Here, we present a 231 Mb draft genome of the centric diatom Conticribra weissflogii CCMP1336. Comparative genomics of C. weissflogii and other Ochrophyta support the existence of unique carbon-concentrating mechanisms and chitin metabolic processes in diatoms. The whole-genome project is available at CNSA (https://db.cngb.org/search/project/CNP0001903/)

Eva C. Sonnenschein

English

Cronfa at Swansea University

Protist 172 (2021) 125845http://www.elsevier.de/protisPublished online date 16 November 2021 ProtistORIGINAL PAPERProtist Genome ReportsThe Draft Genome of the Centric DiatomConticribra weissflogii(Coscinodiscophyceae, Ochrophyta)https://doi.org/10.1016/j.protis.2021.1258451434-4610/ 2021 The Author(s). Published by Elsevier GmbH.This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).2Corresponding author;e-mail evaso@bio.dtu.dk (E.C. Sonnenschein).1These authors contributed equally.Linzhou Li a,b,1, Hongli Wang b,c,1, Sibo Wang b, Yan Xu b,c, Hongping Liang b,c,Huan Liu b, and Eva C. Sonnenschein a,2aDepartment of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads 221, 2800Kgs. Lyngby, DenmarkbState Key Laboratory of Agricultural Genomics, BGI-Shenzhen, Shenzhen 518083, ChinacCollege of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, ChinaSubmitted September 6, 2021; Accepted November 10, 2021Here, we present a 231 Mb draft genome of the centric diatom Conticribra weissflogii CCMP1336. Com-parative genomics of C. weissflogii and other Ochrophyta support the existence of unique carbon-concentrating mechanisms and chitin metabolic processes in diatoms. The whole-genome projectis available at CNSA (https://db.cngb.org/search/project/CNP0001903/). 2021 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BYlicense (http://creativecommons.org/licenses/by/4.0/).Key words: Diatom; genomics; photosynthesis; carbon-concentrating mechanism; chitin synthases.The single-celled eukaryotic diatoms belonging tothe Ochrophyta are crucial for global carbon fixationsimilar to green plants (Falkowski et al. 1998). Whilethe high photosynthetic capacity of C4 CO2-concentrating mechanisms is widespread amongplants, and was initially believed to be absent fromunicellular algae (Hopkinson et al. 2016), it wasidentified in some diatoms, such as Thalassiosirapseudonana (also referred to Cyclotella pseudo-nana) (Kustka et al. 2014) and Conticribra weiss-flogii (Reinfelder 2011) (previously known asThalassiosira weissflogii) (Stachura-Suchoples andWilliams 2009). The diatom-specific C4 pathwayappears unconventional in comparison to landplants, but the precise mechanisms remain to beresolved (Hopkinson et al. 2016).Another unique characteristic of diatoms in com-parison to other Ochrophyta and land plants aretheir silicified cell walls (Martin-Jézéquel et al.2000). Accordingly, they are central to global biosili-2 L. Li et al.cification (Mock et al. 2008) and the diatom frustuleshave gained attention for industrial applications(Rabiee et al. 2021). However, the exact composi-tion of the diatom cell wall remains difficult todecipher. In fungi, chitin is a key component of thecell wall, synthesized by chitin synthases (CHSs).It has been suggested that chitin also plays animportant role in the cell wall of diatoms such as T.pseudonana and C. weissflogii (Cheng et al. 2021;Durkin et al. 2009). Interestingly, chitin is generallyconsidered only to be present in centric rather thanpennate diatoms (Durkin et al. 2009), however,CHSs can be found in both groups (Shao et al.2019).The centric diatomC. weissflogii serves as modelsystem to study these environmentally importantprocesses (Sonnenschein et al. 2021) and is anutrient-rich and robust resource of renewable bio-mass for energy, food and feed. Although a full-length transcriptome of C. weissflogii has recentlybeen released (Cheng et al. 2021), genomic dataare still lacking. Here, we report the draft genomesequence of C. weissflogii strain CCMP1336 andtake a glance at the unique features of the C. weiss-flogii genome in comparison to other Ochrophytagenomes including the CO2-concentrating mecha-nism and the evolutionary origin of diatom CHSs.An axenic culture of C. weissflogii CCMP1336(Fig. 1A) was obtained fromNCMA (National Centerfor Marine Algae and Microbiota, East Boothbay,Maine, USA). C. weissflogii was cultivated in 1.5 Lf/2 (Guillard and Ryther 1962, 1975) culture mediumprepared with 3% Instant Ocean (IO, Aquarium Sys-tems Inc., Sarrebourg, France) in a 2 L Erlenmeyerflask with sterile aeration and incubation at 80 lmolphotons m2 s1 and 18 C for 8 days. The cellswere pelleted by centrifugation (4000  g, 10 minand 4 C), frozen in liquid nitrogen and stored at80 C until freeze-drying.Long fragment DNA was extracted using a mod-ified cetyltrimethylammonium bromide protocol fol-lowed by stLFR (single-tube long fragment reads)library construction. 150 bp paired-end reads weregenerated using MGI-SEQ. Data noise reductionwas performed to remove low-quality and duplicatedreads using SOAPfilter (v2.2) with default parame-ters. Then, the newly constructed reads wereassembled into scaffolds using Supernova (v2.1.1)according to the manufacturer’s protocol. The231 Mb draft genome assembly of C. weissflogiiCCMP1336 (accession number CNP0001903 atCNSA https://db.cngb.org/cnsa/) comprised 31,770scaffolds with a scaffold N50 of 27,127 bp(Fig. 1C). The final, assembled genome size wassimilar to the estimated genome size (228 Mb) aspredicted by GenomeScope 2.0 (Ranallo-Benavidez et al. 2020). Although the assembly isfragmented, it is more complete (contigN50 = 18.3 kb) than previously published genomesof centric diatoms, for example, Cyclotella cryptica(contig N50 = 12 kb) and Thalassiosira oceanica(contig N50 = 3.6 kb) (Lommer et al. 2012; Tralleret al. 2016). BUSCO evaluation (Waterhouse et al.2018) showed that the assembly shared 76% coreeukaryote genes, which was higher than the geno-mic assemblies of other centric diatoms (Supple-mentary Material Table S1). The evaluation of theGC-depth distribution demonstrated a good qualityof the genome assembly based on completenessand accuracy (Fig. 1B).Transposon elements were identified by employ-ing two strategies. For novel repeats, PILER (Edgarand Myers 2005) and RepeatScout (Price et al.2005) were used to search for DNA transposonsand LTR_finder (Xu and Wang 2007) was used tosearch for retrotransposons. For known repeats,RepeatMasker (www. RepeatMasker.org) and Pro-teinMask were used to search repeats based onthe existing database. All candidateswere combinedinto a custom library, and the assembly was exam-ined again based on the custom library. Finally, atotal of 54% transposon elements (TEs) were identi-fied, which is similar to the TE content of C. cryptica(53% of the genome). LTRs (long terminal repeats)and DNA transposon elements comprised approxi-mately 38% and 5% of the genome, respectively.The most common LTR was of the Ty1-copia group,which accounted for 80% of the total LTR. For geneannotation, the BRAKER2 (Brůna et al. 2021) pipe-line was used to automatically train and annotategene models. The pipeline combined evidence ofself-training models with homologous protein infor-mation to obtain more accurate genemodels. A totalof 22,823 gene models were predicted (Fig. 1C).The gene models of C. weissflogii showed similarlengths and numbers as in other centric diatoms,which implies a reliable annotation (SupplementaryMaterial Table S2). To further evaluate the genemodels, BLASTP was used to align them againstseveral protein databases (SwissProt, KEGG,KOG and TrEMBL). InterProScan was performedto identify protein domains. As a result, 80% of thegene models could be aligned to the known proteinand domain databases.Figure 1. The morphology and genome assembly statistics of C. weissflogii. (A) Light micrograph of C.weissflogii imaged with a Nikon Inverted Fluorescence Microscope-EclipseTi2. The chloroplasts are indicatedby the red color. (B) GC-depth plot showing the distribution of the GC content and average reads mapping. (C)Statistics of genome assembly and gene models of C. weissflogii. (D) The phylogenetic tree of Ochrophytagenomes constructed using the maximum-likelihood method by RAxML based on a concatenated method of201 single-copy genes with 500 bootstrap replicates.The Draft Genome of the Diatom Conticribra weissflogii 3To improve the evolutionary understanding ofThalassiosirales, OrthoFinder was applied on pub-lished genomes of Ochrophyta and C. weissflogiiand a phylogenetic tree was constructed based onsingle copy orthogroups (i.e. gene clusters with onlyone gene of each species). Supporting previouswork on the re-classification of former Thalassiosiraspecies (Alverson et al. 2011; Stachura-Suchoplesand Williams 2009), our phylogenetic analysisshowed the distinct placement of C. weissflogii (for-merly Thalassiosira weissflogii) from Cyclotellapseudonana (also Thalassiosira pseudonana) andThalassiosira oceanica (Fig. 1D).To identify differences between diatoms andother Ochrophyta, a heatmap was generated basedon the gene numbers in the orthogroups amongOchrophyta (Fig. 2A). The enrichment of the geneontology (GO) category of photosynthesis-light har-vesting (Fig. 2B and Supplementary MaterialTable S3) could indicate that diatoms may share aunique photosynthetic pathway or CO2-concentrating mechanisms (CCMs). This could sup-port the previous hypothesis that CCMs are morecommon in diatoms than in other ochrophytes(Kustka et al. 2014; Raven and Giordano 2017;(Reinfelder 2011). Furthermore, the orthogroups ofcentric diatoms were enriched in chitin metabolicprocesses supporting the general notion that occursonly in centric, not pennate diatoms (Shao et al.2019). The C. weissflogii-specific orthogroups wereenriched in similar GO categories, as well as carbo-hydrate metabolic processes and signaltransduction.To analyse the phylogenetic relatedness of chitinsynthases (CHSs) in C. weissflogii and diatoms,CHSs from representative fungi and all ochrophytesFigure 2. Comparative genomic analysis of ochrophytes. (A) Heatmap of the proteins of each orthogroup ofOchrophyta sorted according to the phylogenetic tree. The intensity of red indicates the copy number ofproteins. The pink numbers in the phylogenetic tree represent the expansion orthogroups while the bluenumbers represent the contraction orthogroups. (B) The gene ontology enrichment of the genes in diatoms(including centric and pennate diatoms), centric diatoms and C. weissflogii extracted from the nodeshighlighted in Fig. 2A. (C) Phylogenetic tree of chitin synthases. The protein sequences were retrieved from allochrophytes and several representative fungi (Torruella et al. 2015).4 L. Li et al.were retrieved based on the common conserveddomains, including Chitin_synth_1 (PF01644) andChitin_synth_2 (PF03142) (Supplementary MaterialTable S4). The phylogenetic tree was divided intotwo main clades based on the division I and IIdomains (Torruella et al. 2015; Fig. 2C). As previ-ously reported (Torruella et al. 2015), the division Iclade comprised the fungal CHSs 1/2/3 and CHSsfrom pennate diatoms. The division II included thefungal CHSs 4/5/6, CHSs from centric diatomsincluding C. weissflogii, and those from the pennatediatom Fistulifera solaris. While chitin is generallyconsidered to be present in centric, but not in pen-nate diatoms, CHS genes have previously beendetected in pennate diatoms and their function hasbeen investigated (Shao et al. 2019). Why the CHSsof the pennate diatom F. solaris are phylogeneticallycloser to those of centric rather than pennate dia-toms remains to be investigated.In conclusion, this is the first study on the nucleargenome of the model diatom C. weissflogii. Com-parative genomic analyses between diatoms andother ochrophytes indicated the existence of uniqueCCMs and chitin metabolic processes in diatoms.Mining the draft genome for biosynthetic genesusing antiSMASH (Blin et al. 2021) identified five ter-pene, two NRPS-like genes and one Type III PKSgene (Supplementary Material Table S5) suggest-ing a bioactive potential of C. weissflogii, whichcould support its use as a sustainable, biotechnolog-ical production system.Declaration of Competing InterestThe authors declare that they have no known com-peting financial interests or personal relationshipsthat could have appeared to influence the workreported in this paper.The Draft Genome of the Diatom Conticribra weissflogii 5AcknowledgementsThis work was supported by the Danish NationalResearch Foundation to the Center for Microbial Sec-ondary Metabolites (CeMiSt) (DNRF137) and by theNovo Nordisk Foundation (grants NNF20OC0064249and NNFOC0055625). This work is part of the 10KPproject (https://db.cngb.org/10kp/). This work is alsosupported by China National GeneBank (CNGB;https://www.cngb.org/).Appendix A. Supplementary DataSupplementary data to this article can be foundonline at https://doi.org/10.1016/j.protis.2021.125845.ReferencesAlverson AJ, Beszteri B, Julius ML, Theriot EC (2011)The model marine diatom Thalassiosira pseudonana likelydescended from a freshwater ancestor in the genusCyclotella. BMC Evol Biol 11:125Blin K, Shaw S, Kloosterman AM, Charlop-Powers Z, VanWezel GP, Medema MH, Weber T (2021) AntiSMASH 6.0:Improving cluster detection and comparison capabilities.Nucleic Acids Res 49:W29–W35Brůna T, Hoff KJ, Lomsadze A, Stanke M, BorodovskyM (2021) BRAKER2: automatic eukaryotic genomeannotation with GeneMark-EP+ and AUGUSTUSsupported by a protein database. 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The Draft Genome of the Centric Diatom Conticribra weissflogii (Coscinodiscophyceae, Ochrophyta)

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The Draft Genome of the Centric Diatom Conticribra weissflogii (Coscinodiscophyceae, Ochrophyta)

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