Supplemental Materials: Thriving in a tough environment: Insights into the red algae from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta) Table of Contents Methods ......................................................................................................................................................... 4 Culture techniques and antibiotic treatment ............................................................................................. 4 Nucleic acid preparation ........................................................................................................................... 5 Genome sequencing and assembly ........................................................................................................... 6 Genome partitioning ................................................................................................................................. 6 De novo repeat finding and repeat masking ............................................................................................ 10 RNA-Seq and expressed sequence tag (EST) expression analysis ......................................................... 12 Gene model prediction ............................................................................................................................ 12 Comparative analysis of red algal G + C content ........................................................................................ 15 Analysis of genome completeness .............................................................................................................. 16 Chloroplast genome ..................................................................................................................................... 18 Relationships of red algal classes ................................................................................................................ 19 Evolutionary rate comparison ..................................................................................................................... 19 Meiotic genes .............................................................................................................................................. 21 Analysis of Porphyra’s cytoskeleton .......................................................................................................... 22 Photosynthesis, Photoprotection, Stress Genes ........................................................................................... 31 Vitamin E ................................................................................................................................................ 36 Biosynthesis of isoprenoids ......................................................................................................................... 38 Carotenoids ............................................................................................................................................. 38 Sterols ..................................................................................................................................................... 39 Biosynthesis of tetrapyrroles ....................................................................................................................... 39 Circadian clock-related genes ..................................................................................................................... 43 Central carbon metabolism .......................................................................................................................... 45 Cell signaling ............................................................................................................................................... 53 Cell cycle regulation .................................................................................................................................... 59 Detecting microbial pathogens: Putative receptors ..................................................................................... 60 Potential intracellular receptors .............................................................................................................. 61 Potential extracellular receptors .............................................................................................................. 62 Malectin-domain family .......................................................................................................................... 62 1 Carbohydrate-active enzymes (CAZymes) ................................................................................................. 64 Microscopic analysis of the blade of Porphyra umbilicalis ........................................................................ 76 Metal metabolism and Porphyra ................................................................................................................. 78 Macronutrients ............................................................................................................................................ 87 Nitrogen .................................................................................................................................................. 87 Phosphorus. ............................................................................................................................................. 91 Sulfur. ..................................................................................................................................................... 93 Arsenic uptake and metabolism .................................................................................................................. 95 Lipids ........................................................................................................................................................... 95 Inositol phospholipids (phosphoinositides) ............................................................................................ 95 Amino acids ............................................................................................................................................... 100 B Vitamins ................................................................................................................................................. 104 Additional Support and Acknowledgements ............................................................................................. 110 References ................................................................................................................................................. 110 Supplementary Tables and Figures Table S1. Antibiotic treatment schedule for Pum1. ...................................................................................... 5 Table S2: Sequencing for pilot Illumina assembly. ...................................................................................... 6 Figure S3: Principal components analysis of scaffolds ................................................................................. 8 Table S4: Genome assembly summary ....................................................................................................... 10 Table S5: Repeat content of Porphyra genome .......................................................................................... 11 Table S6. Annotation summary statistics .................................................................................................... 13 Figure S7: Histogram of G+C-content (%) in primary transcript sequences in Porphyra gene models. ... 14 Figure S8: Coding G + C- contents in 13 red algal species. ....................................................................... 15 Table S9: Sequence data for G + C studies ................................................................................................. 16 Table S10. Analysis of presence of 41 core informational genes involved in transcription, translation and DNA replication. ......................................................................................................................................... 16 Figure S11. Relatedness of red algal classes ............................................................................................... 19 Figure S12. Maximum likelihood tree of red algae, green algae, and metazoans. ..................................... 20 Table S13. Genome data sets for evolutionary rate comparison ................................................................. 21 Table S14. List of meiotic genes in Porphyra ............................................................................................ 22 Table S15. Table of cytoskeletal genes found in Porphyra ........................................................................ 22 Table S16. Presence/absence of selected cytoskeletal genes in the red algae Porphyra, Cyanidioschyzon, Galdieria, Porphyridium, and Chondrus. ................................................................................................... 24 Figure S17. Phylogenetic analysis of red algal myosins. ........................................................................... 29 Figure S18. Phylogenetic tree of kinesin motor domain sequences ............................................................ 31 2 Table S19. Table of all photosynthetic genes and photoprotection genes .................................................. 32 Table S20: Table of antioxidant and heat-shock proteins. .......................................................................... 34 Table S21.