Physcomitrella patens Reute mCherry as a tool for efficient crossing within and between ecotypes

Abstract

Physcomitrella patens is a monoecious moss that is predominantly selfing in the wild. Laboratory crossing techniques have been established and crosses between the sequenced Gransden ecotype and the genetically divergent Villersexel ecotype were used for genetic mapping. The recently introduced ecotype Reute has a high fertility rate and is genetically more closely related to the Gransden ecotype than the Villersexel ecotype. Reute sexual reproduction phenology is similar to Gransden, which should allow successful crossing. Using the Reute ecotype and an existing Gransden mutant as a test case, we applied a normalised crossing approach to demonstrate crossing potential between these ecotypes. Also, using a standard transformation approach, we generated Reute fluorescent strains expressing mCherry that allow an easy detection of crossed offspring (sporophyte). We show that Reute can be successfully crossed with a self-infertile DR5:DsRed2 mutant generated in the Gransden background. Using newly established Reute fluorescent strains, we show that they can efficiently fertilise Reute as well as Gransden wild type. The resulting progeny display Mendelian 1:1 segregation of the fluorescent marker(s), demonstrating the suitability of such strains for genetic crossing. Overall our results demonstrate that Reute is highly suitable for genetic crossing. The Reute mCherry strain can be used as a suitable background for offspring selection after crossing.

Characterization of evolutionarily conserved key players affecting eukaryotic flagellar motility and fertility using a moss model

“…The ecotype Reute (Re) was shown to develop a significantly higher number of sporophytes per gametophore in comparison to the broadly used ecotype Gransden (Gd, (Hiss et al 2017)). To determine whether the reduced fertility of Gd is based on the female or male reproductive apparatus, crossing analyses between Gd and Re were performed, using the fluorescent marker strain Re-mCherry (Perroud et al 2011, Perroud et al 2019. Re developed on average 100% sporophytes per gametophore, of which 0.79% were crosses ( Fig.…”

“…When no fertilization takes place, P. patens is known to continue gametangiogenesis, leading to the accumulation of multiple archegonia per gametophore (Landberg et al 2013, Sanchez-Vera et al 2017. Under crossing conditions using the male fertile Re-mCherry line (Perroud et al 2019) a close to normal amount of sporophytes (94%) could be observed, with 100% of them being crosses (Fig. 4A, light and dark green), suggesting a defect in the male reproductive apparatus.…”

“…P. patens ecotypes Gd (Rensing et al 2008) and Reute (Hiss et al 2017) were cultivated under the same conditions as described in (Hiss et al 2017) for sporophyte development, and for crossing analysis as described in (Perroud et al 2019). For gametophytic stage phenotyping, protonemal cells were placed on 9cm Petri dishes with solid minimal medium (1% w/v agar, Knop's medium, (Knop 1868)) enclosed with 3M micropore tape and inoculated 7 days for protonema and in total 10 days for gametophore analysis in long-day (LD) conditions (70 μmol m −2 s −1 , 16h light, 8h dark, 22°C).…”

“…S11) was performed at 30 days after watering as described in (Hiss et al 2017) using a Leica S8Apo binocular (Leica, Wetzlar, Germany). Counting of the crosses was performed according to (Perroud et al 2019) using a fluorescence stereomicroscope SteREO LumarV12 (Carl Zeiss, Oberkochen, Germany). For counting of the selfed F1, at least five independent biological replicates with in total 537 to 742 gametophores per sample were analysed.…”

Molecular mechanisms of plant acclimation to changing environments

“…This LOX readily oxidises polyunsaturated fatty acids even in vitro, making it a valuable pioneer enzyme for the study of algal LOXs in general. Similarly, in their study of ecotypes of the moss Physcomitrella, Perroud et al (2019) establish the highly fertile ecotype Reute as a new background for transformation and outcrossing to other ecotypes. They also created a Reute strain expressing the red fluorescent protein mCherry that allows easy detection of offspring from crosses between divergent ecotypes.…”