Comparative Transcriptome of Wild Type and Selected Strains of the Microalgae Tisochrysis lutea Provides Insights into the Genetic Basis, Lipid Metabolism and the Life Cycle

The applied exploitation of microalgae cultures has to date almost exclusively involved the use of wild type strains, deposited over decades in dedicated culture collections. Concomitantly, the concept of improving algae with selection programs for particular specific purposes is slowly emerging. Studying since a decade an economically and ecologically important haptophyte Tisochrysis lutea (Tiso), we took advantage of the availability of wild type (Tiso-Wt) and selected (Tiso-S2M2) strains to conduct a molecular variations study. This endeavour presented substantial challenges: the genome assembly was not yet available, the life cycle unknown and genetic diversity of Tiso-Wt poorly documented. This study brings the first molecular data in order to set up a selection strategy for that microalgae. Following high-throughput Illumina sequencing, transcriptomes of Tiso-Wt and Tiso-S2M2 were de novo assembled and annotated. Genetic diversity between both strains was analyzed and revealed a clear conservation, while a comparison of transcriptomes allowed identification of polymorphisms resulting from the selection program. Of 34,374 transcripts, 291 were differentially expressed and 165 contained positional polymorphisms (SNP, Indel). We focused on lipid over-accumulation of the Tiso-S2M2 strain and 8 candidate genes were identified by combining analysis of positional polymorphism, differential expression levels, selection signature and by study of putative gene function. Moreover, genetic analysis also suggests the existence of a sexual cycle and genetic recombination in Tisochrysis lutea.

[1]  Jean-Michel Claverie,et al.  The Chlorella variabilis NC64A Genome Reveals Adaptation to Photosymbiosis, Coevolution with Viruses, and Cryptic Sex[C][W] , 2010, Plant Cell.

[2]  A. Cembella,et al.  Comparative Genomic and Transcriptomic Characterization of the Toxigenic Marine Dinoflagellate Alexandrium ostenfeldii , 2011, PloS one.

[3]  P Stothard,et al.  The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences. , 2000, BioTechniques.

[4]  L. Audeguin,et al.  Transposable Elements Are a Major Cause of Somatic Polymorphism in Vitis vinifera L. , 2012, PloS one.

[5]  E. Mardis The impact of next-generation sequencing technology on genetics. , 2008, Trends in genetics : TIG.

[6]  A. Chao,et al.  A Two‐Stage Probabilistic Approach to Multiple‐Community Similarity Indices , 2008, Biometrics.

[7]  A. Müller,et al.  Selective and programmed cleavage of GPI-anchored proteins from the surface membrane by phospholipase C. , 2012, Biochimica et biophysica acta.

[8]  Marcel Martin Cutadapt removes adapter sequences from high-throughput sequencing reads , 2011 .

[9]  R. Punnett,et al.  The Theory of the Gene , 1926, Nature.

[10]  Aaron Klug,et al.  The discovery of zinc fingers and their applications in gene regulation and genome manipulation. , 2010, Annual review of biochemistry.

[11]  Xiangqin Cui,et al.  Design and validation issues in RNA-seq experiments , 2011, Briefings Bioinform..

[12]  Jeffrey Philip Obbard,et al.  Enhanced intracellular lipid in Nannochloropsis sp. via random mutagenesis and flow cytometric cell sorting , 2012 .

[13]  Xuan Li,et al.  Optimizing de novo transcriptome assembly from short-read RNA-Seq data: a comparative study , 2011, BMC Bioinformatics.

[14]  Ivan Erill,et al.  Relative Codon Adaptation: A Generic Codon Bias Index for Prediction of Gene Expression , 2010, DNA research : an international journal for rapid publication of reports on genes and genomes.

[15]  P Green,et al.  Base-calling of automated sequencer traces using phred. II. Error probabilities. , 1998, Genome research.

[16]  Leszek Rychlewski,et al.  The Phaeodactylum genome reveals the evolutionary history of diatom genomes , 2008, Nature.

[17]  B. Mishra,et al.  Phylogenomic Study of Lipid Genes Involved in Microalgal Biofuel Production—Candidate Gene Mining and Metabolic Pathway Analyses , 2012, Evolutionary bioinformatics online.

[18]  R. Wijffels,et al.  An Outlook on Microalgal Biofuels , 2010, Science.

[19]  B. Pickersgill Genetic Diversity of Cultivated Tropical Plants , 2006 .

[20]  Nicholas H. Putnam,et al.  The Genome of the Diatom Thalassiosira Pseudonana: Ecology, Evolution, and Metabolism , 2004, Science.

[21]  Teresa M. Mata,et al.  Microalgae for biodiesel production and other applications: A review , 2010 .

[22]  Zhengwei Zhu,et al.  CD-HIT: accelerated for clustering the next-generation sequencing data , 2012, Bioinform..

[23]  Anne Chao User ’ s Guide for Program SPADE ( Species Prediction And Diversity Estimation ) , 2010 .

[24]  A. Di Rienzo,et al.  Detection of the signature of natural selection in humans: evidence from the Duffy blood group locus. , 2000, American journal of human genetics.

[25]  C. Vargas,et al.  New keys to the Past: Current and future DNA studies in Coccolithophores , 2004 .

[26]  C. Delwiche,et al.  The complete mitochondrial genome sequence of the haptophyte Emiliania huxleyi and its relation to heterokonts. , 2004, DNA research : an international journal for rapid publication of reports on genes and genomes.

[27]  Sara L. Zimmer,et al.  The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions , 2007, Science.

[28]  K. Evans,et al.  HIGH LEVELS OF GENETIC DIVERSITY AND LOW LEVELS OF GENETIC DIFFERENTIATION IN NORTH SEA PSEUDO‐NITZSCHIA PUNGENS (BACILLARIOPHYCEAE) POPULATIONS 1 , 2005 .

[29]  C. Darwin On the Origin of Species by Means of Natural Selection: Or, The Preservation of Favoured Races in the Struggle for Life , 2019 .

[30]  Fumiko Ohta,et al.  Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D , 2004, Nature.

[31]  J. Peñuelas,et al.  Environmental change and the option value of genetic diversity. , 2009, Trends in plant science.

[32]  A. Sáez,et al.  Pseudo-cryptic speciation in coccolithophores , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Rebecca L. Taylor,et al.  Cryopreservation of eukaryotic algae – a review of methodologies , 1998, Journal of Applied Phycology.

[34]  Yeqing Sun,et al.  Biotoxicity of nickel oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris. , 2011, Chemosphere.

[35]  R. Jordan,et al.  A revised classification scheme for living haptophytes , 2004 .

[36]  Haematococcus as a promising cell factory to produce recombinant pharmaceutical proteins , 2012, Molecular Biology Reports.

[37]  N. V. Joshi,et al.  Evolutionary genetics: From molecules to morphology , 2000, Journal of Genetics.

[38]  Jean-Paul Cadoret,et al.  Les microalgues - Usines cellulaires productrices de molécules commerciales recombinantes , 2008 .

[39]  N. Davidovich,et al.  Light is a key factor in triggering sexual reproduction in the pennate diatom Haslea ostrearia. , 2009, FEMS microbiology ecology.

[40]  C. Vargas,et al.  On the description of Tisochrysis lutea gen. nov. sp. nov. and Isochrysis nuda sp. nov. in the Isochrysidales, and the transfer of Dicrateria to the Prymnesiales (Haptophyta) , 2013, Journal of Applied Phycology.

[41]  V. Loeschcke,et al.  A test of quantitative genetic theory using Drosophila– effects of inbreeding and rate of inbreeding on heritabilities and variance components , 2005, Journal of evolutionary biology.

[42]  M. Gerstein,et al.  RNA-Seq: a revolutionary tool for transcriptomics , 2009, Nature Reviews Genetics.

[43]  K. Misawa,et al.  Phylogenetic positions of Glaucophyta, green plants (Archaeplastida) and Haptophyta (Chromalveolata) as deduced from slowly evolving nuclear genes. , 2009, Molecular phylogenetics and evolution.

[44]  Gregory Butler,et al.  OrfPredictor: predicting protein-coding regions in EST-derived sequences , 2005, Nucleic Acids Res..

[45]  J. Varela,et al.  Characterization of Dunaliella salina strains by flow cytometry: a new approach to select carotenoid hyperproducing strains , 2008 .

[46]  J. Cadoret,et al.  Integrative taxonomy of the Pavlovophyceae (Haptophyta): a reassessment. , 2011, Protist.

[47]  D. Mann,et al.  Experimental studies on sexual reproduction in diatoms. , 2004, International review of cytology.

[48]  D. Loo,et al.  Biology of human sodium glucose transporters. , 2011, Physiological reviews.

[49]  B. Whitton Ecology of Cyanobacteria II , 2012, Springer Netherlands.

[50]  F. Not,et al.  Holococcolithophore‐heterococcolithophore (Haptophyta) life cycles: Flow cytometric analysis of relative ploidy levels , 2004 .

[51]  J. Cadoret,et al.  Enhancement of neutral lipid productivity in the microalga Isochrysis affinis Galbana (T‐Iso) by a mutation‐selection procedure , 2012, Biotechnology and bioengineering.

[52]  P. Groot,et al.  Fatty acid activation: specificity, localization, and function. , 1976, Advances in lipid research.

[53]  J. Yim,et al.  Mutant selection of Hahella chejuensis KCTC 2396 and statistical optimization of medium components for prodigiosin yield-up , 2008, The Journal of Microbiology.

[54]  N. Friedman,et al.  Trinity: reconstructing a full-length transcriptome without a genome from RNA-Seq data , 2011, Nature Biotechnology.

[55]  K. Karrer,et al.  Analysis of Genomic G + C Content, Codon Usage, Initiator Codon Context and Translation Termination Sites In Tetrahymena Thermophila , 1999, The Journal of eukaryotic microbiology.

[56]  Jean-Paul Cadoret,et al.  La production de biocarburant lipidique avec des microalgues : promesses et défis , 2008 .

[57]  P. Spolaore,et al.  Commercial applications of microalgae. , 2006, Journal of bioscience and bioengineering.

[58]  D. H. Reed When it comes to inbreeding: slower is better , 2009, Molecular ecology.

[59]  P. Watkins Fatty acid activation. , 1997, Progress in lipid research.

[60]  H. Guzman,et al.  Quick estimation of intraspecific variation of fatty acid composition in Dunaliella salina using flow cytometry and Nile Red , 2012, Journal of Applied Phycology.

[61]  Gerald G Singh,et al.  Meta-analysis reveals negative yet variable effects of ocean acidification on marine organisms. , 2010, Ecology letters.

[62]  V. Chepurnov,et al.  Classical Breeding in Diatoms: Scientific Background and Practical Perspectives , 2011 .

[63]  H. Claustre,et al.  Extreme diversity in noncalcifying haptophytes explains a major pigment paradox in open oceans , 2009, Proceedings of the National Academy of Sciences.

[64]  A. Kalia,et al.  Functional diversity of ankyrin repeats in microbial proteins. , 2010, Trends in microbiology.

[65]  I. Ross,et al.  Selection, breeding and engineering of microalgae for bioenergy and biofuel production. , 2012, Trends in biotechnology.

[66]  Robert D. Finn,et al.  InterPro in 2011: new developments in the family and domain prediction database , 2011, Nucleic acids research.

[67]  J. A. Campo,et al.  Outdoor cultivation of microalgae for carotenoid production: current state and perspectives , 2007, Applied Microbiology and Biotechnology.

[68]  Qian Wang,et al.  GFOLD: a generalized fold change for ranking differentially expressed genes from RNA-seq data , 2012, Bioinform..

[69]  V. Loeschcke,et al.  Efficiency of selection, as measured by single nucleotide polymorphism variation, is dependent on inbreeding rate in Drosophila melanogaster , 2009, Molecular ecology.

[70]  Robert E. Synovec,et al.  Data Analysis Methods , 2012 .

[71]  Juliane C. Dohm,et al.  Substantial biases in ultra-short read data sets from high-throughput DNA sequencing , 2008, Nucleic acids research.

[72]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[73]  D. Mann,et al.  Highly differentiated populations of the freshwater diatom Sellaphora capitata suggest limited dispersal and opportunities for allopatric speciation. , 2009, Protist.

[74]  Tai-Huang Huang,et al.  GDSL family of serine esterases/lipases. , 2004, Progress in lipid research.

[75]  Joachim Hermisson,et al.  Mutation-selection balance: ancestry, load, and maximum principle. , 2002, Theoretical population biology.

[76]  Joel Cracraft,et al.  Assembling the tree of life , 2004 .

[77]  Jean-Paul Cadoret,et al.  Microalgae, Functional Genomics and Biotechnology , 2012 .

[78]  E. Wright,et al.  The sodium/glucose cotransport family SLC5 , 2003, Pflügers Archiv.