An indexed, mapped mutant library enables reverse genetics studies of biological processes in Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii is a leading single-celled model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods which: (1) Enable easy propagation on agar and cryogenic maintenance of tens of thousands of C. reinhardtii strains; (2) Identify mutant insertion sites and physical coordinates in such collections; (3) Validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1,935 mapped mutants, representing disruptions in 1,562 genes. We further characterized randomly selected mutants, and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this librar...