PYROPIA YEZOENSIS MUTANT WITH HIGH NITROGEN AVAILABILITY ACTA BOT. CROAT. 79 (2), 2020 1 On-line Suppl. Tab. 1. Sequences of primer sets used for RT-qPCR to validate results of transcriptome analysis. Contig name Primer sequence Py2K_unigene_122397 F 5´-GAAGATTACCAAGAAGAGCGGC-3´ R 5´-ACTTTGTCTCGTCCTTCCACTC-3´ Py2K_unigene_173936 F 5´-GCTACAAGATCAAGCCATTCGT-3´ R 5´-TCAGAACCACCAACAAACTCAC-3´ Py2K_unigene_43753 F 5´-CAAGACGGACCATAATCGATACG-3´ R 5´-AAGGCACCCAAGTACGATGAAT-3´ PyWT_unigene_402 F 5´-AACAGCCATCAATTACCGTCAC-3´ R 5´-ACAGTCTTCCAGGTTTCAGTCT-3´ Py2K_unigene_145511 F 5´-GCCATGGTCGAGTTCCTCTA-3´ R 5´-GATGACACCACCCGAGTAGAA-3´ Py2K_unigene_90598 F 5´-GCCATAGGATTAGTAGTAGTTGCA-3´ R 5´-CTTGCAACTCACCAACTTTTCG-3´ Py2K_unigene_146357 F 5´-TCTGAAGCCTATGTTGAGAGCA-3´ R 5´-TCTTGAACTCATACCACTCCGT-3´ Py2K_unigene_159094 F 5´-ATTTTATGCCGTGGTTAGTGGG-3´ R 5´-AGGGAATTTATCAAACACGCCA-3´ Py2K_unigene_115858 F 5´-CCATTCCGTCACTGTACAACAC-3´ R 5´-CCCATTCTTCTGTAGGTCTTGC-3´ Py2K_unigene_159070 F 5´-GGTGTGGACGAGCGACAATACAG-3´ R 5´-CGGACGGGGGGTAAGCAAAAGAAG-3´ On-line Suppl. Tab. 2. Summary statistics of raw and clean reads in Pyropia yezoensis wild type (PyWT) and mutant (Py2K). Sample Raw reads Clean reads Reads (no.) Bases (total) Avg. length (bp) GC (%) Reads (no.) % of raw data GC (%) PyWT WT1 75,146,820 11,347,169,820 151 45.2 49,337,074 65.65 45.08 WT2 69,260,700 10,458,365,700 151 44.41 50,952,456 73.57 44.03 WT3 64,414,678 9,726,616,378 151 44.41 45,643,162 70.86 43.27 Py2K 2K1 65,639,484 9,911,562,084 151 44.59 54,125,262 82.46 44.31 2K2 70,666,770 10,670,682,270 151 43.16 56,377,874 79.78 42.85 2K3 79,066,906 11,939,102,806 151 44.02 53,554,388 67.73 44.56 On-line Suppl. Tab. 3. List of upregulated genes in mutant Pyropia yezoensis (Py2K) annotated as ‘antioxidant’. TRINITY name Log2-Fold Description Identity Pfam description TRINITY_Py2K_DN131974_c0_g1 7.97 Chloroplastic putative glutathione peroxidase 7 [Arabidopsis thaliana] 52.17% Glutathione peroxidase TRINITY_Py2K_DN254273_c0_g1 4.28 Glutathione peroxidase-like peroxiredoxin gpx1 [Schizosaccharomyces pombe] 57.14% Glutathione peroxidase TRINITY_Py2K_DN63293_c0_g1 6.42 Glutathione S-transferase [Aplysia californica] 32.94% Glutathione S-transferase, N-terminal domain TRINITY_Py2K_DN59285_c0_g4 2.53 1-Cys peroxiredoxin [Dictyostelium discoideum] 49.28% C-terminal domain of 1-Cys peroxiredoxin TRINITY_Py2K_DN64707_c0_g1 7.55 Glutathione synthetase [Bordetella pertussis] 66.12% Glutathione synthetase, ATP-grasp domain TRINITY_Py2K_DN59959_c0_g1 3.74 Chloroplastic 2-Cys peroxiredoxin BAS1 [Hordeum vulgare] 38.39% C-terminal domain of 1-Cys peroxiredoxin TRINITY_Py2K_DN51886_c0_g1 4.60 Catalase [Pyropia yezoensis] 91.20% Catalase TRINITY_Py2K_DN66210_c7_g3 2.71 Thioredoxin [Porphyra purpurea] 40.00% Thioredoxin TRINITY_Py2K_DN65307_c1_g2 4.79 Thioredoxin [Ictalurus punctatus] 46.79% Thioredoxin TRINITY_Py2K_DN59105_c0_g1 4.97 Mitochondrial aldehyde dehydrogenase [Pongo abelii] 55.51% Aldehyde dehydrogenase family PARK S, CHOI J 2 ACTA BOT. CROAT. 79 (2), 2020 On-line Suppl. Tab. 4. List of differentially expressed genes in Pyropia yezoensis mutant (Py2K) related to the Shikimate pathway. TRINITY name Log2-Fold Description Identity Pfam description TRINITY_Py2K_DN61445_c1_g1 2.76 Chorismate synthase [Ruegeria pomeroyi] 83.33% Chorismate_synt TRINITY_Py2K_DN61820_c0_g2 4.87 3-dehydroquinate synthase [Thioalkalivibrio sulfidiphilus] 65.69% 3-dehydroquinate synthase TRINITY_Py2K_DN61821_c0_g1 4.02 Shikimate kinase [Pseudomonas aeruginosa] 60.00% Shikimate kinase TRINITY_Py2K_DN63681_c0_g1 6.94 Shikimate dehydrogenase [Methylobacillus flagellatus] 60.44% Shikimate dehydrogenase substrate binding domain On-line Suppl. Tab. 5. List of differentially expressed genes in Pyropia yezoensis mutant (Py2K) related to nitrogen assimilation and re- assimilation, ribosome biogenesis, and amino acid biosynthesis. TRINITY name log2Fold Description Identity Pfam description Nitrogen assimilation TRINITY_Py2K_DN59400_c0_g2 4.22 High affinity nitrate transporter 2.5 [Pyropia yezoensis] 99.20% Major Facilitator Super- family TRINITY_PyWT_DN101823_c0_g1 2.76 Chloroplastic ferredoxin-nitrite reductase [Arabidopsis thaliana] 28.94% Nitrite and sulfite reductase 4Fe-4S domain TRINITY_Py2K_DN61283_c1_g2 2.87 Glutamine synthetase [Cryptococcus neoformans var. neoformans serotype D] 56.34% Glutamine synthetase, beta-Grasp domain Ribosome biogenesis TRINITY_Py2K_DN141935_c0_g1 7.05 Casein kinase II subunit beta [Arabidopsis thaliana] 61.28% Casein kinase II regulatory subunit TRINITY_Py2K_DN278700_c0_g1 6.53 Serine/threonine-protein kinase rio1 [Capsaspora owczarzaki ATCC 30864] 92.40% Calcineurin-like phospho- esterase TRINITY_Py2K_DN56816_c1_g1 6.97 GTP-binding nuclear protein Ran [Sphaeroforma arctica JP610] 74.50% ADP-ribosylation factor family TRINITY_Py2K_DN303240_c0_g1 6.68 13 kDa ribonucleoprotein-associ- ated protein [Nicotiana tomento- siformis] 76.00% Ribosomal protein L7Ae/ L30e/S12e/Gadd45 family TRINITY_Py2K_DN63805_c1_g4 5.18 Oligoribonuclease [Pseudomonas aeruginosa] 67.98% Exonuclease TRINITY_Py2K_DN19438_c0_g1 6.26 NTF2-related export protein [Caenorhabditis elegans] 28.57% Nuclear transport factor 2 (NTF2) domain TRINITY_Py2K_DN56108_c0_g1 6.42 Guanine nucleotide-binding protein-like 3 homolog [Dictyostelium discoideum] 45.45% GNL3L/Grn1 putative GTPase TRINITY_Py2K_DN292600_c0_g1 7.81 Nucleolar protein 56 [Schizosaccharomyces pombe] 50.62% NOP5NT (NUC127) domain TRINITY_Py2K_DN30246_c0_g1 5.84 Ribosomal RNA small subunit methyltransferase NEP1 [Dictyostelium discoideum] 61.22% EMG1/NEP1 methyltrans- ferase Purine ring catabolism TRINITY_Py2K_DN50780_c0_g1 6.37 Probable allantoinase 1 [Dictyostelium discoideum] 40.86% OHCU decarboxylase TRINITY_Py2K_DN63532_c0_g1 4.69 Probable xanthine dehydrogenase subunit A [Bacillus subtilis] 27.03% XdhC and CoxI family Biosynthe- sis of amino acids TRINITY_Py2K_DN203287_c0_g1 2.61 D-amino-acid N-acetyltransfer- ase [Saccharomyces cerevisiae] 37.93% Acetyltransferase (GNAT) family TRINITY_Py2K_DN251932_c0_g1 3.69 Cystathionine gamma-lyase [Dictyostelium discoideum] 57.89% Cys/Met metabolism PLP-dependent enzyme TRINITY_Py2K_DN33143_c0_g2 6.30 Argininosuccinate synthase [Achlya hypogyna] 58.80% Arginosuccinate synthase PYROPIA YEZOENSIS MUTANT WITH HIGH NITROGEN AVAILABILITY ACTA BOT. CROAT. 79 (2), 2020 3 On-line Suppl. Tab. 6. List of differentially expressed genes in Pyropia yezoensis mutant (Py2K) related to the electron transport chain. TRINITY name log2Fold Description Identity Pfam description TRINITY_Py2K_DN188135_c0_g1 2.41 NADH dehydrogenase ubiquinone iron- sulfur protein 8-B [Arabidopsis thaliana] 61.19% 4Fe-4S binding domain TRINITY_Py2K_DN63881_c0_g1 3.02 Succinate dehydrogenase assembly factor 2 [Dictyostelium discoideum] 39.77% Flavinator of succinate dehydrogenase TRINITY_Py2K_DN63855_c0_g2 2.50 Mitochondrial succinate dehydrogenase ubiquinone flavoprotein subunit [Oryza sativa subsp. Japonica] 74.75% Fumarate reductase flavoprotein C-term TRINITY_Py2K_DN65270_c1_g1 5.93 Alternative oxidase [Mangifera indica] 59.60% Alternative oxidase TRINITY_Py2K_DN38046_c0_g1 6.50 Cytochrome b-c1 complex subunit Rieske-1 [Arabidopsis thaliana] 65.22% Ubiquinol cytochrome reductase transmembrane region TRINITY_Py2K_DN65488_c2_g5 8.24 Ubiquinol-cytochrome c reductase iron- sulfur subunit [Allochromatium vinosum] 56.68% Ubiquitinol-cytochrome C reductase Fe-S subunit TAT signal TRINITY_Py2K_DN28456_c0_g1 2.46 Cytochrome c [Pectinaria gouldii] 71.96% Cytochrome c TRINITY_Py2K_DN63840_c0_g2 2.11 Cytochrome c oxidase subunit 1 [Marchantia polymorpha] 76.81% Cytochrome C and Quinol oxidase polypeptide TRINITY_Py2K_DN3622_c0_g1 2.29 Mitochondrial ATP synthase subunit beta [Fistulifera solaris] 84.10% ATP synthase alpha/beta family, beta-barrel domain TRINITY_Py2K_DN61343_c0_g1 5.44 Mitochondrial isocitrate dehydrogenase [Candida tropicalis] 67.74% Isocitrate/isopropylmalate dehydrogenase TRINITY_Py2K_DN129036_c0_g1 5.79 Putative cytochrome b5 [Neurospora crassa] 45.45% Cytochrome b5-like Heme/ Steroid binding domain TRINITY_Py2K_DN62822_c0_g1 2.55 Mitochondrial manganese-dependent superoxide dismutase [Dictyostelium discoideum] 58.26% Iron/manganese superoxide dismutases, alpha-hairpin domain On-line Suppl. Fig. 1. Random amplified polymorphic DNA (RAPD) analysis of Pyropia yezoensis wild-type (PyWT) and mutant (Py2K). RAPD analysis was conducted to compare PyWT and Py2K using 20 RAPD primer sets (OPA 1-20 primer sets). Representative ampli- fications with OPA 4, 7, 11, 12, 15 17 and 20 are shown. Arrows indicate the bands that were different between PyWT and Py2K. Wt, wild-type; Mt, mutant. PARK S, CHOI J 4 ACTA BOT. CROAT. 79 (2), 2020 On-line Suppl. Fig. 2. Relationship among samples based on log2-transformed count data between Pyropia yezoensis wild-type (PyWT) and mutant (Py2K) with hierarchically clustered heatmap using Pearson’s correlation coefficients. On-line Suppl. Fig. 3. Reverse transcription quantitative PCR (RT-qPCR) results for verification of transcriptome analysis results. All experiments were performed in triplicate and error bar indicated standard deviation. PYROPIA YEZOENSIS MUTANT WITH HIGH NITROGEN AVAILABILITY ACTA BOT. CROAT. 79 (2), 2020 5 On-line Suppl. Fig. 4. Description of differentially expressed genes (DEGs) in the shikimate biosynthesis pathway related to phenol bio- synthesis in Py2K. The shikimic acid pathway was described by Santos-Sánchez et al. (2019). The green arrows indicate upregulated genes in Py2K. PEP – phosphoenolpyruvic acid, D-erythores-4-P – D-erythores-4-phosphate, DAHP – 3-deoxy-D-arabion-heptulosonic acid 7-phosphate, DHQ – 3-dehydroquinic acid, DHS – 3-dehydroshikimic acid, S3P – shikimic acid 3-phosphate, EPSP – 5-enolpyruvylshi- kimic acid 3-phosphate, DAHPS – 3-deoxy-D-arabion-heptulosonic acid 7-phosphate synthase, DHQS – 3-dehydroquinic acid synthase, SDH – shikimate dehydrogenase, SK – shikimate kinase, EPSPS – 5-enolpyruvylshikimic acid 3-phosphate synthase, CS – chorismite synthase.