Expanding the toolkit for ploidy manipulation in Chlamydomonas reinhardtii.

Whole-genome duplications, widely observed in plant lineages, have significant evolutionary and ecological impacts. Yet, our current understanding of the direct implications of ploidy shifts on short- and long-term plant evolution remains fragmentary, necessitating further investigations across multiple ploidy levels. Chlamydomonas reinhardtii is a valuable model organism with profound potential to study the impact of ploidy increase on the longer term in a laboratory environment. This is partly due to the ability to increase the ploidy level. We developed a strategy to engineer ploidy in C. reinhardtii using noninterfering, antibiotic, selectable markers. This approach allows us to induce higher ploidy levels in C. reinhardtii and is applicable to field isolates, which expands beyond specific auxotroph laboratory strains and broadens the genetic diversity of parental haploid strains that can be crossed. We implement flow cytometry for precise measurement of the genome size of strains of different ploidy. We demonstrate the creation of diploids, triploids, and tetraploids by engineering North American field isolates, broadening the application of synthetic biology principles in C. reinhardtii. However, our newly formed triploids and tetraploids show signs of rapid aneuploidization. Our study greatly facilitates the application of C. reinhardtii to study polyploidy, in both fundamental and applied settings.