Selection of a histidine auxotrophic

Area of Science:

• Plant molecular biology
• Genetics and genomics
• Biotechnology

Background:

• Marchia polymorpha is a key model liverwort with growing molecular resources.
• Development of novel genetic tools is crucial for advancing research in this species.

Purpose of the Study:

• To create a histidine auxotrophic strain of M. polymorpha.
• To develop a novel auxotrophic selective marker gene for M. polymorpha.

Main Methods:

• CRISPR/Cas9 genome editing was used to mutate the IMIDAZOLEGLYCEROL-PHOSPHATE DEHYDRATASE (IGPD) gene, creating a histidine auxotrophic mutant (igpd).
• A modified IGPD gene (IGPDm) was engineered as a selective marker, resistant to CRISPR/Cas9 targeting.
• The igpd mutant was complemented by transformation with the IGPDm gene.

Main Results:

• A histidine auxotrophic M. polymorpha strain (igpd) was successfully generated, requiring histidine for growth.
• The IGPDm gene functioned as an effective auxotrophic selective marker, enabling complementation of the igpd mutant.
• Transgenic M. polymorpha lines were produced using the IGPDm marker in the igpd background, eliminating the need for antibiotic selection.

Conclusions:

• The histidine auxotrophic strain igpd and the IGPDm selective marker represent valuable new molecular tools for M. polymorpha research.
• These tools facilitate gene editing and the development of transgenic lines in M. polymorpha without antibiotic resistance markers.