Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii.
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...
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2021
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oai:doaj.org-article:59d15a396265411ab7cb8cd094b796d12021-12-02T20:03:03ZDiscovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii.1553-73901553-740410.1371/journal.pgen.1009725https://doaj.org/article/59d15a396265411ab7cb8cd094b796d12021-09-01T00:00:00Zhttps://doi.org/10.1371/journal.pgen.1009725https://doaj.org/toc/1553-7390https://doaj.org/toc/1553-7404Large-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.Setsuko WakaoPatrick M ShihKatharine GuanWendy SchackwitzJoshua YeDhruv PatelRobert M ShihRachel M DentMansi ChovatiaAditi SharmaJoel MartinChia-Lin WeiKrishna K NiyogiPublic Library of Science (PLoS)articleGeneticsQH426-470ENPLoS Genetics, Vol 17, Iss 9, p e1009725 (2021) |
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DOAJ |
| collection |
DOAJ |
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EN |
| topic |
Genetics QH426-470 |
| spellingShingle |
Genetics QH426-470 Setsuko Wakao Patrick M Shih Katharine Guan Wendy Schackwitz Joshua Ye Dhruv Patel Robert M Shih Rachel M Dent Mansi Chovatia Aditi Sharma Joel Martin Chia-Lin Wei Krishna K Niyogi Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii. |
| description |
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. |
| format |
article |
| author |
Setsuko Wakao Patrick M Shih Katharine Guan Wendy Schackwitz Joshua Ye Dhruv Patel Robert M Shih Rachel M Dent Mansi Chovatia Aditi Sharma Joel Martin Chia-Lin Wei Krishna K Niyogi |
| author_facet |
Setsuko Wakao Patrick M Shih Katharine Guan Wendy Schackwitz Joshua Ye Dhruv Patel Robert M Shih Rachel M Dent Mansi Chovatia Aditi Sharma Joel Martin Chia-Lin Wei Krishna K Niyogi |
| author_sort |
Setsuko Wakao |
| title |
Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii. |
| title_short |
Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii. |
| title_full |
Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii. |
| title_fullStr |
Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii. |
| title_full_unstemmed |
Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii. |
| title_sort |
discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of chlamydomonas reinhardtii. |
| publisher |
Public Library of Science (PLoS) |
| publishDate |
2021 |
| url |
https://doaj.org/article/59d15a396265411ab7cb8cd094b796d1 |
| work_keys_str_mv |
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