title: An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii.
abstract: The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (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 1935 mapped mutants, representing disruptions in 1562 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 library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305.
authors.forename: Xiaobo
authors.identifier: http://orcid.org/0000-0003-3951-9646
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305.
authors.forename: Ru
authors.identifier: http://orcid.org/0000-0002-4860-7800
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305.
authors.forename: Weronika
authors.identifier: http://orcid.org/0000-0002-8388-1303
authors.initials: W
authors.lastname: Patena
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305.
authors.forename: Spencer S
authors.initials: SS
authors.lastname: Gang
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305.
authors.forename: Sean R
authors.initials: SR
authors.lastname: Blum
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305.
authors.forename: Nina
authors.initials: N
authors.lastname: Ivanova
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Yue
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305.
authors.forename: Jacob M
authors.identifier: http://orcid.org/0000-0001-5139-8707
authors.initials: JM
authors.lastname: Robertson
authors.affiliation: Department of Plant Biology, University of Minnesota, St. Paul, Minnesota 55108.
authors.forename: Paul A
authors.identifier: http://orcid.org/0000-0003-0028-7753
authors.initials: PA
authors.lastname: Lefebvre
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095.
authors.forename: Sorel T
authors.identifier: http://orcid.org/0000-0001-7090-5719
authors.initials: ST
authors.lastname: Fitz-Gibbon
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305.
authors.forename: Arthur R
authors.initials: AR
authors.lastname: Grossman
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305 mjonikas@carnegiescience.edu.
authors.forename: Martin C
authors.identifier: http://orcid.org/0000-0002-9519-6055
authors.initials: MC
authors.lastname: Jonikas
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000235:genetics* / Q000502:physiology; D002736:Chloroplasts / Q000378:metabolism; D002874:Chromosome Mapping; D005227:Fatty Acids / Q000378:metabolism; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D008055:Lipids / Q000032:analysis; D016254:Mutagenesis, Insertional; D009154:Mutation; D010641:Phenotype; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D020543:Proteome; D059386:Reverse Genetics; D017422:Sequence Analysis, DNA; D014280:Triglycerides / Q000378:metabolism*
chemical_list: D005227:Fatty Acids; D008055:Lipids; D010940:Plant Proteins; D020543:Proteome; D014280:Triglycerides