Stajich phyloseminar 2011 06-29
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  • R. stolonifera infecting the strawberries\n\nMacro and microscales\n\n
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  • 1. Fungal phylogenomics: Getting lost in the moldy forestJason E StajichDept of Plant Pathology & MicrobiologyUniversity of California, Riverside http://lab.stajich.org http://fungalgenomes.org/blog http://fungiDB.org twitter{stajichlab,hyphaltip,fungalgenomes,fungidb}
  • 2. Fungi have diverse forms, ecology, and associations Cryptococcus neoformans X. Lin Coprinopsis cinerea Ellison & Stajich Aspergillus niger. N Read Glomus sp. Univ Sydney Rozella allomycis. James et alPuccinia graminis J. F. Hennen Batrachochytrium dendrobatidis Laccaria bicolor Martin et al. Neurospora crassa. Hickey & Reed Phycomyces blakesleansus T. Ootaki J. LongcoreUstilago maydis Kai Hirdes Amanita phalloides. M Wood Xanthoria elegans. Botany POtD Rhizopus stolonifera. Blastocadiela simplex Stajich & Taylor
  • 3. Fungi are an old group of organisms <:,./,7 40(78(6(, (Eutherian 31(,.(6(, $7/,6(,mammal origins $%&()*(%+%, ,-./$~ 150-180 Mya) !"#$%%& 312/%+%(02&(/, C:,)/(&:,+%(02&(/, $=:/%&7::=:,FI%/1F $=&((02&(/%., +%GG77./%,/7+F/%))=7) -./(0(*1/1((02&(/%., >((*,5(02&(/%., E())F(GFG:,57::=0 ?%&@A7::(02&(/%., ;:(07(02&(/, $%/(/%&F)*(,.5%, <=&&%.%(02&(/%., !"#$%$&()&*" /(F0%/(/%&F&(.%+%,F 9)/%:,5%.(02&(/%., H75=:,F)7*/, 45,%&(02&(/%., D,*1%.(02&(/%., +#)&()&*" $7%(/%&F)*(,.5%,F/(F B,&&1,(02&(/%., 7A/7.,:F07%()*(7) <76%6(02&(/%., !"## !### "## # $%::%(.)F(GF27,) Stajich et al. Current Biol 2009
  • 4. Awash in Fungal Genomes• Mid-2011: Genomes from 150+ species sequenced and ~400 more in progress/pipeline• Several multi-strain resequencing projects (Neurospora - Ellison PNAS 2011), Saccharomyces (Liti Nature 2009), Coccidoides (Neafsey Genome Res 2010) and many in progress/proposed.• Many of sequenced genomes were focused around plant, animal pathogens, and some specific evolutionary questions.• Now are starting to fill out the tree more to capture the diversity of kingdom and also for studies of molecular evolution among many related species.• Also efforts targeting specific questions - pathogens and their relatives; wood rotting fungi; flagellated and non-flagellated forms; extremophiles; comparison of growth forms (e.g. yeast forms from phylogenetically very different species)
  • 5. Genome sizes of fungi are 8Mb-100Mb = easy(ish) tosequence Melampsora, Blumeria, Puccinia Genome size of fungi (many Transposable 100 ! ! ! ! elements) 80 ! 60 Mushrooms Francis Martin Flickr:/fmartin1954/2338045504/ http://en.wikipedia.org/wiki/File:Barleypowderymildew.jpg 40 Ashbya, Malassezia, Candida other yeasts 20 http://www.biologie.uni-osnabrueck.de/Genetik/index.php?menuid=12
  • 6. Need to cover more of the phylogenetic diversity: 1000 Fungal genomes project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lue = completed or in progress, Red= proposed for Tier One sampling,Green = remaining unsampled families Numbers or Percent of Families in each clade and their current or proposed genome sampling
  • 7. Tools to access this data• Genome databases at sequencing centers and NCBI• Comprehensive and integrated systems are not as well developed in Fungi, but some examples of excellent tools highlighted at http://tools.fungalgenomes.org/• Ensembl Fungi, MicrobesOnline, JGI’s Mycocosm, Comparative Fungal Genomics, and some targeted to specific clades e.g. Saccharomyces (SGD), Aspergillus (AspGD), Candida (CGD)• We have launched FungiDB - http://fungidb.org to support integrating functional genomics data for data mining as well as standard ‘Gene’ pages for genes.
  • 8. Searching with FungiDB:A strategy for drug targets C.neoformans genes with - no hits in other eukaryotes (excepting fungi), bacteria - has S.cerevisiae orthologs that have EC terms OR orthologs in other fungi with GO metabolic terms associated
  • 9. Searching with FungiDB: A strategy for reannotation2885 C.neoformans genes with desc “"conserved hypotheticalprotein” but 1327 can be assigned name from ortholog in any fungus with EC #, GO terms, or Pfam domains
  • 10. Gene page and genome browserSynteny Views Data mining interface F ngiD !"#$%&$(#)*+,-.(-)".,(-
  • 11. Using comparative genomics towards understandpathogen evolution• How do traits like pathogenecity evolve?• Can comparative genomics indicate meaningful differences that can lead to understanding the basis for pathogenesis?• Contrasting genomes of pathogens with non-pathogens can suggest recent genomic changes that might be testable in the lab• Gene duplication is thought to be important source for evolutionary innovation - What role might gene family size change play in adaptation?
  • 12. Models for comparing gene family size changes• A model for gene family size change that incorporates the birth-death process of gene families which follow a power law distribution (Hahn et al, Genome Res 2005)• Implemented in a program called CAFE to find unexpectedly large lineage or clade- specific changes in gene family sizes (Hahn Lab @Indiana Univ) (De Bie et al, Bioinformatics 2006)• Can screen genome family sizes across multiple species to find expectedly large changes (based on counts) which can be verified using gene tree-species tree reconciliation approaches like Notung (Chen, Durand, Farach-Colton, J Comp Bio 2000)
  • 13. Short LifeCoccidioides Granuloma• Fungal pathogen Doctorfungus.com Spherule M. McGinnis genomics: Gene families Endospores Long Life and appetite differences
  • 14. Human pathogen Coccidioides • Coccidioides (Valley fever) - 2 species C. immitis and C. posadasii • Is a primary human pathogen - infects healthy people (most human pathogenic fungi are opportunistic) • Endemic in US Southwest, Mexico • Requires laboratory BSL3 and is a Select Agent • Genomes of 2 species (Sharpton et al Genome Res 2009) and then 18 strains (Neafsey et al Genome Res 2010) • Comparative analyses of Coccidoides spp
  • 15. Human pathogen Coccidioides Life cycle S/Hypha Spherule Endospores
  • 16. Coccidioides ecology Soil? Short Life Granuloma D octorfungus. com M. McGinnis Spherule Endospores Long Life pocket mice?
  • 17. Two species of Coccidioides are allopatric C.immitisC.posadasii EVOLUTION Fisher et al, 2000
  • 18. Studying the evolution of a pathogen• Comparing sequences from two Coccidioides species, closely related outgroup, and more distant ougroups species:• Evidence for recent positive selection• What gene family differences can be identified that distinguish phenotypic groups (mammalian pathogens from non- pathogens)• Evidence for recent introgression which contains candidate genes for pathogenesis
  • 19. Gene family changes• Another mechanism for adaptation may be changes in copy number of a gene family • Gene duplication is a source of novelty allowing for changes in the function of one copy if the other maintains original function • Expansions of copy number may also be an easy way to get more protein for a particular process• How important is copy number change in adaptation?
  • 20. Genome samples from fungi Dictyostelium Monosiga Choanoflagellida Caenorhabditis Metazoa Drosophila Homo Batrachochytrium ‘Chytrid’ Spiromyces Zygomycota KickxellomycotinaOpisthokont ‘Chytrid’ Olpidium Rhizopus Mucormycotina Fungi Glomus Glomeromycota Puccinia Cryptococcus Basidiomycota Coprinopsis Schizosaccharomyces Taphrinomycotina Yarrowia Saccharomyces Ascomycota Candida Saccharomycotina Morchella Cochliobolus Cladonia Pezizomycotina Aspergillus Coccidioides Magnaporthe Pezizomycotina Neurospora Fusarium Botryotinia
  • 21. Aspergillus clavatus Aspergillus fumigatus Aspergillus flavus Animal Pathogen Aspergillus oryzae (Opportunistic) Aspergillus terreus Eurotiales Aspergillus niger Animal Pathogen Aspergillus nidulans (Primary) Penicillium marneffei Blastomyces dermatitidisEurotiomycetes Plant Pathogen Histoplasma capsulatum 186AR Histoplasma capsulatum 217B Histoplasma capsulatum WU24 Paracoccidioides brasiliensis Onygenales Coccidioides immitis Coccidioides posadasii Uncinocarpus reesii Fusarium graminearum Sclerotinia sclerotiorum 200 100 0 Mya
  • 22. Few protein domains for eating plant material in animal pathogens Animal Pathogen Peptidase_M35 Peptidase_M36 (Opportunistic) Peptidase_S8 Pec_lyase_C Subtilisin_N Cellulase Cutinase TannaseLoss of plant CBM_1 NPP1 APH Animal Pathogensaprophytic (Primary) enzymes Anid 6 6 6 2 4 13 2 3 3 0 9 Plant Pathogen Afum 17 5 5 2 5 10 2 5 2 1 9 Ater 15 6 6 2 8 13 2 6 2 1 29 Hcap 0 0 0 0 2 2 2 6 1 0 20 Uree 0 0 0 0 1 2 15 19 4 2 33 Cimm 0 0 0 0 1 1 13 16 7 2 38 Cpos 0 0 0 0 1 1 14 16 7 2 32 Ncra 18 1 1 4 3 6 3 6 2 0 6 Sharpton, Stajich, et al, Genome Fgra 12 7 9 9 12 8 11 24 1 1 15 Res. 2009
  • 23. Domains for eating animal material? Animal Pathogen Peptidase_M35 Peptidase_M36 (Opportunistic) Peptidase_S8 Pec_lyase_C Subtilisin_N Cellulase Cutinase Tannase CBM_1 NPP1 Animal Pathogen APH (Primary) Anid 6 6 6 2 4 13 2 3 3 0 9 Plant Pathogen Afum 17 5 5 2 5 10 2 5 2 1 9 Ater 15 6 6 2 8 13 2 6 2 1 29 Hcap 0 0 0 0 2 2 2 6 1 0 20 Uree 0 0 0 0 1 2 15 19 4 2 33 Cimm 0 0 0 0 1 1 13 16 7 2 38 Cpos 0 0 0 0 1 1 14 16 7 2 32 Ncra 18 1 1 4 3 6 3 6 2 0 6 Fgra 12 7 9 9 12 8 11 24 1 1 15 Sharpton, Stajich, et al, Genome Res. 2009
  • 24. Keratinases in Onygenales SignalP Subtilisin_N • Onygenales are Keratinophilic • Domains: Peptidase S8, Subtilisin domains • Large expansion of putative keratinases in Onygenales
  • 25. Peptidase S8 expansionI in Onygenales 14 copies in Coccidioides 1 in Histoplasma IIIII
  • 26. Peptidase S8 expansionI in Onygenales 14 copies in Coccidioides 1 in Histoplasma IIIII
  • 27. Population genomics• Revealed no loci with evidence for balancing selection - so little evidence for long standing host- pathogen battle as in Plasmodium.• Sliding window FST analysis revealed some regions of reduced divergence and followup revealed a regions of recent intergression. Directionality looks to be mostly from Cp and into Ci.• One gene implicated in pathogenesis, MEP4, is found in introgressed region
  • 28. Towards identifying genes underlying adaptation Sharpton et al. Genome Res 2009 Neafsey et al, Genome Res 2010
  • 29. Towards identifying genes underlying adaptation• Coccidioides is found in desert soil and associated with animals - perhaps has a long term animal association without inducing response from the host. Sharpton et al. Genome Res 2009 Neafsey et al, Genome Res 2010
  • 30. Towards identifying genes underlying adaptation• Coccidioides is found in desert soil and associated with animals - perhaps has a long term animal association without inducing response from the host.• Loss of genes involved in plant product metabolism suggests nutritional shift in Onygenales from relatives in Eurotiales Sharpton et al. Genome Res 2009 Neafsey et al, Genome Res 2010
  • 31. Towards identifying genes underlying adaptation• Coccidioides is found in desert soil and associated with animals - perhaps has a long term animal association without inducing response from the host.• Loss of genes involved in plant product metabolism suggests nutritional shift in Onygenales from relatives in Eurotiales• Expansion of only a few gene families, seem to be involved in metabolism - none are Coccidioides specific though. Sharpton et al. Genome Res 2009 Neafsey et al, Genome Res 2010
  • 32. Towards identifying genes underlying adaptation• Coccidioides is found in desert soil and associated with animals - perhaps has a long term animal association without inducing response from the host.• Loss of genes involved in plant product metabolism suggests nutritional shift in Onygenales from relatives in Eurotiales• Expansion of only a few gene families, seem to be involved in metabolism - none are Coccidioides specific though.• From population genetics analysis - failed to find regions under balancing selection suggesting there is not a long-term arms race between host and pathogen. Sharpton et al. Genome Res 2009 Neafsey et al, Genome Res 2010
  • 33. Towards identifying genes underlying adaptation• Coccidioides is found in desert soil and associated with animals - perhaps has a long term animal association without inducing response from the host.• Loss of genes involved in plant product metabolism suggests nutritional shift in Onygenales from relatives in Eurotiales• Expansion of only a few gene families, seem to be involved in metabolism - none are Coccidioides specific though.• From population genetics analysis - failed to find regions under balancing selection suggesting there is not a long-term arms race between host and pathogen.• Evidence for introgression between the species and perhaps imported novel alleles that are important for adaptation in both species to their animal hosts. Sharpton et al. Genome Res 2009 Neafsey et al, Genome Res 2010
  • 34. Batrachochytrium dendrobatidis (Bd)- a major cause of amphibian decline Bd is a Chytridhttp://cisr.ucr.edu/chytrid_fungus.html fungus $%&()*(%+%, Fungi !"#$%%& 312/%+%(02&(/, B7,)/(&7,+%(02&(/, $<&((02&(/%., -./(0(*1/1((02&(/%., =((*,5(02&(/%., >%&?@977(02&(/%., 87(09(02&(/, :<&&%.%(02&(/%., Basidiomycota 6)/%7,5%.(02&(/%., 45,%&(02&(/%., C,*1%.(02&(/%., Ascomycota A,&&1,(02&(/%., :9;%;(02&(/%., !"## !### "## # Rosenblum et al PNAS 2008 $%77%(.)D(ED29,)
  • 35. Bd genome sequence projects• 2 strains sequenced at JGI and Broad; allow whole genome comparison between strains• Found that genome is diploid, but with large regions with loss of heterozygocity (LOH)• Identified gene family changes that might suggest mechanisms of pathogenecity• Greater understanding of what the early fungus was like• With collaborators we are sequencing 20 more strains for population genomics to better understand population dynamics, trace origin of diversity, and understand the LOH as independent or shared events.
  • 36. Bd grows intercellularlyFormally described by Joyce Longcore (U Maine) (Mycologia 1999) Rick Speare, Lee Berger, Alax Haytt James Cook University, Townsville, Australia http://www.jcu.edu.au/school/phtm/PHTM/frogs/chpr1/fc13.htm
  • 37. Phylogenomic profiling• For each gene in the the target (Bd) genome, look to see which have homologs in other fungi, animals, plants• Classify genes by its profile as to when it must have arisen based on identified homologs• Also compare well-studied organisms (S. cerevisiae, N. crassa) to see which genes were missing• For Bd/Chytrids several trends appeared • Missing: some cell wall genes, spindle-pole body • Present in Bd but not other non-chytrid fungi: Flagella, some signaling pathways, effector like proteins • Some expansions of gene families
  • 38. Fungal cell wall evolution- a view from earliest branchesErgosterol Yeast Cell Wall What is the fungal cell wall made of? Sugar polymers Some fungi also have Selitrennikoff CP, AEM 2001 α (1,3) glucan
  • 39. Evolution of β (1,3) glucan synthase
  • 40. Evolution of FKS1 !"& $ 789:%;<=-)>#36) C7D<%+0!#&6E 789:%;<=-)>3.)( [ β (1,3) glucan !"%( 45,$%&623 GLS1 $ /4?G%&#3)$& synthetase ] $ $ C7D<%+F!#.3E C7D<%+1!&#3E FKS1,2 Ascomycota $ $ 789:%;<=-)>3)3) 55-1%&6)6-$& $ 55-1%&.3(3$& !" $ @A8B%@A&-6#) BasidiomycotaNo Chytridiomycota !"(% !"() 54/1%&6(&2$& !"#$%-&#.2 $ !"&) !"#$%&()6 !"#$%&() Rhizopus !"% !"#$%&.#)# /0/1%&3&26$& Allomyces !"( $ $ /0/1%&&($& /0/1%&&(6$& /0/1%&&&2#$& $ /0/1%-&6.3$& (Blastocladiomycota) *?$1%&###($& $ *+,-%$&&&. *+,-%$&&3# $ *?$1%&#.#$& $ !"%& *+,-%$&&3#2 !" *?$1%&#.3$& $ *?$1%&6.32$& $ *+,-%$&-#&2. $ $ *?$1%&6#(2$& *+,-%$&&2).. Oomycetes $ *?$1%-.3$& $ *+,-%$&&&(.2 $ $ *?$1%&(&)$& *+,-%$&&&(6 Callose synthase
  • 41. No 1,3 Beta-glucan?
  • 42. B. dendrobatidis cell wall biochemical analysis Cellulose β (1,3)- β(1,6)- α(1,3)- β(1,4)- Chitin glucan glucan glucan glucan X X X ✓ ✓ A putative cellulose synthase gene can be found in genome of Bd but also found in N. crassa and other Fungi with JP Latgé, M. Fisher
  • 43. B. dendrobatidis cell wall biochemical analysis Cellulose β (1,3)- β(1,6)- α(1,3)- β(1,4)- Chitin glucan glucan glucan glucan X X X ✓ ✓ A putative cellulose synthase gene can be found in genome of Bd but also found in N. crassa and other Fungi with JP Latgé, M. Fisher 100X higher MIC for Caspofungin than C. albicans with N.A.R Gow & M. Fisher
  • 44. Expansion of CBM18 domain - Chitin binding gene family 0 100 200 300 800 900 1000 400 500 600 700 Length - aa BD_03764 C C C DE 471-aa TL BD_05538 O E D DE DL * 746-aa BD_05531 O D 439-aa LL ** BD_07015 O E D DE 503-aa BD_08806 O D DE 503-aa * ** BD_05161 O D DE 444-aa Deactylase Like BD_05540 O E D DE 478-aa BD_05533 D DE 368-aa BD_05536 D DE 321-aa BD_06123 A TYR * 635-aa BD_06122 A TYR 673-aa Tyrosinase Like BD_06121 A B B B A TYR 771-aa * BD_00259 K K K K K H G 577-aa Lectin Like 1033-aa BD_01761 F M I X L J J J J H G * BD_00264 F N M H H G 621-aa ** BD_00271 F N M H H G 577-aa * * BD_00287 F N M M H H G 636-aa BD_00289 N I I 334-aa Abramyan & Stajich, submitted
  • 45. BD_06121 - A TL .60 A BD_06121 - A DL * BD_06123 - A BD_06122 - A LL * B BD_06121 - B ** * BD_06121 - B BD_06121 - B * = 1.0 * C BD_03764 - C * BD_03764 - C ** ≥ .90 BD_03764 - C BD_05538 - D BD_08806 - D .85 * BD_05540 - D BD_07015 - D * D ** BD_05161 - D * BD_05536 - D BD_05531 - D BD_05533 - D * E BD_05538 - E * BD_05540 - E BD_07015 - E BD_00271 - F F * BD_00264 - F * BD_00287 - F BD_01761 - F XDomain tree shows ** G ** * BD_01761 - X BD_00264 - G BD_00287 - G BD_00271 - G ** BD_00259 - Gclade-specific grouping ** * BD_01761 - G BD_00271 - H BD_00264 - H BD_00287 - H H BD_00287 - H BD_00259 - H * BD_00264 - H BD_00271 - HDomains evolved from mostly *I BD_01761 - H BD_01761 - I BD_00289 - Itandem duplications with some BD_00289 - I BD_01761 - J *J BD_01761 - Jintergene duplications. BD_01761 - J BD_01761 - J *K * BD_00259 - K ** BD_00259 - K BD_00259 - K ** L * BD_00259 - K BD_00259 - K BD_01761 - L BD_00287 - M * BD_00271 - M .81 M ** BD_00264 - M BD_01761 - M .86 BD_00287 - M BD_00289 - N ** N BD_00264 - N * BD_00271 - N BD_00287 - N BD_08806 - O O * BD_05538 - O * BD_05531 - O BD_05161 - O ** * BD_07015 - O BD_05540 - O SP_07646 SP_07647 SP_03958 Abramyan & Stajich, submitted
  • 46. CBM18 expansion• Largest copy number of CBM18 in all fungi, and most number of domains (11) in a single locus• Evidence for positive selection among copies of the domains based on codon analyses• CBM18 thought to bind chitin, could be involved in binding its own chitin to cloak the cells from the host immunity• Could also bind chitin-related molecules in animals attach more firmly to the animal cells Abramyan & Stajich, submitted
  • 47. Tyrosinase expansions in Bd and other fungi AllomycesSynthesis of melanin, H.Whistler via Liu et al BMC Evol Biol 2006 pigments and otherpolyphenols Hajime Muraguchi via Stajich et al PNAS 2010 E.Medina
  • 48. But are these changes important?• Just observing big families in a genome is nice, but does it mean that changes are really related to recent adaptation? • The branches on some gene trees are short, indicating duplications are recent• A better is to polarize the changes to the Bd branch by having a closer species than 50-100 Mya...• So is there a species closer to Bd we can use? ...
  • 49. Homolaphylictis polyrhiza JEL142 is a close(ish) relative F. graminearum 1 N. crassa 1 B. cinerea 454 genome sequencing 1 U. reesii C. immitisMAKER genome annotation 1 1 1 T. rubrum A. benhamiae OrthoMCL gene families 1 1 M. canis 0.58 A. nidulans 1 P. tritici-repentis C. cinerea 1 C. neoformans 1 * U. maydis 1 P. graminis 1 P. blakesleeanus A. macrogynus S. punctatus 1 H. polyrhiza JEL142 1 B. dendrobatidis ! "! # Joneson et al, Submitted
  • 50. Contrasting some family sizes among the Chytrids M36 S41 Tyrosinase CBM18 Allomyces 31 0 16 3 Spizellomyces 3 3 3 3 Hp 3-5 3 15 5 Bd 38 32 12 14-16 Fungalysin Serine protease Serine protease expansions in Fungalysins - thought to be Coccidoides and relatives maybekeratinases (break down keratin in related to breaking down on an amphibian skin...) animal matter Joneson et al, Submitted
  • 51. H. polyrhiza won’t grow on frog skin; doesn’t causemortality in frogs Hp- frog skin intact Bd- frog skin degraded Joneson et al, Submitted
  • 52. A) GLEANP3 001910 B) 1 GLEANP3 010940 GLEANP3 058130 0.8 GLEANP3 058330 GLEANP3 058280Fungalysin GLEANP3 038010 0.6 GLEANP3 035340 M36 GLEANP3 045390 0.4 GLEANP3 016580up in sporangia GLEANP3 040970 0.2 GLEANP3 030000 no expression GLEANP3 068250 0 zoospore sporangia GLEANP3 037130 diff GLEANP3 027740 GLEANP3 075460up in zoospore GLEANP3 019170 1 GLEANP3 075190 GLEANP3 066500 0.8 GLEANP3 075130 GLEANP3 041170 0.6 GLEANP3 035280 GLEANP3 018720 GLEANP3 048430 0.4 GLEANP3 046850 Aspergillus fumigatus outgroup 0.2 Rhizopus oryzae outgroup GLEANP3 078890 0 0.1 zoospore sporangia Rosenblum et al PNAS 2008
  • 53. Bd summary• Comparisons to close species can polarize some differences in lineage specific expansions • Findings of M36, S41 expansions seem to be recent to Bd and could be a functional link to pathogenesis • Expansion of Tyronsinase from counts is less compelling, but gene tree analyses suggests recent expansions on Bd-Hp branch • Lack of FKS1 in early Chytridiomycota lineages suggests a more recent origin of this gene than origin of the Fungi and predicts timing of some changes in fungal cell wall composition • CBM18 expansion may be related to adhesion, future experiments to test this• Future: We are employing a population genomic approach, resequencing 24 strains of Bd and hope to understand more about origins and variation in the genome from these data.
  • 54. Summary• Unraveling the evolution of pathogens can be tricky, more so when mode of pathogenesis is not obvious (e.g. not just a toxin gene)• Comparative genomics at the scale of gene and gene families can suggest changes that may be important in adaptation of a species.• Connecting these molecular changes to pathogenecity is still needed to understand the role these expansions may play - but provides rich experimental fodder for the laboratory.
  • 55. AcknowledgementsCoccidioides B. dendrobatidisTom Sharpton {UCSF} John Taylor {UC Berkeley} John Abramyan {UCR}Dan Neafsey {Broad Institute} Edgar Medina {Universidad de Los Andes,Bridget Barker, Marc Orbach, Steve Rounsley {U Colombia}Arizona} Christina Cuomo {Broad Institute} Tim James {U Michigan} Suzanne Joneson, Tom Poorten, Erica Stajich lab @UCR lab Rosenblum {U Idaho} Steven Ahrendt Igor Grigoriev {JGI} Divya Sain H. polyrhiza Yizhou Wang Suzanne Joneson, Erica Rosenblum {U Idaho} Yi Zhou Shin-Han shiu {Michigan State} Jessica De Anda Burroughs Wellcome Fund Grant Programs Joyce Longcore {U Maine} Annie Nguyen Tim James {U Michigan} W.M. KeckBiomedical Sciences Foundation Career Awards for Medical Scientists http://lab.stajich.org Five-year awards for physician scientists provide $700,000 to bridge advanced postdoctoral/fellowship training and the early years of faculty service. This award addresses the on-going problem of increasing http://mobedac.org the number of physician scientists and will help facilitate the transition to a career in research. Proposals must be in the area of basic biomedical, disease-oriented, translational, or molecular, genetic, or pharmacological epidemiology research. Collaborative Research Travel Grants http://fungidb.org Provide up to $15,000 in support for researchers from degree-granting institutions to travel to a labo-