-called resultant law of gravitropism ('Resultantengesetz'; Jahrbuch der wissenschaftlichen Botanik, 71, 325, 1929; Der Geotropismus der Pflanzen, Gustav Fischer, Jena, Germany, 1932), which predicts that gravitropic organs orient in a centrifuge rotor parallel to the stimulus vector resulting from the centrifugal acceleration and gravity. Sporangiophores of wild-type strain C171 carAcarR and of several gravitropism mutants were subjected for 7 h to centrifugal accelerations in a dynamic range between 0.01 and 3 × g. The stimulus-response curves that were obtained for C171 carA carR, C2 carA geo and C148 carA geo madC were complex and displayed two response components: a low-acceleration component between 0.01 and 0.5 × g and a high-acceleration component above 0.5 × g. The low acceleration component is characterised by bending angles exceeding those predicted by the resultant law and kinetics faster than that of the second component; in contrast, the high-acceleration component is characterised by bending slightly below the predicted level and kinetics slower than that of the first component. Sporangiophores of the wild-type C171 centrifuged horizontally displayed the opposite behaviour, i.e. low accelerations diminished and high accelerations slightly enhanced bending. Further proof for the existence of the two response components was provided by the phenotype of gravitropism mutants that either lacked the first response component or which caused its overexpression. The tropism mutant C148 with defective madC gene, which codes for a RasGap protein (Fungal Genetics Reports, 60 (Suppl.), Abstract # 211, 2013), displayed hypergravitropism and concomitant deviations from the resultant law that were twice as high as in the wild-type C171. Gravitropism mutants with defects in the genes madF, madG and madJ lacked the low-response component below 0.5 × g. Our data are at variance with the so-called resultant law and imply that gravitropic orientation cannot depend exclusively on the classical sine stimulus (i.e. acting perpendicularly on the side walls); it rather must also be controlled by the cosine stimulus acting parallel to the longitudinal axis of the gravisensing organ. Our studies indicate that the threshold for the cosine response is the same as that of the sine response, and thus close to 0.01 × g.
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