In:
PLOS Genetics, Public Library of Science (PLoS), Vol. 17, No. 9 ( 2021-9-7), p. e1009725-
Abstract:
Large-scale mutant libraries have been indispensable for genetic studies, and the development of next-generation genome sequencing technologies has greatly advanced efforts to analyze mutants. In this work, we sequenced the genomes of 660 Chlamydomonas reinhardtii acetate-requiring mutants, part of a larger photosynthesis mutant collection previously generated by insertional mutagenesis with a linearized plasmid. We identified 554 insertion events from 509 mutants by mapping the plasmid insertion sites through paired-end sequences, in which one end aligned to the plasmid and the other to a chromosomal location. Nearly all (96%) of the events were associated with deletions, duplications, or more complex rearrangements of genomic DNA at the sites of plasmid insertion, and together with deletions that were unassociated with a plasmid insertion, 1470 genes were identified to be affected. Functional annotations of these genes were enriched in those related to photosynthesis, signaling, and tetrapyrrole synthesis as would be expected from a library enriched for photosynthesis mutants. Systematic manual analysis of the disrupted genes for each mutant generated a list of 253 higher-confidence candidate photosynthesis genes, and we experimentally validated two genes that are essential for photoautotrophic growth, CrLPA3 and CrPSBP4 . The inventory of candidate genes includes 53 genes from a phylogenomically defined set of conserved genes in green algae and plants. Altogether, 70 candidate genes encode proteins with previously characterized functions in photosynthesis in Chlamydomonas , land plants, and/or cyanobacteria; 14 genes encode proteins previously shown to have functions unrelated to photosynthesis. Among the remaining 169 uncharacterized genes, 38 genes encode proteins without any functional annotation, signifying that our results connect a function related to photosynthesis to these previously unknown proteins. This mutant library, with genome sequences that reveal the molecular extent of the chromosomal lesions and resulting higher-confidence candidate genes, will aid in advancing gene discovery and protein functional analysis in photosynthesis.
Type of Medium:
Online Resource
ISSN:
1553-7404
DOI:
10.1371/journal.pgen.1009725
DOI:
10.1371/journal.pgen.1009725.g001
DOI:
10.1371/journal.pgen.1009725.g002
DOI:
10.1371/journal.pgen.1009725.g003
DOI:
10.1371/journal.pgen.1009725.g004
DOI:
10.1371/journal.pgen.1009725.g005
DOI:
10.1371/journal.pgen.1009725.t001
DOI:
10.1371/journal.pgen.1009725.t002
DOI:
10.1371/journal.pgen.1009725.s001
DOI:
10.1371/journal.pgen.1009725.s002
DOI:
10.1371/journal.pgen.1009725.s003
DOI:
10.1371/journal.pgen.1009725.s004
DOI:
10.1371/journal.pgen.1009725.s005
DOI:
10.1371/journal.pgen.1009725.s006
DOI:
10.1371/journal.pgen.1009725.s007
DOI:
10.1371/journal.pgen.1009725.s008
DOI:
10.1371/journal.pgen.1009725.s009
DOI:
10.1371/journal.pgen.1009725.s010
Language:
English
Publisher:
Public Library of Science (PLoS)
Publication Date:
2021
detail.hit.zdb_id:
2186725-2
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