bioRxiv preprint doi: https://doi.org/10.1101/2021.07.27.453968; this version posted July 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Complete Mitochondrial and Plastid DNA Sequences of the Freshwater Green Microalga Medakamo hakoo

Mari Takusagawa1,2, Shoichi Kato3, Sachihiro Matsunaga3,4, Shinichiro Maruyama5,6, Yayoi Tsujimoto-Inui3, Hisayoshi Nozaki7, Fumi Yagisawa8,9, Mio Ohnuma10, Haruko Kuroiwa11, Tsuneyoshi Kuroiwa11 & Osami Misumi2#

1Department of Botany, Graduate School of Science, Kyoto University, Kyoto, Japan 2Department of Biology, Faculty of Science, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi, Japan 3Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Japan 4Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan 5Department of Ecological Developmental Adaptability Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan. 6Graduate School of Humanities and Sciences, Ochanomizu University, Bunkyo-ku, Tokyo 112-8610, Japan. 7Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan 8Center for Research Advancement and Collaboration, University of the Ryukyus, Okinawa, Japan 9Graduate School of Engineering and Science, University of the Ryukyus, Okinawa, Japan 10National Institute of Technology (KOSEN), Hiroshima College, Hiroshima, Japan 11Department of Chemical and Biological Science, Faculty of Science, Japan Women’s University, Tokyo, Japan

Running Head: The complete organellar genomes of the green alga Medakamo hakoo

# Address correspondence to Osami Misumi, [email protected]

bioRxiv preprint doi: https://doi.org/10.1101/2021.07.27.453968; this version posted July 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Abstract Here we report the complete organellar genome sequences of Medakamo hakoo, a green alga identified in freshwater in Japan. It has 90.8-kb plastid and 36.5-kb mitochondrial genomes containing 80 and 33 putative protein coding genes, respectively, representing the smallest organellar genome among currently known core Trebouxiophyceae.

Body The genomes of various organisms have been sequenced, and knowledge has been accumulated about not only biodiversity but also universal genes that are conserved among all organisms. These findings will be updated as new species are discovered; therefore, exploring new species will continue to be important. Recently, we discovered a green alga, Medakamo hakoo, in freshwater in Japan (1, 2). Based on the preliminary microphotometric observation, we predicted that it would have the smallest organellar and nuclear genomes among green plants, which prompted us to investigate its genomes. Whole-genome sequencing was performed with the PacBio RS II system (Pacific Biosciences, Menlo Park, CA, USA), and the read sequences were assembled de novo with the RS HGAP Assemble.3 program run by SMRT Analysis software 2.3.0 (Pacific Biosciences). Genes encoded by the chloroplast/mitochondrial genomes were annotated using GeSeq (3) with the following organellar genomes as references: Chlorella vulgaris (NC_001865/NC_045362), Coccomyxa subellipsoidea C-169 (NC_015084/NC_015316), and Botryococcus braunii (NC_025545/NC_027722) because M. hakoo belongs to Trebouxiophyceae, as described in Kato et al. (submitted). StructRNAfinder (4) and RNAweasel (5) were used to predict rRNA. We obtained two contigs representing the organellar genomes (Fig.1). The chloroplast genome (cpDNA) is 90,872 bp and has a GC content of 41.4%. We functionally annotated 80 protein-coding genes, including 31

photosynthesis-related genes (components of photosystem I and II, the cytochrome b6f complex, and ATP synthase subunits) and 29 tRNA and 2 rRNA genes. The cpDNA of M. hakoo is slightly smaller than that of its most closely related alga, Choricystis parasitica (6); they share most genes but not their order. The mitochondrial genome (mtDNA) of M. hakoo is 36,544 bp and has a GC content of 35.2%. It is the smallest in size and the gene density appears high compared with other Trebouxiophyceae mtDNAs. It comprises 25 tRNAs, 3 rRNAs, and 33 predicted protein-coding genes, including 18 respiratory chain complex genes and ATP synthase, as well as tatC and a homing endonuclease gene. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.27.453968; this version posted July 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Another notable characteristic of the mtDNA was that almost all the genes were localized to a single strand. This mtDNA characteristic is also observed among core Trebouxiales, including Trebouxiophyceae sp. MX-AZ01 (7) and Coccomyxa subellipsoidea C-169 (8) and Botoryococcus braunii (9,10); however, the group II introns of the trnH (gug), and trnW (cca) genes conserved in these species (10) were not predicted in M. hakoo. Normally in Trebouxiophyceae, rpl10 gene was located next to rps19 (10), but M. hakoo had another ORF that was not similar to other known genes. As expected, the size of the M. hakoo organellar genome was relatively small, and the gene density was high. It will be important for future algal research to focus on how mitochondrial genes encoded in a single strand are expressed (whether they form operons), and conversely how the separately encoded chloroplast rRNA genes, which should normally form an operon structure, exhibit coordinated transcription.

Public availability and accession numbers. The cpDNA and mtDNA genome sequences were deposited in DNA Data Bank of Japan (https://www.ddbj.nig.ac.jp/index-e.html) with accession numbers LC604816 and LC604817, respectively.

Acknowledgments This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (16H04813 and 19H03260) to T.K. and by JST-CREST (Grant Number JPMJCR20S6) to S.Matsunaga. The authors thank Mr. Brody Frink (Kyoto University) for editing a draft of this manuscript.

Figure legend Fig. 1. Complete chloroplast and mitochondrial genomes of Medakamo hakoo. The both genomes were plotted using OrganellarGenomeDRAW (11). The inner gray circle illustrates the G+C content of the genome.

References 1. Kuroiwa T, Ohnuma M, Nozaki H, Imoto Y, Misumi O, Kuroiwa H. 2015. Cytological Evidence of Cell-Nuclear Genome Size of a New Ultra-Small Unicellular Freshwater Green Alga, “Medakamo hakoo” strain M-hakoo 311 I. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.27.453968; this version posted July 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Comparison with Cyanidioschyzon merolae and Ostreococcus tauri. Cytologia 80:143-150

2. Kuroiwa T, Ohnuma M, Imoto Y, Misumi O, Nagata N, Miyakawa I, Fujishima M, Yagisawa F, Kuroiwa H. 2016. Genome Size of the Ultrasmall Unicellular Freshwater Green Alga, Medakamo hakoo 311, as Determined by Staining with 4′,6-Diamidino-2-phenylindole after Microwave Oven Treatments: II. Comparison with Cyanidioschyzon merolae, Saccharomyces cerevisiae (n, 2n), and Chlorella variabilis. Cytologia 81:69-76

3. Tillich M, Lehwark P, Pellizzer T, Ulbricht-Jones ES, Fischer A, Bock R, Greiner S 2017. GeSeq – versatile and accurate annotation of organelle genomes. Nucleic Acids Research 45: W6-W11

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5. Lang BF, Laforest MJ, Burger G. 2007. Mitochondrial introns: a critical view. Trends Genet. 23:119-25

6. Lemieux C, Otis C, Turmel M. 2014. Chloroplast phylogenomic analysis resolves deep-level relationships within the green algal class Trebouxiophyceae. BMC Evol Biol. 14:211

7. Servín-Garcidueñas LE, Martínez-Romero E. 2012. Complete Mitochondrial and Plastid Genomes of the Green Microalga Trebouxiophyceae sp. Strain MX-AZ01 Isolated from a Highly Acidic Geothermal Lake. Eukaryotic Cell, 11:1417-1418

8. Smith DR, Burki F, Yamada T, Grimwood J, Grigoriev IV, et al. 2011. The GC-Rich Mitochondrial and Plastid Genomes of the Green Alga Coccomyxa Give Insight into the Evolution of Organelle DNA Nucleotide Landscape. PLOS ONE 6(8): e23624 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.27.453968; this version posted July 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

9. Blifernez-Klassen O, Wibberg D, Winkler A, Blom J, Goesmann A, Kalinowski J, Kruse O. 2016. Complete chloroplast and mitochondrial genome sequences of the hydrocarbon oil-producing green microalga Botryococcus braunii Race B (Showa). Genome Announc. 4: e00524-16

10. Martínez-Alberola F, Barreno E, Casano LM, Gasulla F, Molins A, del Campo EM. 2019. Dynamic evolution of mitochondrial genomes in Trebouxiophyceae, including the first completely assembled mtDNA from a lichen-symbiont microalga (Trebouxia sp. TR9). Scientific Reports 9:8209

11. Greiner S, Lehwark P, Bock R. 2019. OrganellarGenomeDRAW (OGDRAW) version 1.3.1: expanded toolkit for the graphical visualization of organellar genomes. Nucleic Acids Research 47: W59-W64 trnR-CCG

trnM-CAU trnR-UCU

ycf3

psbM

psbN

trnS-GCU psbH LSU psbT

psbB

minD trnR-ACG

psbA

psaA trnR-ACG atpE ORF atpB psaB trnN-GUU rps3* rps19 chlL trnN-GUU cemA* trnT-UGU GIY-HE rps14 chlN trnC-GCA rps4

rrnL chlB atp1

rpl16

bioRxiv preprint doi: https://doi.org/10.1101/2021.07.27.453968; this version posted July 27, 2021. The copyright holder for this preprint rps13 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. petA trnA-UGC trnI-GAU nad1 rps10 rpoB* trnP-UGG trnG-UCC ycf1* trnH-GUG trnL-UAG trnV-UAC trnS-UGA rrn5 nad4 trnK-UUU trnM-CAU cysT rpoC1* rpl32 cox1 tilS trnF-GAA rps12 ycf47 psbZ rps7 rps11 rpoC2* trnA-UGC Medakamo hakoo trnS-GCU Medakamo hakoo trnK-UUU trnF-GAA cox2 trnQ-UUG chloroplast genome trnM-CAU mitochondrial genome trnD-GUC trnM-CAU psbI ftsH trnL-UAA cox3 90,934 bp trnL-CAA ccsA 36,544 bp trnY-GUA rrnS

rrnS atp6

trnH-GUG nad4L psbJ psbL rps2 trnI-GAU psbF psbE rpl12 cob rps9 trnD-GUC trnQ-UUG nad2 trnG-UCC rpoA trnR-UCU nad3 rps11 trnL-UAA

atpI atp9 rps8 trnW-CCA rpl36 atpH trnP-UGG rpl5 infA atp4 rps2 atpF rpl14 rpl16 atp8 rps3 trnE-UUC trnW-CCA rpl5 atpA trnY-GUA rps19 nad6 nad9 trnS-UGA rpl2

CCG-Gnrt rpl23

nad5 petB petD psaJ psbC psbK trnL-GAGpsaM rps12 ycf12 rps7 psbD nad7 tufA ycf4

rpl19 cysA

clpP accD LHE petL rpl20 petG photosystem I chlI rps18 trnL-UAG photosystem II tatC

Casp rbcL cytochrome b/f complex trnC-GCA

ATP synthase rps14 complex I (NADH dehydrogenase) psaI RubisCO large subunit rps4 complex III (ubichinol cytochrome c reductase) trnE-UUC ycf20 trnV-UAC trnMf-CAU RNA polymerase trnT-UGU complex IV (cytochrome c oxidase) ribosomal proteins (SSU) trnM-CAU ATP synthase ribosomal proteins (LSU) ribosomal proteins (SSU) transfer RNAs ribosomal proteins (LSU) ribosomal RNAs other genes clpP, matK ORFs other genes transfer RNAs hypothetical chloroplast reading frames (ycf) ribosomal RNAs