High-throughput sequencing of the chloroplast and mitochondrion of Chlamydomonas reinhardtii to generate improved de novo assemblies, analyze expression patterns and transcript speciation, and evaluate diversity among laboratory strains and wild isolates

doi:10.1111/tpj.13788

Title: High-throughput sequencing of the chloroplast and mitochondrion of Chlamydomonas reinhardtii to generate improved de novo assemblies, analyze expression patterns and transcript speciation, and evaluate diversity among laboratory strains and wild isolates

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Abstract

Chlamydomonas reinhardtii is a unicellular chlorophyte alga that is widely studied as a reference organism for understanding photosynthesis, sensory and motile cilia, and for development of an algal-based platform for producing biofuels and bio-products. Its highly repetitive, ~205-kbp circular chloroplast genome and ~15.8-kbp linear mitochondrial genome were sequenced prior to the advent of high-throughput sequencing technologies. Here, high coverage shotgun sequencing was used to assemble both organellar genomes de novo. These new genomes correct dozens of errors in the prior genome sequences and annotations. Gen-ome sequencing coverage indicates that each cell contains on average 83 copies of the chloroplast genome and 130 copies of the mitochondrial genome. Using protocols and analyses optimized for organellar tran-scripts, RNA-Seq was used to quantify their relative abundances across 12 different growth conditions. Forty-six percent of total cellular mRNA is attributable to high expression from a few dozen chloroplast genes. RNA-Seq data were used to guide gene annotation, to demonstrate polycistronic gene expression, and to quantify splicing of psaA and psbA introns. In contrast to a conclusion from a recent study, we found that chloroplast transcripts are not edited. Unexpectedly, cytosine-rich polynucleotide tails were observed at the 3’-end of all mitochondrial transcripts. A comparative genomicsmore »« less

Authors:

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Department of Chemistry and Biochemistry, University of California, Los Angeles CA 90095 USA
Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles CA 90095 USA
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland WA 99352 USA

Publication Date:: 2018-01-07

Research Org.:: Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

Sponsoring Org.:: USDOE

OSTI Identifier:: 1455414

Report Number(s):: PNNL-SA-135188
Journal ID: ISSN 0960-7412; KP1704020

DOE Contract Number:: AC05-76RL01830

Resource Type:: Journal Article

Journal Name:: The Plant Journal

Additional Journal Information:: Journal Volume: 93; Journal Issue: 3; Journal ID: ISSN 0960-7412

Publisher:: Society for Experimental Biology

Country of Publication:: United States

Language:: English

Subject:: plastid; organelles; RNA-Seq; transcript editing; trans-splicing; rpoC1; rps2; Wendy transposon; ftsH; ycf1.

Citation Formats


                    Gallaher, Sean D., Fitz-Gibbon, Sorel T., Strenkert, Daniela, Purvine, Samuel O., Pellegrini, Matteo, and Merchant, Sabeeha S. High-throughput sequencing of the chloroplast and mitochondrion of Chlamydomonas reinhardtii to generate improved de novo assemblies, analyze expression patterns and transcript speciation, and evaluate diversity among laboratory strains and wild isolates.  United States: N. p., 2018. 
        Web.  doi:10.1111/tpj.13788.

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                    Gallaher, Sean D., Fitz-Gibbon, Sorel T., Strenkert, Daniela, Purvine, Samuel O., Pellegrini, Matteo, & Merchant, Sabeeha S. High-throughput sequencing of the chloroplast and mitochondrion of Chlamydomonas reinhardtii to generate improved de novo assemblies, analyze expression patterns and transcript speciation, and evaluate diversity among laboratory strains and wild isolates.  United States.  doi:10.1111/tpj.13788.

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                    Gallaher, Sean D., Fitz-Gibbon, Sorel T., Strenkert, Daniela, Purvine, Samuel O., Pellegrini, Matteo, and Merchant, Sabeeha S. Sun .  
        "High-throughput sequencing of the chloroplast and mitochondrion of Chlamydomonas reinhardtii to generate improved de novo assemblies, analyze expression patterns and transcript speciation, and evaluate diversity among laboratory strains and wild isolates".  United States.  doi:10.1111/tpj.13788.

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@article{osti_1455414,

  title        = {High-throughput sequencing of the chloroplast and mitochondrion of Chlamydomonas reinhardtii to generate improved de novo assemblies, analyze expression patterns and transcript speciation, and evaluate diversity among laboratory strains and wild isolates},

  author       = {Gallaher, Sean D. and Fitz-Gibbon, Sorel T. and Strenkert, Daniela and Purvine, Samuel O. and Pellegrini, Matteo and Merchant, Sabeeha S.},

  abstractNote = {Chlamydomonas reinhardtii is a unicellular chlorophyte alga that is widely studied as a reference organism for understanding photosynthesis, sensory and motile cilia, and for development of an algal-based platform for producing biofuels and bio-products. Its highly repetitive, ~205-kbp circular chloroplast genome and ~15.8-kbp linear mitochondrial genome were sequenced prior to the advent of high-throughput sequencing technologies. Here, high coverage shotgun sequencing was used to assemble both organellar genomes de novo. These new genomes correct dozens of errors in the prior genome sequences and annotations. Gen-ome sequencing coverage indicates that each cell contains on average 83 copies of the chloroplast genome and 130 copies of the mitochondrial genome. Using protocols and analyses optimized for organellar tran-scripts, RNA-Seq was used to quantify their relative abundances across 12 different growth conditions. Forty-six percent of total cellular mRNA is attributable to high expression from a few dozen chloroplast genes. RNA-Seq data were used to guide gene annotation, to demonstrate polycistronic gene expression, and to quantify splicing of psaA and psbA introns. In contrast to a conclusion from a recent study, we found that chloroplast transcripts are not edited. Unexpectedly, cytosine-rich polynucleotide tails were observed at the 3’-end of all mitochondrial transcripts. A comparative genomics analysis of eight laboratory strains and 11 wild isolates of C. reinhardtii identified 2658 variants in the organellargenomes, which is 1/10th as much genetic diversity as is found in the nucleus.},

  doi          = {10.1111/tpj.13788},

  journal      = {The Plant Journal},

  issn         = {0960-7412},

  number       = 3,

  volume       = 93,

  place        = {United States},

  year         = {2018},

  month        = {1}

}

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DOI: 10.1111/tpj.13788

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