Abstract
BackgroundThe liverwort Marchantia polymorpha occupies a crucial position in land plant evolution and provides the opportunity to investigate adaptations to a terrestrial plant life style. Marchantia reverse genetic analyses have thus far been conducted by employing a homologous recombination approach, which yields an efficiency of around 3%. Availability of the characterized and suitable endogenous MpEF1α promoter prompted us to establish the TALEN gene targeting technique for Marchantia.ResultsHere, two different TALEN techniques, using custom and self-assembled TALEN constructs, were applied and compared. The MpNOP1 gene was selected as a candidate gene, as the respective knockout mutant has been shown to lack air chamber formation, representing an easily traceable phenotype. We demonstrate that both TALEN approaches are successful in Marchantia yielding high gene targeting efficiencies of over 20%. Investigation of selected G1 up to G4 generations proved the stability of the knockout mutants. In 392 analyzed T1 plants, no additional phenotypes were observed and only one chimeric knockout plant was detected after an extended cultivation period. Interestingly, two out of the 24 sequenced mutants harbored indels causing in-frame mutations and revealed novel Mpnop1-related phenotypes. This demonstrates the potential to detect crucial amino acids and motives of targeted proteins, which is of special interest for essential genes where full knockouts are lethal. The FastTALE™ TALEN assembly kit enables the rapid assembly and ligation of the TALEN arms within half a day. For transformations, custom and assembled constructs were subcloned into Marchantia binary vectors possessing the MpEF1α promoter.ConclusionConsidering time, costs and practicability, the assembly TALEN approach represents a rapid and highly efficient gene targeting system to generate Marchantia knockout mutants, which can be further adapted for future advanced genome-editing applications.
Highlights
The liverwort Marchantia polymorpha occupies a crucial position in land plant evolution and provides the opportunity to investigate adaptations to a terrestrial plant life style
We demonstrate that Marchantia knockout mutants can be generated with an efficiency of over 20% and anticipate that this technique will further advance the use of this novel model organism to investigate the evolution of land plant gene functions
Different strategies, such as custom TALENs supplied by a company or self-assembled TALENs generated by kits can be employed to generate the two TALEN arms
Summary
The liverwort Marchantia polymorpha occupies a crucial position in land plant evolution and provides the opportunity to investigate adaptations to a terrestrial plant life style. Whereas ancient polyploidization events increased the genome content in the moss model organism Physcomitrella patens [8], evidence for increased chromosome numbers is lacking for liverworts such as Marchantia that have likely experienced a low rate of chromosomal evolution. These findings, together with the relatively small genome size of about 280 Mb [9] and an easy cultivation in the laboratory spurred the development of molecular techniques to establish Marchantia as a basal land plant model organism.
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