Construction of an Indexed Mutant Library to Enable 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, the use of this organism has been limited by our inability to easily generate mutants in specific genes of interest. To overcome this limitation, we developed approaches for high-throughput generation, propagation, cryogenic maintenance and mapping of random insertional mutants and were able to construct an indexed, stably maintained mutant library. Combinatorial pooling of the insertional mutants coupled with deep sequencing of genomic sequences flanking the insertion cassette (20-21 bp of genomic sequence generated by a procedure called ChlaMmeSeq) allowed us to map the site of insertion for each mutant strain. To overcome mapping errors due to short insertions of DNA between the insertion cassette and the flanking genomic sequence, the DNA sequence flanking insertion sites was confirmed to >500bp by using a novel procedure called Retrieval of Insertion site Sequences in Chlamydomonas (RISC). The identified sites of insertion were divided into high- and low-confidence sets containing 1,536 mutants and 5,242 mutants, respectively. The high-confidence mutants cover 8% of the genes in C. reinhardtii genome. To demonstrate the power of this library for reverse genetics, we analyzed mutants disrupted in proteins found in the algal lipid droplet proteome, and identified a novel gene required for triacylglycerol synthesis and normal cytokinesis. Mutants in this library are available for distribution to the public through the Chlamydomonas Resource Center. Based on these results, we have also generated a larger scale library, which will cover approximately 80% of Chlamydomonas genes with at least one allele. Insertion site mapping for this library is in progress.