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New and old regions for Barcoding

New and old regions for Barcoding . Teun Boekhout CBS. Problems with Mitochondrial Genomes. Taphrinomycotina versus Saccharomycotina versus Pezizomycotina Cryptococcus gattii. (3). (1). (2). Pezizomycotina. Saccharomycotina. Saccharomycotina. Pezizomycotina. Taphrinamycotina.

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New and old regions for Barcoding

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  1. New and old regions for Barcoding Teun Boekhout CBS

  2. Problems with Mitochondrial Genomes • Taphrinomycotina versus Saccharomycotina versus Pezizomycotina • Cryptococcus gattii

  3. (3) (1) (2) Pezizomycotina Saccharomycotina Saccharomycotina Pezizomycotina Taphrinamycotina Taphrinamycotina Saccharomycotina Taphrinamycotina Pezizomycotina Mitochondrial support Most nuclear genes No support Three published scenario’s on fission yeasts

  4. Cryptococcus gattii • 4 monophyletic lineages (phylogenetic species) • 6 nuclear concordant nuclear gene phylogenies • Mitochondrial versus nuclear genes incongruent • [ATP 6 & MtLrRNA] vs [CNLAC, TEF1α, RPB1, RPB2, ITS, IGS1] • Mitochondrial loci incongruent • Mitochondrial recombination

  5. CBS6998 CBS1622/CBS6992 100 ATP6 haplotype 3 MtLrRNA haplotype 2 M3 CBS6290 63 60 48A/56A 64 CBS8273/RV20186T 74 Nucleus CBS6289 93 Mitochondrion ATP6 haplotype 4 MtLrRNA haplotype 2 M1 WM830 MtLrRNA ATP6 WM276 60 blue = AFLP4 green = AFLP6 purple = AFLP4 like yellow = AFLP6 like red = chimera AFLP4/AFLP6 WM176 99 5032738 CBS7229T/RV54130 60 E566 WM179R ATP6 haplotype 1 MtLrRNA haplotype 2 M2 CBS919T 64 CBS7748 CBS883T 10 ATP6 haplotype 2 MtLrRNA haplotype 1 M5 ATP6 haplotype 2 MtLrRNA haplotype 4 M4 Nuclear AFLP/MLST 4 with varying mitochondria

  6. WM714 WM714 H99 H99 Cryptococcus neoformans 125.91 125.91 CBS6886 CBS6886 JEC20 JEC20 RAM2 ICB184 ICB184 HEC11102 A1MR265 CBS1930 A1MR406 CBS6956 A1M368 CBS7750 CBS1930 CBS8684 CBS6956 A1MR269 AFLP6 VGII MLST 6 CBS8684 A1MR368 WM178 WM178 A1MR409 A1MR406 95 HEC11102 A1MR409 A1MF2866 A1MF2932 A1MF2932 A1MR265 A1MR269 A1MR271 A1MR271 A1MF2866 100 CBS7750 RAM2 (M5) (M5) WM179 WM179 (M5) (M5) E566 E566 (M4) 63 84 (M4) CBS919 CBS883 (M4) (M4) CBS883 CBS7748 (M4) (M4) CBS7748 CBS919 65 (M3) (M3) CBS1622 CBS1622 (M3) (M3) AFLP4 VGI MLST4 CBS6992 CBS6992 (M2) (M2) CBS7229 CBS7229 (M2) (M2) RV54130 RV54130 (M2) 100 (M2) WM176 WM176 (M2) (M2) 75 5032738 5032738 (M2) (M2) WM276 WM276 (M2) (M2) WM830 WM830 62 AFLP7 VGIV MLST7 B5748 97 B5748 B5742 B5742 WM779 M27055 M27055 WM779 (M1) (M1) CBS6290 CBS6290 (M1) 93 (M1) 56A 56A (M1) (M1) 74 CBS8273 AFLP4 VGI MLST4 CBS8273 (M1) (M1) 48A 48A (M1) (M1) CBS6289 CBS6289 (M1) (M1) CBS6998 CBS6998 (M1) (M1) RV20186 RV20186 CBS6993 CBS6996 WM161 380C WM728 CBS6955 94 AFLP5 VGIII MLST5 CBS5758 CBS6993 81 CBS8755 384C WM726 WM161 b a 380C CBS5758 82 CBS6955 WM726 ATP6 MtLrRNA 0.1 CN043 1 CBS8755 CBS6996 CN043 98 384C WM728

  7. The ‘old’ alternative • rDNA universally present • ITS + D1/D2 LSU rDNA (Kurtzman > 20years) • Basidio + Asco yeasts • Yeast book chapters: both needed • Clinical yeasts (C. glabrata / C. nivariensis / C. bracarensis) • Large datasets available • Proven useful for ID (Kurtzman, Fell, CBS, et al.) • Luminex technology (ID, M. Diaz, Miami) • Cryptococcus, Trichosporon, Candida, Malassezia

  8. Alternatives 2 • ‘Unique’ fungal pathways • E.g. Ergosterol pathway (membranes) • Concatenated ERG genes resolve TOL • ERG7 (lanosterol synthase) fungi/animals • ERG1 (squalene epoxidase) fungi/animals/plants • ERG11 (lanosterol C14 demethylase) fungi/animals

  9. Alternatives 3 • Comparative Genomics • 33 Fungal Genomes • 4852 KOGs • 70 single protein Kogs (paralogs!) • 32 gene KOs not viable (S. cerevisae) > essential genes • Reference tree based on 531 KOGs • KOG2671 showed highest correlation (0.97) with 531 KOG tree • Cophenetic correlation between individual protein NJ trees > 0.5 > 64 KOGs remaining • Development phylogenetic signal

  10. 64 concatenated proteins Sac. cerevisiae RM11-1a * Sac. cerevisiae S288c * Sac. paradoxus * Sac. mikatae * * Sac. kudriavzevii * > 80% * Sac. bayanus * Sac. castellii IA Can. glabrata * Ash. gossypii Kluyveromyces lactis Sac. kluyveri * Can. guilliermondii * Debaromyces hansenii * * Can. albicans I Can. lusitaniae Yarrowia lipolytica Chaetomium globosum * Neurospora crassa * IB1 * * Magnaporthe grisea Fusarium graminearum * IB IB2 Sclerotinia sclerotiorum * Botritys cinerea * Asp. fumigatus * IB3 * Asp. nidulans * Coccidioides immitis IB4 Stagonospora nodorum IC * Schizosaccharomyces pombe Coprinopsis cinereus * IIA Phanerochaete chrysosporium II * Cry. neoformans var. neoformans JEC21 * * Cry. neoformans var. grubii H99 IIB Ustilago maydis III IIIA Rhizopus oryzae Caenorhabditis elegans 0.1

  11. Development of phylogenetic signal Mainly Information storage and Processing & Cellular processes and signaling

  12. Potential Barcoding candidates • KOG2671 Putative RNA methylase • KOG0340 ATP-dependent RNA helicase • KOG4089 Predicted mitochondrial ribosomal protein L23 • KOG0173 20S proteasome • KOG2728 Uncharacterized conserved protein • Etc. Etc. Top 5 orthologues with high discriminatory potential

  13. My suggestions • There may be alternative genes • D1/D2 + ITS 1+2! • Panfungal / Paneukaryotic / Single copy / PCRable • Need to be investigated further for Barcoding (and TOL) potential • Option: Multilocus Barcoding ? • Webtool: Different loci per (higher) taxon • Selection depends on taxonomic resolution

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