Adventures in Bioinformatics (2012)
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The document discusses the identification and analysis of the NB-LRR gene family in potatoes, highlighting a novel predictive model that identified and located 438 NB-LRRs in the potato genome, surpassing previous annotations. It also addresses the emergence of Dickeya species as significant bacterial plant pathogens and the development of specific diagnostic primers for accurate detection. Lastly, it outlines methodologies for phylogenetic analysis of potato transcription factors and their relevance in plant response mechanisms.
![Potato:
NB-‐LRRs:
Introduc1on
l Potato
genome
sequenced
by
PGSC
(2011)
l NB-‐LRR
predic5ons
thought
to
be
incomplete
l NB-‐LRR
l Large
plant
gene
family
l Modular:
[nuclear
binding:NB]-‐[leucine-‐rich
receptor:LRR]
l Several
subclasses
(modular
varia5on)
l R
(resistance)
genes
are
a
subset
of
NB-‐LRRs](https://image.slidesharecdn.com/20121130cmsjhi-140905095940-phpapp02/85/Adventures-in-Bioinformatics-2012-3-320.jpg)
Adventures in Bioinformatics (2012)
- 1. Adventures in Bioinforma1cs (2012) Leighton Pritchard
- 2. Summary l Potato: NB-‐LRRs l Bacteria: diagnos5cs l Potato: phylogene5cs l Bacteria: genomics
- 3. Potato: NB-‐LRRs: Introduc1on l Potato genome sequenced by PGSC (2011) l NB-‐LRR predic5ons thought to be incomplete l NB-‐LRR l Large plant gene family l Modular: [nuclear binding:NB]-‐[leucine-‐rich receptor:LRR] l Several subclasses (modular varia5on) l R (resistance) genes are a subset of NB-‐LRRs
- 4. Potato: NB-‐LRRs: Method l Computa5onal iden5fica5on l Modular/domain varia5on is an issue l Work within predicted gene complement (improved annota5on) ‘good’ score ‘bad’ score Not NB-‐LRR: NB-‐LRR: No false nega5ves No false posi5ves
- 5. Potato: NB-‐LRRs: Method l Mo5f iden5fica5on/composi5on l Train MEME (psp-‐gen) on posi5ve and nega5ve examples to build model Not NB-‐LRR: NB-‐LRR: l Use MAST to iden5fy score thresholds and predic5ve performance l Could dis5nguish between posi5ve and nega5ve example sets absolutely on basis of MAST reported E-‐value ‘good’ score ‘bad’ score
- 6. Potato NB-‐LRRs: Results l Applied model to l predicted gene complement l gene models extended by 3kbp to iden5fy addi5onal domains l manual correc5on l mapped to genome l clusters iden5fied
- 7. Potato: NB-‐LRRs: Results l NB-‐LRR model used to build gene enrichment bead ‘array’ l Iden5fied 338 addi5onal candidate NB-‐LRRs
- 8. Potato NB-‐LRRs: Summary l Developed novel predic5ve model for NB-‐LRRs l Iden5fied and located 438 NB-‐LRRs on the potato genome (~10% more than published annota5on) l Classified phylogene5cally and on domain composi5on l Models used to build enrichment arrays to iden5fy 338 novel NB-‐LRRs Jupe F, Pritchard L, Etherington GJ, MacKenzie K, Cock PJ, et al. (2012) Iden5fica5on and localisa5on of the NB-‐LRR gene family within the potato genome. BMC Genomics 13: 75. doi:10.1186/1471-‐2164-‐13-‐75.
- 9. Bacteria: diagnos1cs: Introduc1on I l Dickeya spp. l Major pan-‐European bacterial plant pathogen l Emerging threat: Dickeya “solani” l Exis5ng diagnos5c primers did not discriminate D.“solani” „ ADE/pel: all Dickeya „ Laurila, Nassar, Toth primers not specific at species level l Use drag genomes to develop diagnos5c primers? Toth IK, der Wolf van JM, Saddler G, Lojkowska E, Hélias V, et al. (2011) Dickeya species: an emerging problem for potato produc5on in Europe. Plant Pathol: doi:10.1111/j.1365-‐3059.2011.02427.x.
- 10. Bacteria: diagnos1cs: Method DraG genomes (several species): Design >1000 primers per genome (Primer3) Pritchard L, Holden NJ, Bielaszewska M, Karch H, Toth IK (2012) Alignment-‐free design of highly discriminatory diagnos5c primer sets for Escherichia coli O104:H4 outbreak strains. PLoS ONE 7: e34498. doi:10.1371/journal.pone.0034498. l Design primers in bulk: l specific thermodynamics and amplicon size
- 11. Bacteria: diagnos1cs: Method Classify (colour) primers by predicted ability to amplify only a subset of genome sequences in silico: Group I (genus) Group II Group (species) III Group (species) IV Group (species) V Amplifica5on of nega5ve samples: discard Simultaneous design of primers specific to all subgroups of the input sequence set Validate in vitro: unseen data (performance es5mates) Pritchard L, Holden NJ, Bielaszewska M, Karch H, Toth IK (2012) Alignment-‐free design of highly discriminatory diagnos5c primer sets for Escherichia coli O104:H4 outbreak strains. PLoS ONE 7: e34498. doi:10.1371/journal.pone.0034498.
- 13. Bacteria: diagnos1cs: Method l Automated process, pipeline (open source) available at hnps://github.com/widdowquinn/find_differen5al_primers l (Forked by two other developers) l Found a problem in GenBank! l Cross-‐amplifying primers suggested that species assignment of reference genomes was incorrect Pritchard L, Humphris S, Saddler GS, Parkinson NM, Bertrand V, et al. (2012) Detec5on of phytopathogens of the genus Dickeya using a PCR primer predic5on pipeline for drag bacterial genome sequences. Plant Pathol doi:10.1111/j. 1365-‐3059.2012.02678.x.
- 14. Bacteria: diagnos1cs: Method l Automated process, pipeline available at hnps://github.com/widdowquinn/find_differen5al_primers l (Forked by two other developers) l Found a problem in GenBank! l Cross-‐amplifying primers suggested that species assignment of reference genomes was incorrect l Confirmed by recA maximum likelihood tree
- 15. Bacteria: diagnos1cs: Results I l Specificity of D.“solani” (and D. dianthicola) primers confirmed in vitro Pritchard L, Humphris S, Saddler GS, Parkinson NM, Bertrand V, et al. (2012) Detec5on of phytopathogens of the genus Dickeya using a PCR primer predic5on pipeline for drag bacterial genome sequences. Plant Pathol doi:10.1111/j. 1365-‐3059.2012.02678.x.
- 16. Bacteria: diagnos1cs: Introduc1on II l E. coli EHEC O104:H4 outbreak, Europe 2011 l Unprecedented: l Scale of outbreak (3950 infected, >50 deaths, economic impact and interna5onal import restric5ons) l Rapid, open produc5on of sequence data l Crowdsourcing of assembly and annota5on via collabora5ve revision control site: GitHub hPps://github.com/ehec-‐outbreak-‐crowdsourced/BGI-‐data-‐analysis/wiki Rohde H, Qin J, Cui Y, Li D, Loman NJ, et al. (2011) Open-‐source genomic analysis of Shiga-‐toxin-‐producing E. coli O104:H4. N Engl J Med 365: 718–724. doi:10.1056/NEJMoa1107643.
- 17. Bacteria: diagnos1cs: Introduc1on II l A changing paradigm? Beyond serotyping: l 4 PCRs to serotype: (O-‐an5gen, flagellar locus, tellurite resistance, shigatoxin) Kwan et al. (2011) hnp://precedings.nature.com/documents/6663/version/1
- 18. Bacteria: diagnos1cs: Results II l Direct experimental valida5on of primer candidates (Münster): l ‘Posi5ve’ set = 21 clinical outbreak isolates l ‘Nega5ve’ set = 32 HUSEC / EPEC isolates l Posi5ve control = LB 226692 l Extremely good diagnos5c performance l Specific at outbreak isolate (sub-‐species) level
- 19. Bacteria: diagnos1cs: Results II posi1ve nega1ve + -‐ Pritchard L, Holden NJ, Bielaszewska M, Karch H, Toth IK (2012) Alignment-‐free design of highly discriminatory diagnos5c primer sets for Escherichia coli O104:H4 outbreak strains. PLoS ONE 7: e34498. doi:10.1371/journal.pone.0034498.
- 20. Bacteria: diagnos1cs: Summary II l Developed novel rapid primer design technique l Drag genome design (default assemblies) l Simultaneous design of primers specific to all subgroups where possible l Can specify internal probes for TaqMan l New primers for Dickeya spp. diagnos5cs l In ac5ve use in Europe l Outbreak isolate-‐specific primers for E.coli O104:H4 outbreak l Specific diagnosis with two PCRs l Discriminatory primers to dis5nguish Phytophthora effector gene family members for diversity studies (not shown)
- 21. Potato: phylogene1cs: Introduc1on l Potato NAC transcrip5on factors are targeted by Phytophthora effectors l Plant-‐specific, >100 family representa5ves in each organism l Implicated in many plant responses, not just defence/stress l Defini5ve N-‐terminal domain (NAM domain), unique fold Olsen AN, Ernst HA, Leggio LL, Skriver K (2005) NAC transcrip5on factors: structurally dis5nct, func5onally diverse. Trends Plant Sci 10: 79–87. doi:10.1016/j.tplants.2004.12.010
- 22. Potato: phylogene1cs: Introduc1on l Variable lengths with highly diverse C-‐terminal structure l NAM domains are DNA-‐binding and associated with 5ssue-‐specific expression l Some NACs have transmembrane (TM) domains in the C-‐terminus: (including our interactors) (other structural classes) Jensen MK, Kjaersgaard T, Nielsen MM, Galberg P, Petersen K, et al. (2010) The Arabidopsis thaliana NAC transcrip5on factor family: structure-‐ func5on rela5onships and determinants of ANAC019 stress signalling. Biochem J 426: 183–196. doi:10.1042/BJ20091234.
- 23. Potato: phylogene1cs: Methods l Two potato target sequences of interest l Two N. benthamiana homologues: what about other organisms? l 2253 proteins from nr: HMM search with NAM domain model l 137 proteins from PGSC potato annota5on l 104 proteins from tomato annota5on l 2552 proteins total l 2200 non-‐redundant NAM-‐containing proteins
- 24. Potato: phylogene1cs: Methods 1. Predict presence of transmembrane (TM) domain (TMHMM) 2. Restrict sequence to NAM domain (only common region) 3. Remove outliers <40% amino acid iden5ty: 1700 large sequence set 4. Cluster sequences (MCL) to iden5fy reduced set including potato target sequences: 406 sequences 5. Align 406 sequence set (M-‐COFFEE, HMMalign) 6. Back-‐translate to nucleo5de sequence (more work than you might think…)
- 25. 100 100 99 100 100 100 100 72 100 29 100 100 100 100 100 0.3 Oryza sativa Indica Group - gi|125564636|gb|EAZ10016.1|/116-246 Hordeum vulgare subsp. vulgare - gi|326496190|dbj|BAJ90716.1|/6-145 Brachypodium distachyon - gi|357154515|ref|XP_003576809.1|/6-142 Sorghum bicolor - gi|242045528|ref|XP_002460635.1|/6-135 Sorghum bicolor - gi|242040125|ref|XP_002467457.1|/6-134 Brachypodium distachyon - gi|357141157|ref|XP_003572109.1|/6-135 Oryza sativa Japonica Group - gi|110289615|gb|AAP55107.2|/6-134 Arabidopsis lyrata subsp. lyrata - gi|297846226|ref|XP_002890994.1|/6-139 Arabidopsis thaliana - gi|42562467|ref|NP_174529.2|/6-141 Arabidopsis lyrata subsp. lyrata - gi|297794579|ref|XP_002865174.1|/6-135 Arabidopsis thaliana - gi|15237469|ref|NP_199471.1|/6-135 Arabidopsis thaliana - gi|15236721|ref|NP_193532.1|/6-136 Arabidopsis lyrata subsp. lyrata - gi|297800286|ref|XP_002868027.1|/6-136 Vitis vinifera - gi|147765514|emb|CAN78113.1|/6-135 Vitis vinifera - gi|359474569|ref|XP_002280894.2|/6-135 Ricinus communis - gi|255557623|ref|XP_002519841.1|/6-134 Populus trichocarpa - gi|224126509|ref|XP_002329572.1|/6-134 Populus trichocarpa - gi|224138076|ref|XP_002326512.1|/6-134 Solyc06g061080.2.1/6-134 Petunia x hybrida - gi|21105751|gb|AAM34774.1|AF509874_1/6-134 Solyc08g077110.2.1/6-134 PGSC0003DMP400030569/6-134 Medicago truncatula - gi|357482369|ref|XP_003611470.1|/6-148 Medicago truncatula - gi|357476695|ref|XP_003608633.1|/6-137 Glycine max - gi|356563453|ref|XP_003549977.1|/6-136 Glycine max - gi|356512014|ref|XP_003524716.1|/6-136 Sorghum bicolor - gi|242091638|ref|XP_002436309.1|/30-161 Zea mays - gi|293333166|ref|NP_001169690.1|/11-142 Brachypodium distachyon - gi|357119028|ref|XP_003561248.1|/14-150 Oryza sativa Japonica Group - gi|115465864|ref|NP_001056531.1|/18-149 Zea mays - gi|293332711|ref|NP_001169185.1|/6-140 Sorghum bicolor - gi|242080633|ref|XP_002445085.1|/12-146 Brachypodium distachyon - gi|357144944|ref|XP_003573468.1|/9-143 Oryza sativa Japonica Group - gi|115474875|ref|NP_001061034.1|/9-143 Hordeum vulgare subsp. vulgare - gi|326526251|dbj|BAJ97142.1|/9-143 Hordeum vulgare subsp. vulgare - gi|295881154|gb|ADG56507.1|/3-134 Sorghum bicolor - gi|242032581|ref|XP_002463685.1|/4-155 Hordeum vulgare subsp. vulgare - gi|326508056|dbj|BAJ86771.1|/12-139 Brachypodium distachyon - gi|357159131|ref|XP_003578349.1|/8-135 Zea mays - gi|212274312|ref|NP_001130458.1|/9-136 Sorghum bicolor - gi|242049706|ref|XP_002462597.1|/9-136 Oryza sativa Indica Group - gi|218202386|gb|EEC84813.1|/10-144 Oryza sativa Japonica Group - gi|115479907|ref|NP_001063547.1|/10-144 Oryza sativa Japonica Group - gi|222641843|gb|EEE69975.1|/10-134 Hordeum vulgare subsp. vulgare - gi|326530494|dbj|BAJ97673.1|/13-150 Oryza sativa Japonica Group - gi|115449815|ref|NP_001048559.1|/16-153 Oryza sativa Indica Group - gi|125541671|gb|EAY88066.1|/16-153 Brachypodium distachyon - gi|357137711|ref|XP_003570443.1|/14-151 Zea mays - gi|223949493|gb|ACN28830.1|/24-161 Sorghum bicolor - gi|242063552|ref|XP_002453065.1|/19-156 Zea mays - gi|293336942|ref|NP_001168544.1|/26-154 Sorghum bicolor - gi|242079615|ref|XP_002444576.1|/32-160 Oryza sativa Japonica Group - gi|115477845|ref|NP_001062518.1|/21-149 Oryza sativa Indica Group - gi|125562576|gb|EAZ08024.1|/21-149 Brachypodium distachyon - gi|357144217|ref|XP_003573214.1|/29-157 Hordeum vulgare subsp. vulgare - gi|326509519|dbj|BAJ91676.1|/22-150 Triticum aestivum - gi|292659258|gb|ADE34584.1|/22-150 Glycine max - gi|356576364|ref|XP_003556302.1|/4-132 Vitis vinifera - gi|147802301|emb|CAN70406.1|/9-137 Vitis vinifera - gi|225448908|ref|XP_002265550.1|/13-141 Vitis vinifera - gi|147802300|emb|CAN70405.1|/13-141 Glycine max - gi|356576366|ref|XP_003556303.1|/34-162 Glycine max - gi|356535507|ref|XP_003536286.1|/33-161 Populus trichocarpa - gi|224109864|ref|XP_002315336.1|/39-167 Arabidopsis thaliana - gi|15237698|ref|NP_196060.1|/28-170 Arabidopsis lyrata subsp. lyrata - gi|297829588|ref|XP_002882676.1|/27-155 Arabidopsis thaliana - gi|18398893|ref|NP_566375.1|/27-155 Arabidopsis lyrata subsp. lyrata - gi|297829586|ref|XP_002882675.1|/27-155 Arabidopsis thaliana - gi|18398891|ref|NP_566374.1|/27-155 Arabidopsis thaliana - gi|238479717|ref|NP_001154602.1|/27-188 Malus x domestica - gi|302399033|gb|ADL36811.1|/9-137 Ricinus communis - gi|255583748|ref|XP_002532627.1|/11-139 Populus trichocarpa - gi|224109872|ref|XP_002315338.1|/5-133 Populus trichocarpa - gi|224100709|ref|XP_002311983.1|/10-138 Arabidopsis lyrata subsp. lyrata - gi|297829590|ref|XP_002882677.1|/9-137 Arabidopsis thaliana - gi|21593389|gb|AAM65338.1|/9-137 Arabidopsis thaliana - gi|12322791|gb|AAG51388.1|AC011560_20/9-137 Arabidopsis lyrata subsp. lyrata - gi|297806417|ref|XP_002871092.1|/9-137 Arabidopsis thaliana - gi|15237699|ref|NP_196061.1|/9-137 Arabidopsis thaliana - gi|6456751|gb|AAF09254.1|AF201456_1/9-137 Petunia x hybrida - gi|21105746|gb|AAM34772.1|AF509872_1/10-138 Solyc11g008000.1.1/10-139 PGSC0003DMP400054118/10-139 Solyc05g055470.2.1/10-138 PGSC0003DMP400040418/1-129 Petunia x hybrida - gi|21105744|gb|AAM34771.1|AF509871_1/23-151 PGSC0003DMP400040416/22-150 Solyc05g055480.2.1/17-145 Petunia x hybrida - gi|21105736|gb|AAM34767.1|AF509867_1/35-163 PGSC0003DMP400054120/30-158 Solyc11g008010.1.1/97-225 Oryza sativa Japonica Group - gi|115480567|ref|NP_001063877.1|/6-134 Sorghum bicolor - gi|242045530|ref|XP_002460636.1|/6-134 Vitis vinifera - gi|296088017|emb|CBI35300.3|/6-134 Ricinus communis - gi|255569031|ref|XP_002525485.1|/6-134 Populus trichocarpa - gi|224125708|ref|XP_002329698.1|/6-134 Populus trichocarpa - gi|224120448|ref|XP_002318332.1|/6-134 Glycine max - gi|356561847|ref|XP_003549188.1|/6-134 Glycine max - gi|356531066|ref|XP_003534099.1|/6-134 PGSC0003DMP400054092/6-134 Solyc03g098190.2.1/6-134 Arabidopsis lyrata subsp. lyrata - gi|297830402|ref|XP_002883083.1|/6-134 Arabidopsis thaliana - gi|15229511|ref|NP_188400.1|/6-134 Arabidopsis thaliana - gi|15237939|ref|NP_197228.1|/6-134 Arabidopsis lyrata subsp. lyrata - gi|297811871|ref|XP_002873819.1|/6-134 Arabidopsis lyrata subsp. lyrata - gi|297832960|ref|XP_002884362.1|/6-134 Arabidopsis thaliana - gi|186509710|ref|NP_186970.2|/6-134 Arabidopsis thaliana - gi|6714418|gb|AAF26106.1|AC012328_9/6-126 Arabidopsis thaliana - gi|4585977|gb|AAD25613.1|AC005287_15/3-131 Arabidopsis lyrata subsp. lyrata - gi|297853370|ref|XP_002894566.1|/6-134 Vitis vinifera - gi|147854237|emb|CAN83436.1|/6-134 Medicago truncatula - gi|357451351|ref|XP_003595952.1|/6-134 Glycine max - gi|356557052|ref|XP_003546832.1|/6-134 Glycine max - gi|356528635|ref|XP_003532905.1|/6-134 Ricinus communis - gi|255573304|ref|XP_002527580.1|/6-134 Populus trichocarpa - gi|224098826|ref|XP_002311281.1|/6-134 Populus trichocarpa - gi|224112333|ref|XP_002316156.1|/6-134 Arabidopsis thaliana - gi|15218843|ref|NP_176766.1|/6-134 Arabidopsis lyrata subsp. lyrata - gi|297841167|ref|XP_002888465.1|/6-134 Brachypodium distachyon - gi|357120932|ref|XP_003562178.1|/6-134 Oryza sativa Indica Group - gi|125542167|gb|EAY88306.1|/6-134 Sorghum bicolor - gi|242042457|ref|XP_002468623.1|/6-134 Zea mays - gi|226531876|ref|NP_001146336.1|/6-134 Solyc02g081270.2.1/6-134 PGSC0003DMP400037231/6-134 Solyc02g036430.1.1/6-134 Vitis vinifera - gi|225466227|ref|XP_002267333.1|/6-134 Ricinus communis - gi|255571259|ref|XP_002526579.1|/6-134 Populus trichocarpa - gi|224135105|ref|XP_002327567.1|/5-133 Populus trichocarpa - gi|224079646|ref|XP_002305903.1|/6-134 Medicago truncatula - gi|357491761|ref|XP_003616168.1|/6-134 Glycine max - gi|356499060|ref|XP_003518362.1|/5-133 Glycine max - gi|356551985|ref|XP_003544352.1|/5-133 Medicago truncatula - gi|357460717|ref|XP_003600640.1|/6-134 Glycine max - gi|356569398|ref|XP_003552888.1|/6-134 Glycine max - gi|356537835|ref|XP_003537430.1|/6-134 Picea sitchensis - gi|116786496|gb|ABK24128.1|/6-136 Picea sitchensis - gi|148907008|gb|ABR16648.1|/6-136 Physcomitrella patens subsp. patens - gi|168025227|ref|XP_001765136.1|/5-132 Physcomitrella patens subsp. patens - gi|168001016|ref|XP_001753211.1|/1-128 Physcomitrella patens subsp. patens - gi|168001860|ref|XP_001753632.1|/6-133 Physcomitrella patens subsp. patens - gi|168016362|ref|XP_001760718.1|/6-131 Picea sitchensis - gi|148909054|gb|ABR17630.1|/7-147 Picea sitchensis - gi|116793533|gb|ABK26780.1|/6-134 Setaria italica - gi|326369339|gb|ADZ55681.1|/6-134 Oryza sativa Japonica Group - gi|115464001|ref|NP_001055600.1|/6-134 Brachypodium distachyon - gi|357133590|ref|XP_003568407.1|/6-134 Hordeum vulgare subsp. vulgare - gi|188593543|dbj|BAG32519.1|/6-134 Oryza sativa Indica Group - gi|125550359|gb|EAY96181.1|/6-134 Brachypodium distachyon - gi|357168397|ref|XP_003581627.1|/5-133 Hordeum vulgare subsp. vulgare - gi|326492385|dbj|BAK01976.1|/6-134 Sorghum bicolor - gi|242077812|ref|XP_002448842.1|/6-134 Zea mays - gi|293336322|ref|NP_001169920.1|/6-134 Vitis vinifera - gi|296088554|emb|CBI37545.3|/6-134 Petunia x hybrida - gi|21105732|gb|AAM34765.1|AF509865_1/4-132 Solyc03g078120.2.1/4-132 PGSC0003DMP400049938/4-132 Solyc11g005920.1.1/4-132 Malus x domestica - gi|302399003|gb|ADL36796.1|/6-134 Malus x domestica - gi|302399015|gb|ADL36802.1|/8-136 Arabidopsis lyrata subsp. lyrata - gi|297797411|ref|XP_002866590.1|/6-134 Arabidopsis thaliana - gi|15237609|ref|NP_201211.1|/6-134 Arabidopsis lyrata subsp. lyrata - gi|297811023|ref|XP_002873395.1|/6-134 Arabidopsis thaliana - gi|15242390|ref|NP_196495.1|/6-134 Ricinus communis - gi|255552430|ref|XP_002517259.1|/5-133 Populus trichocarpa - gi|224130574|ref|XP_002328323.1|/7-135 Populus trichocarpa - gi|224095914|ref|XP_002310505.1|/7-135 Medicago truncatula - gi|357464643|ref|XP_003602603.1|/7-135 Glycine max - gi|356516356|ref|XP_003526861.1|/5-133 Glycine max - gi|356508975|ref|XP_003523228.1|/5-133 Glycine max - gi|356525677|ref|XP_003531450.1|/3-131 Glycine max - gi|356512837|ref|XP_003525122.1|/3-131 Medicago truncatula - gi|357519943|ref|XP_003630260.1|/6-134 Medicago truncatula - gi|357516809|ref|XP_003628693.1|/6-134 Medicago truncatula - gi|7716952|gb|AAF68626.1|AF254124_1/6-134 Selaginella moellendorffii - gi|302756881|ref|XP_002961864.1|/1-126 Zea mays - gi|226493114|ref|NP_001147448.1|/9-137 Sorghum bicolor - gi|242056921|ref|XP_002457606.1|/9-137 Oryza sativa Japonica Group - gi|115435780|ref|NP_001042648.1|/9-137 Oryza sativa Indica Group - gi|218187925|gb|EEC70352.1|/9-137 Brachypodium distachyon - gi|357129971|ref|XP_003566632.1|/9-137 Hordeum vulgare subsp. vulgare - gi|371925001|tpe|CBZ41162.1|/9-137 Triticum aestivum - gi|289551912|gb|ADD10614.1|/9-137 Petunia x hybrida - gi|21105740|gb|AAM34769.1|AF509869_1/12-140 Solyc12g056790.1.1/22-150 PGSC0003DMP400029635/22-150 Petunia x hybrida - gi|21105742|gb|AAM34770.1|AF509870_1/19-147 NbNac2_1/29-157 Solyc04g072220.2.1/32-160 StNac2_5/32-160 PGSC0003DMP400054265/31-159 Vitis vinifera - gi|225463127|ref|XP_002265611.1|/20-148 Glycine max - gi|356535729|ref|XP_003536396.1|/19-147 Glycine max - gi|356576117|ref|XP_003556180.1|/19-147 Populus trichocarpa - gi|224116242|ref|XP_002331996.1|/2-129 Populus trichocarpa - gi|224120762|ref|XP_002330945.1|/237-361 Populus trichocarpa - gi|224082532|ref|XP_002306731.1|/16-144 Populus trichocarpa - gi|224066587|ref|XP_002302150.1|/16-144 Arabidopsis lyrata subsp. lyrata - gi|297846266|ref|XP_002891014.1|/10-136 Arabidopsis thaliana - gi|14334572|gb|AAK59465.1|/10-136 Arabidopsis thaliana - gi|18399168|ref|NP_564440.1|/16-144 Arabidopsis lyrata subsp. lyrata - gi|297846402|ref|XP_002891082.1|/16-144 Arabidopsis lyrata subsp. lyrata - gi|297846400|ref|XP_002891081.1|/16-144 Arabidopsis thaliana - gi|21618244|gb|AAM67294.1|/16-144 Arabidopsis thaliana - gi|18399166|ref|NP_564439.1|/16-144 Arabidopsis thaliana - gi|145324118|ref|NP_001077648.1|/16-156 Glycine max - gi|356562269|ref|XP_003549394.1|/9-144 Arabidopsis lyrata subsp. lyrata - gi|297807857|ref|XP_002871812.1|/13-130 Populus trichocarpa - gi|224132570|ref|XP_002321355.1|/8-141 Malus x domestica - gi|302398995|gb|ADL36792.1|/8-118 Oryza sativa Japonica Group - gi|115481670|ref|NP_001064428.1|/18-129 100 Oryza sativa Indica Group - gi|218184374|gb|EEC66801.1|/31-142 Arabidopsis thaliana - gi|15233215|ref|NP_191081.1|/11-151 Arabidopsis lyrata subsp. lyrata - gi|297819350|ref|XP_002877558.1|/46-187 Arabidopsis lyrata subsp. lyrata - gi|297819354|ref|XP_002877560.1|/223-364 Arabidopsis lyrata subsp. lyrata - gi|297819354|ref|XP_002877560.1|/5-145 Populus trichocarpa - gi|224080239|ref|XP_002306067.1|/7-131 Populus trichocarpa - gi|224135051|ref|XP_002321971.1|/10-134 Populus trichocarpa - gi|224120980|ref|XP_002318467.1|/13-137 Populus trichocarpa - gi|224120988|ref|XP_002318469.1|/13-137 Populus trichocarpa - gi|224120984|ref|XP_002318468.1|/13-137 Vitis vinifera - gi|147832851|emb|CAN63961.1|/30-157 Vitis vinifera - gi|225464868|ref|XP_002272914.1|/17-144 Medicago truncatula - gi|357453161|ref|XP_003596857.1|/18-145 Glycine max - gi|356542037|ref|XP_003539478.1|/17-145 Glycine max - gi|356547116|ref|XP_003541963.1|/17-144 PGSC0003DMP400034078/40-167 Solyc11g068750.1.1/82-209 Solyc06g074170.2.1/32-159 PGSC0003DMP400010437/31-158 Populus trichocarpa - gi|224104873|ref|XP_002313601.1|/19-146 Ricinus communis - gi|255565737|ref|XP_002523858.1|/17-145 Arabidopsis lyrata subsp. lyrata - gi|297826043|ref|XP_002880904.1|/14-141 Arabidopsis thaliana - gi|15225866|ref|NP_180298.1|/14-141 Arabidopsis lyrata subsp. lyrata - gi|297812351|ref|XP_002874059.1|/13-140 Arabidopsis thaliana - gi|18420448|ref|NP_568414.1|/21-148 Eutrema halophilum - gi|312282343|dbj|BAJ34037.1|/24-151 Arabidopsis thaliana - gi|15229931|ref|NP_190015.1|/14-141 Arabidopsis lyrata subsp. lyrata - gi|297818896|ref|XP_002877331.1|/10-137 Ricinus communis - gi|255555833|ref|XP_002518952.1|/9-142 Malus x domestica - gi|302399031|gb|ADL36810.1|/9-145 Vitis vinifera - gi|147780480|emb|CAN73478.1|/9-139 Populus trichocarpa - gi|224115462|ref|XP_002317040.1|/6-139 Populus trichocarpa - gi|224061527|ref|XP_002300524.1|/8-141 Arabidopsis thaliana - gi|186516558|ref|NP_001119122.1|/9-137 Arabidopsis lyrata subsp. lyrata - gi|297798362|ref|XP_002867065.1|/9-137 Arabidopsis lyrata subsp. lyrata - gi|297851744|ref|XP_002893753.1|/24-152 Arabidopsis thaliana - gi|42562475|ref|NP_174582.3|/24-152 Vitis vinifera - gi|147837829|emb|CAN73792.1|/84-212 Malus x domestica - gi|302398999|gb|ADL36794.1|/9-137 PGSC0003DMP400046923/18-147 Solyc06g073050.2.1/19-148 NbNac1_1/28-157 Petunia x hybrida - gi|21358787|gb|AAM47025.1|/27-156 Solyc03g080090.2.1/27-156 PGSC0003DMP400001112/27-156 StNac1_5/28-157 Arabidopsis thaliana - gi|30689531|ref|NP_197847.3|/13-142 Arabidopsis lyrata subsp. lyrata - gi|297812653|ref|XP_002874210.1|/13-142 Arabidopsis thaliana - gi|15229161|ref|NP_190522.1|/13-142 Arabidopsis lyrata subsp. lyrata - gi|297819600|ref|XP_002877683.1|/13-142 Ricinus communis - gi|255560596|ref|XP_002521312.1|/9-137 Populus trichocarpa - gi|224118488|ref|XP_002317831.1|/11-139 Populus trichocarpa - gi|224134993|ref|XP_002321956.1|/11-139 Medicago truncatula - gi|357490459|ref|XP_003615517.1|/25-153 Medicago truncatula - gi|357464259|ref|XP_003602411.1|/230-358 Medicago truncatula - gi|357464261|ref|XP_003602412.1|/22-150 Glycine max - gi|255646322|gb|ACU23644.1|/22-150 Glycine max - gi|356516174|ref|XP_003526771.1|/22-150 Vitis vinifera - gi|225453680|ref|XP_002268892.1|/14-144 Vitis vinifera - gi|296089035|emb|CBI38738.3|/64-192 Vitis vinifera - gi|147843195|emb|CAN80539.1|/9-137 Vitis vinifera - gi|147787054|emb|CAN62333.1|/14-159 Vitis vinifera - gi|147843196|emb|CAN80540.1|/669-799 Vitis vinifera - gi|296089034|emb|CBI38737.3|/14-144 Vitis vinifera - gi|147787053|emb|CAN62332.1|/14-144 Vitis vinifera - gi|147843196|emb|CAN80540.1|/9-139 Vitis vinifera - gi|296089035|emb|CBI38738.3|/682-812 Vitis vinifera - gi|147843196|emb|CAN80540.1|/1150-1280 Vitis vinifera - gi|296089033|emb|CBI38736.3|/9-139 Ricinus communis - gi|255548101|ref|XP_002515107.1|/14-139 Ricinus communis - gi|255548117|ref|XP_002515115.1|/12-128 Ricinus communis - gi|255548119|ref|XP_002515116.1|/20-150 Populus trichocarpa - gi|224068220|ref|XP_002302683.1|/30-151 Populus trichocarpa - gi|224130712|ref|XP_002320909.1|/12-143 Populus trichocarpa - gi|224063969|ref|XP_002301326.1|/14-145 Populus trichocarpa - gi|224063971|ref|XP_002301327.1|/11-142 Populus trichocarpa - gi|224127506|ref|XP_002320091.1|/16-140 Populus trichocarpa - gi|224130136|ref|XP_002320761.1|/16-140 Ricinus communis - gi|255558041|ref|XP_002520049.1|/13-134 Ricinus communis - gi|255558043|ref|XP_002520050.1|/5-124 Populus trichocarpa - gi|224127202|ref|XP_002329425.1|/10-136 Populus trichocarpa - gi|224127206|ref|XP_002329426.1|/6-134 Jatropha curcas - gi|337743347|gb|AEI73170.1|/3-124 Ricinus communis - gi|255548099|ref|XP_002515106.1|/3-128 Ricinus communis - gi|255548097|ref|XP_002515105.1|/3-141 Populus trichocarpa - gi|224127510|ref|XP_002320092.1|/11-142 Populus trichocarpa - gi|224063973|ref|XP_002301328.1|/11-142 Populus trichocarpa - gi|224130708|ref|XP_002320908.1|/6-138 Populus trichocarpa - gi|224068212|ref|XP_002302682.1|/11-142 Medicago truncatula - gi|358346547|ref|XP_003637328.1|/7-149 Glycine max - gi|356560207|ref|XP_003548385.1|/11-141 Glycine max - gi|356522462|ref|XP_003529865.1|/12-142 Glycine max - gi|356560211|ref|XP_003548387.1|/4-136 Glycine max - gi|356560215|ref|XP_003548389.1|/5-139 Glycine max - gi|356520204|ref|XP_003528754.1|/5-139 Glycine max - gi|356520206|ref|XP_003528755.1|/5-139 Medicago truncatula - gi|358346553|ref|XP_003637331.1|/193-323 Medicago truncatula - gi|358347538|ref|XP_003637813.1|/53-186 Medicago truncatula - gi|358346553|ref|XP_003637331.1|/12-142 Medicago truncatula - gi|358346559|ref|XP_003637334.1|/11-141 Medicago truncatula - gi|358346870|ref|XP_003637487.1|/12-128 Medicago truncatula - gi|358346543|ref|XP_003637326.1|/12-142 Populus trichocarpa - gi|224068206|ref|XP_002302681.1|/2-128 Populus trichocarpa - gi|224130704|ref|XP_002320907.1|/2-130 Populus trichocarpa - gi|224145744|ref|XP_002336258.1|/2-133 Ricinus communis - gi|255548095|ref|XP_002515104.1|/4-138 Arabidopsis lyrata subsp. lyrata - gi|297795805|ref|XP_002865787.1|/4-145 Arabidopsis lyrata subsp. lyrata - gi|297843024|ref|XP_002889393.1|/2-138 Arabidopsis thaliana - gi|15229292|ref|NP_187093.1|/2-138 Arabidopsis lyrata subsp. lyrata - gi|297833156|ref|XP_002884460.1|/3-135 Arabidopsis thaliana - gi|15234225|ref|NP_192064.1|/4-136 Arabidopsis lyrata subsp. lyrata - gi|297810005|ref|XP_002872886.1|/4-136 Arabidopsis lyrata subsp. lyrata - gi|297810007|ref|XP_002872887.1|/5-138 Arabidopsis thaliana - gi|15234216|ref|NP_192061.1|/4-137 Arabidopsis thaliana - gi|145332955|ref|NP_001078343.1|/4-137 Arabidopsis lyrata subsp. lyrata - gi|297792793|ref|XP_002864281.1|/3-134 Arabidopsis lyrata subsp. lyrata - gi|297848416|ref|XP_002892089.1|/3-139 Arabidopsis thaliana - gi|110742030|dbj|BAE98952.1|/3-139 Arabidopsis thaliana - gi|15223276|ref|NP_171609.1|/3-139 Arabidopsis lyrata subsp. lyrata - gi|297848426|ref|XP_002892094.1|/3-133 Arabidopsis thaliana - gi|334182236|ref|NP_171727.2|/3-133 Arabidopsis lyrata subsp. lyrata - gi|297848422|ref|XP_002892092.1|/3-133 Arabidopsis thaliana - gi|15217677|ref|NP_171725.1|/3-130 Arabidopsis lyrata subsp. lyrata - gi|297848420|ref|XP_002892091.1|/11-146 Arabidopsis lyrata subsp. lyrata - gi|297848424|ref|XP_002892093.1|/3-133 Arabidopsis thaliana - gi|145334985|ref|NP_171726.2|/3-133 100 100 100 100 100 100 100 99 100 100 100 100 100 100 98 100 90 56 6 99 100 100 100 58 98 100 99 69 91 99 100 100 100 100 100 98 99 98 84 100 15 100 100 100 100 100 71 100 99 90 11 96 100 100 83 100 100 99 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 47 100 100 100 100 100 100 100 96 100 100 100 100 15 100 100 100 100 100 99 98 100 100 99 100 100 64 100 77 100 94 100 99 100 100 100 100 100 100 100 100 100 100 90 100 100 100 100 100 100 100 100 100 100 100 67 100 100 100 100 100 100 100 100 98 15 96 100 100 100 100 100 50 77 100 100 100 6 100 100 96 100 100 7 98 100 100 94 80 5 96 100 100 100 100 100 100 99 100 18 100 100 100 100 100 100 100 100 96 100 100 100 100 14 21 100 97 100 100 100 100 100 100 96 100 100 100 3 100 100 100 100 100 100 100 100 100 100 70 100 100 100 69 100 56 100 100 18 100 1 100 95 100 100 100 74 4 11 100 100 91 14 14 87 100 8 100 100 90 100 32 88 42 95 42 100 100 100 37 71 100 17 31 100 100 87 100 6 95 100 12 100 14 100 100 100 100 88 100 100 100 71 100 6 100 100 100 14 100 100 96 100 29 2 100 100 96 100 100 100 98 100 66 52 97 95 100 6 19 100 100 57 100 95 7 6 100 97 18 100 7 97 100 27 94 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Potato: phylogene1cs: Methods l Construct neighbour-‐joining (NJ) tree from 406 sequence set including target sequences (TOPALi) l Infer ‘best’ evolu5onary model from NJ tree structure (jModelTest) l Construct maximum-‐likelihood tree with bootstrap (RaxML)
- 26. Potato: phylogene1cs: Results 100 100 69 98 98 32 100 91 100 0.3 NTL1 0.0 0.5 1.0 1.5 2.0 2.5 100 97 58 100 97 56 90 100 100 100 57 100 100 100 67 100 100 100 96 100 100 100 52 100 100 100 100 100 100 100 99 100 100 18 100 100 100 100 17 99 100 100 100 100 100 100 100 99 100 100 83 100 100 100 100 100 100 100 100 100 100 100 17 100 99 96 100 100 100 100 100 100 100 100 100 97 100 100 19 100 94 100 100 100 100 100 6 47 96 70 100 100 100 100 100 42 100 6 100 100 100 100 100 100 4 100 100 100 100 77 100 100 96 100 100 100 90 100 95 7 99 100 100100 100 100 100 100 29 100 3 100 100 100 66 6 71 42 100 98 001 77 98 100 15 18 100 100 100 99 100 12 100 100 100 98 72 100 100 100 95 88 100 74 100 50 100 2 100 100 100 100 100 100 100 100 100 14 100 96 100 31 100 100 100 6 96 100 100 6 100 100 100 100 88 6 99 7 100 100 100 56 8 100 7 100 100 100 100 100 100 94 100 100 99 100 100 100 15 100 80 27 100 100 100 91 100 64 11 100 95 100 100 100 100 90 100 100 100 98 100 100 100 99 100 100 21 100 100 100 100 99 14 100 14 100 100 97 100 100 100 100 99 100 5 100 99 100 87 100 15 100 29 100 100 96 96 100 100 100 18 100 100 95 71 100 100 100 100 100 100 100 100 100 100 94 100 100 100 14 100 100 11 100 98 100 69 95 87 100 1 96 90 100 100 100 100 100 37 100 98 100 14 84 100 NAC1 NAC2 NTL13 NTL12 NTL11 NTL10 NTL9 NTL8 NTL7 NTL5 NTL6 NTL4 NTL3 NTL2 Arabidopsis Solanaceae Cereals l Target sequences: NAC1, NAC2 l Arabidopsis NACs: NTL1-‐NTL13 l TM-‐containing NACs in red l Divergence pre-‐At/Sol split l Novel At NAC classes l At NAC family expansion l TM-‐containing NACs cluster l Cereals underrepresented
- 27. gi|358347538|ref|XP_003637813.1|/53-186 gi|296089034|emb|CBI38737.3|/14-144 gi|296089033|emb|CBI38736.3|/9-139 gi|358346553|ref|XP_003637331.1|/12-142 gi|358346553|ref|XP_003637331.1|/193-323 gi|147843196|emb|CAN80540.1|/1150-1280 gi|147787054|emb|CAN62333.1|/14-159 gi|296089035|emb|CBI38738.3|/64-192 gi|224115462|ref|XP_002317040.1|/6-139 gi|302399031|gb|ADL36810.1|/9-145 gi|30689531|ref|NP_197847.3|/13-142 gi|297851744|ref|XP_002893753.1|/24-152 gi|186516558|ref|NP_001119122.1|/9-137 gi|224134993|ref|XP_002321956.1|/11-139 gi|224118488|ref|XP_002317831.1|/11-139 gi|357464259|ref|XP_003602411.1|/230-358 gi|357464261|ref|XP_003602412.1|/22-150 gi|255646322|gb|ACU23644.1|/22-150 gi|356516174|ref|XP_003526771.1|/22-150 gi|302398999|gb|ADL36794.1|/9-137 gi|255560596|ref|XP_002521312.1|/9-137 NbNac1_1/28-157 PGSC0003DMP400001112/27-156 Solyc03g080090.2.1/27-156 PGSC0003DMP400046923/18-147 Solyc06g073050.2.1/19-148 gi|224127506|ref|XP_002320091.1|/16-140 gi|224130136|ref|XP_002320761.1|/16-140 gi|356560215|ref|XP_003548389.1|/5-139 gi|356520204|ref|XP_003528754.1|/5-139 gi|296089035|emb|CBI38738.3|/682-812 gi|147837829|emb|CAN73792.1|/84-212 gi|21358787|gb|AAM47025.1|/27-156 gi|115474875|ref|NP_001061034.1|/9-143 gi|326526251|dbj|BAJ97142.1|/9-143 gi|242080633|ref|XP_002445085.1|/12-146 gi|293332711|ref|NP_001169185.1|/6-140 gi|357144944|ref|XP_003573468.1|/9-143 gi|293336942|ref|NP_001168544.1|/26-154 gi|110289615|gb|AAP55107.2|/6-134 gi|242040125|ref|XP_002467457.1|/6-134 gi|224063971|ref|XP_002301327.1|/11-142 gi|224068206|ref|XP_002302681.1|/2-128 gi|358346547|ref|XP_003637328.1|/7-149 gi|356522462|ref|XP_003529865.1|/12-142 gi|326496190|dbj|BAJ90716.1|/6-145 gi|357141157|ref|XP_003572109.1|/6-135 gi|125564636|gb|EAZ10016.1|/116-246 gi|357154515|ref|XP_003576809.1|/6-142 gi|242063552|ref|XP_002453065.1|/19-156 gi|357137711|ref|XP_003570443.1|/14-151 gi|326509519|dbj|BAJ91676.1|/22-150 gi|115449815|ref|NP_001048559.1|/16-153 gi|223949493|gb|ACN28830.1|/24-161 gi|292659258|gb|ADE34584.1|/22-150 gi|125541671|gb|EAY88066.1|/16-153 gi|357144217|ref|XP_003573214.1|/29-157 gi|115477845|ref|NP_001062518.1|/21-149 gi|326530494|dbj|BAJ97673.1|/13-150 gi|242079615|ref|XP_002444576.1|/32-160 gi|115465864|ref|NP_001056531.1|/18-149 gi|242045528|ref|XP_002460635.1|/6-135 gi|242091638|ref|XP_002436309.1|/30-161 gi|293333166|ref|NP_001169690.1|/11-142 gi|357159131|ref|XP_003578349.1|/8-135 gi|242049706|ref|XP_002462597.1|/9-136 gi|212274312|ref|NP_001130458.1|/9-136 gi|115479907|ref|NP_001063547.1|/10-144 gi|326508056|dbj|BAJ86771.1|/12-139 gi|218202386|gb|EEC84813.1|/10-144 gi|357119028|ref|XP_003561248.1|/14-150 gi|295881154|gb|ADG56507.1|/3-134 gi|222641843|gb|EEE69975.1|/10-134 gi|242032581|ref|XP_002463685.1|/4-155 gi|297848420|ref|XP_002892091.1|/11-146 gi|334182236|ref|NP_171727.2|/3-133 gi|224127202|ref|XP_002329425.1|/10-136 gi|15217677|ref|NP_171725.1|/3-130 gi|224127206|ref|XP_002329426.1|/6-134 gi|297848422|ref|XP_002892092.1|/3-133 gi|337743347|gb|AEI73170.1|/3-124 gi|255558043|ref|XP_002520050.1|/5-124 gi|297848426|ref|XP_002892094.1|/3-133 gi|145334985|ref|NP_171726.2|/3-133 gi|255548117|ref|XP_002515115.1|/12-128 gi|224130712|ref|XP_002320909.1|/12-143 gi|255558041|ref|XP_002520049.1|/13-134 gi|224068212|ref|XP_002302682.1|/11-142 gi|255548101|ref|XP_002515107.1|/14-139 gi|255548119|ref|XP_002515116.1|/20-150 gi|224063973|ref|XP_002301328.1|/11-142 gi|224130708|ref|XP_002320908.1|/6-138 gi|224127510|ref|XP_002320092.1|/11-142 gi|224068220|ref|XP_002302683.1|/30-151 gi|255548099|ref|XP_002515106.1|/3-128 Solyc12g056790.1.1/22-150 gi|224130704|ref|XP_002320907.1|/2-130 gi|224082532|ref|XP_002306731.1|/16-144 PGSC0003DMP400029635/22-150 gi|15223276|ref|NP_171609.1|/3-139 gi|110742030|dbj|BAE98952.1|/3-139 gi|224066587|ref|XP_002302150.1|/16-144 gi|168001016|ref|XP_001753211.1|/1-128 gi|302756881|ref|XP_002961864.1|/1-126 gi|297792793|ref|XP_002864281.1|/3-134 gi|297810007|ref|XP_002872887.1|/5-138 gi|15234216|ref|NP_192061.1|/4-137 gi|145332955|ref|NP_001078343.1|/4-137 gi|356576117|ref|XP_003556180.1|/19-147 gi|116793533|gb|ABK26780.1|/6-134 gi|168025227|ref|XP_001765136.1|/5-132 PGSC0003DMP400054265/31-159 gi|21105742|gb|AAM34770.1|AF509870_1/19-147 gi|168001860|ref|XP_001753632.1|/6-133 gi|168016362|ref|XP_001760718.1|/6-131 gi|148909054|gb|ABR17630.1|/7-147 Solyc04g072220.2.1/32-160 NbNac2_1/29-157 gi|7716952|gb|AAF68626.1|AF254124_1/6-134 gi|225463127|ref|XP_002265611.1|/20-148 gi|356535729|ref|XP_003536396.1|/19-147 gi|224116242|ref|XP_002331996.1|/2-129 Potato: phylogene1cs: Summary l Largest phylogene5c survey of NAC transcrip5on factors l Observed expansion and greatest diversity in Arabidopsis l TM domains conserved in the same clade across species l Iden5fied supported clades for two target sequences in potato l NACs not strongly represented in cereals – single large clade captures most known sequences l Recombina5on and domain composi5on clearly an issue… gi|356562269|ref|XP_003549394.1|/9-144 gi|224132570|ref|XP_002321355.1|/8-141 gi|218184374|gb|EEC66801.1|/31-142 gi|297819354|ref|XP_002877560.1|/223-364 gi|297795805|ref|XP_002865787.1|/4-145 gi|297819354|ref|XP_002877560.1|/5-145 gi|297819350|ref|XP_002877558.1|/46-187 gi|224135051|ref|XP_002321971.1|/10-134 gi|224120980|ref|XP_002318467.1|/13-137 gi|224120988|ref|XP_002318469.1|/13-137 gi|224080239|ref|XP_002306067.1|/7-131 gi|224120984|ref|XP_002318468.1|/13-137 gi|15233215|ref|NP_191081.1|/11-151 gi|15225866|ref|NP_180298.1|/14-141 gi|289551912|gb|ADD10614.1|/9-137 gi|371925001|tpe|CBZ41162.1|/9-137 gi|218187925|gb|EEC70352.1|/9-137 gi|115435780|ref|NP_001042648.1|/9-137 gi|226493114|ref|NP_001147448.1|/9-137 gi|242056921|ref|XP_002457606.1|/9-137 gi|357129971|ref|XP_003566632.1|/9-137 gi|297818896|ref|XP_002877331.1|/10-137 gi|18420448|ref|NP_568414.1|/21-148 gi|297812351|ref|XP_002874059.1|/13-140 gi|297826043|ref|XP_002880904.1|/14-141 Solyc11g068750.1.1/82-209 Solyc06g074170.2.1/32-159 gi|312282343|dbj|BAJ34037.1|/24-151 gi|15229931|ref|NP_190015.1|/14-141 PGSC0003DMP400034078/40-167 gi|302399033|gb|ADL36811.1|/9-137 gi|125542167|gb|EAY88306.1|/6-134 gi|15218843|ref|NP_176766.1|/6-134 gi|255571259|ref|XP_002526579.1|/6-134 gi|356542037|ref|XP_003539478.1|/17-145 gi|356547116|ref|XP_003541963.1|/17-144 gi|356499060|ref|XP_003518362.1|/5-133 gi|357453161|ref|XP_003596857.1|/18-145 gi|147832851|emb|CAN63961.1|/30-157 gi|116786496|gb|ABK24128.1|/6-136 gi|297841167|ref|XP_002888465.1|/6-134 gi|148907008|gb|ABR16648.1|/6-136 gi|356551985|ref|XP_003544352.1|/5-133 gi|6714418|gb|AAF26106.1|AC012328_9/6-126 gi|297811871|ref|XP_002873819.1|/6-134 gi|297832960|ref|XP_002884362.1|/6-134 gi|186509710|ref|NP_186970.2|/6-134 gi|356569398|ref|XP_003552888.1|/6-134 gi|15237939|ref|NP_197228.1|/6-134 gi|356537835|ref|XP_003537430.1|/6-134 gi|226531876|ref|NP_001146336.1|/6-134 gi|357120932|ref|XP_003562178.1|/6-134 gi|242042457|ref|XP_002468623.1|/6-134 gi|357460717|ref|XP_003600640.1|/6-134 gi|357491761|ref|XP_003616168.1|/6-134 PGSC0003DMP400037231/6-134 gi|224079646|ref|XP_002305903.1|/6-134 gi|225466227|ref|XP_002267333.1|/6-134 Solyc02g036430.1.1/6-134 Solyc02g081270.2.1/6-134 gi|224135105|ref|XP_002327567.1|/5-133 gi|224109872|ref|XP_002315338.1|/5-133 gi|147802301|emb|CAN70406.1|/9-137 gi|224109864|ref|XP_002315336.1|/39-167 Solyc11g008010.1.1/97-225 gi|357451351|ref|XP_003595952.1|/6-134 gi|4585977|gb|AAD25613.1|AC005287_15/3-131 gi|356528635|ref|XP_003532905.1|/6-134 gi|356557052|ref|XP_003546832.1|/6-134 gi|224112333|ref|XP_002316156.1|/6-134 gi|255573304|ref|XP_002527580.1|/6-134 gi|224098826|ref|XP_002311281.1|/6-134 PGSC0003DMP400054092/6-134 gi|296088017|emb|CBI35300.3|/6-134 gi|356531066|ref|XP_003534099.1|/6-134 gi|255569031|ref|XP_002525485.1|/6-134 gi|224120448|ref|XP_002318332.1|/6-134 gi|224125708|ref|XP_002329698.1|/6-134 gi|297830402|ref|XP_002883083.1|/6-134 gi|115480567|ref|NP_001063877.1|/6-134 gi|297853370|ref|XP_002894566.1|/6-134 gi|147854237|emb|CAN83436.1|/6-134 gi|297800286|ref|XP_002868027.1|/6-136 gi|15237469|ref|NP_199471.1|/6-135 gi|297794579|ref|XP_002865174.1|/6-135 gi|15236721|ref|NP_193532.1|/6-136 Solyc06g061080.2.1/6-134 gi|42562467|ref|NP_174529.2|/6-141 gi|21105751|gb|AAM34774.1|AF509874_1/6-134 PGSC0003DMP400030569/6-134 gi|356563453|ref|XP_003549977.1|/6-136 gi|224138076|ref|XP_002326512.1|/6-134 gi|356512014|ref|XP_003524716.1|/6-136 gi|242045530|ref|XP_002460636.1|/6-134 gi|357476695|ref|XP_003608633.1|/6-137 Solyc03g098190.2.1/6-134 gi|224126509|ref|XP_002329572.1|/6-134 gi|297829588|ref|XP_002882676.1|/27-155 gi|18398891|ref|NP_566374.1|/27-155 gi|238479717|ref|NP_001154602.1|/27-188 gi|12322791|gb|AAG51388.1|AC011560_20/9-137 gi|6456751|gb|AAF09254.1|AF201456_1/9-137 gi|21105744|gb|AAM34771.1|AF509871_1/23-151 PGSC0003DMP400054118/10-139 gi|297833156|ref|XP_002884460.1|/3-135 gi|15229292|ref|NP_187093.1|/2-138 gi|188593543|dbj|BAG32519.1|/6-134 gi|115464001|ref|NP_001055600.1|/6-134 gi|297846226|ref|XP_002890994.1|/6-139 gi|297829590|ref|XP_002882677.1|/9-137 gi|21593389|gb|AAM65338.1|/9-137 gi|15237699|ref|NP_196061.1|/9-137 gi|255583748|ref|XP_002532627.1|/11-139 gi|224100709|ref|XP_002311983.1|/10-138 gi|356535507|ref|XP_003536286.1|/33-161 Solyc05g055480.2.1/17-145 gi|125550359|gb|EAY96181.1|/6-134 gi|357464643|ref|XP_003602603.1|/7-135 gi|224130574|ref|XP_002328323.1|/7-135 PGSC0003DMP400040418/1-129 PGSC0003DMP400040416/22-150 Solyc11g008000.1.1/10-139 gi|357133590|ref|XP_003568407.1|/6-134 gi|296088554|emb|CBI37545.3|/6-134 gi|224095914|ref|XP_002310505.1|/7-135 gi|15242390|ref|NP_196495.1|/6-134 gi|18399168|ref|NP_564440.1|/16-144 gi|224120762|ref|XP_002330945.1|/237-361 gi|21105740|gb|AAM34769.1|AF509869_1/12-140 gi|255557623|ref|XP_002519841.1|/6-134 gi|356561847|ref|XP_003549188.1|/6-134 gi|357482369|ref|XP_003611470.1|/6-148 gi|15229511|ref|NP_188400.1|/6-134 gi|15237698|ref|NP_196060.1|/28-170 gi|18398893|ref|NP_566375.1|/27-155 gi|297806417|ref|XP_002871092.1|/9-137 gi|147802300|emb|CAN70405.1|/13-141 PGSC0003DMP400054120/30-158 Solyc05g055470.2.1/10-138 gi|21105736|gb|AAM34767.1|AF509867_1/35-163 gi|357168397|ref|XP_003581627.1|/5-133 gi|356508975|ref|XP_003523228.1|/5-133 gi|356516356|ref|XP_003526861.1|/5-133 gi|297846402|ref|XP_002891082.1|/16-144 StNac2_5/32-160 gi|21105746|gb|AAM34772.1|AF509872_1/10-138 gi|326492385|dbj|BAK01976.1|/6-134 PGSC0003DMP400049938/4-132 gi|357519943|ref|XP_003630260.1|/6-134 gi|297846400|ref|XP_002891081.1|/16-144 gi|356576366|ref|XP_003556303.1|/34-162 gi|242077812|ref|XP_002448842.1|/6-134 gi|302399003|gb|ADL36796.1|/6-134 gi|297811023|ref|XP_002873395.1|/6-134 gi|15237609|ref|NP_201211.1|/6-134 gi|326369339|gb|ADZ55681.1|/6-134 Solyc11g005920.1.1/4-132 gi|255552430|ref|XP_002517259.1|/5-133 gi|356512837|ref|XP_003525122.1|/3-131 gi|14334572|gb|AAK59465.1|/10-136 gi|356525677|ref|XP_003531450.1|/3-131 gi|297797411|ref|XP_002866590.1|/6-134 gi|297846266|ref|XP_002891014.1|/10-136 gi|302399015|gb|ADL36802.1|/8-136 Solyc03g078120.2.1/4-132 gi|21105732|gb|AAM34765.1|AF509865_1/4-132 gi|293336322|ref|NP_001169920.1|/6-134 gi|356576364|ref|XP_003556302.1|/4-132 Solyc08g077110.2.1/6-134 gi|297829586|ref|XP_002882675.1|/27-155 gi|225464868|ref|XP_002272914.1|/17-144 gi|224104873|ref|XP_002313601.1|/19-146 gi|255565737|ref|XP_002523858.1|/17-145 PGSC0003DMP400010437/31-158 gi|297807857|ref|XP_002871812.1|/13-130 gi|302398995|gb|ADL36792.1|/8-118 gi|297843024|ref|XP_002889393.1|/2-138 gi|15234225|ref|NP_192064.1|/4-136 gi|297810005|ref|XP_002872886.1|/4-136 gi|297848416|ref|XP_002892089.1|/3-139 gi|297848424|ref|XP_002892093.1|/3-133 gi|255548097|ref|XP_002515105.1|/3-141 gi|224063969|ref|XP_002301326.1|/14-145 gi|255548095|ref|XP_002515104.1|/4-138 gi|356560211|ref|XP_003548387.1|/4-136 gi|356520206|ref|XP_003528755.1|/5-139 gi|356560207|ref|XP_003548385.1|/11-141 gi|358346559|ref|XP_003637334.1|/11-141 gi|358346870|ref|XP_003637487.1|/12-128 gi|225453680|ref|XP_002268892.1|/14-144 gi|147787053|emb|CAN62332.1|/14-144 gi|147843196|emb|CAN80540.1|/9-139 gi|147780480|emb|CAN73478.1|/9-139 gi|255555833|ref|XP_002518952.1|/9-142 gi|224061527|ref|XP_002300524.1|/8-141 gi|297798362|ref|XP_002867065.1|/9-137 gi|42562475|ref|NP_174582.3|/24-152 gi|297812653|ref|XP_002874210.1|/13-142 gi|15229161|ref|NP_190522.1|/13-142 gi|297819600|ref|XP_002877683.1|/13-142 gi|357490459|ref|XP_003615517.1|/25-153 gi|145324118|ref|NP_001077648.1|/16-156 StNac1_5/28-157 gi|125562576|gb|EAZ08024.1|/21-149 gi|18399166|ref|NP_564439.1|/16-144 gi|21618244|gb|AAM67294.1|/16-144 gi|357516809|ref|XP_003628693.1|/6-134 gi|225448908|ref|XP_002265550.1|/13-141 gi|147765514|emb|CAN78113.1|/6-135 gi|359474569|ref|XP_002280894.2|/6-135 gi|115481670|ref|NP_001064428.1|/18-129 gi|224145744|ref|XP_002336258.1|/2-133 gi|358346543|ref|XP_003637326.1|/12-142 gi|147843196|emb|CAN80540.1|/669-799 gi|147843195|emb|CAN80539.1|/9-137 0.01
- 28. Bacteria: genomics: Introduc1on I l Dickeya spp. l Major pan-‐European bacterial plant pathogen l Emerging threat: Dickeya “solani” l Four Dickeya genomes in Genbank l Three misiden5fied: (D.dadanGi, D,chrysanthemi, D.zeae, D.paradisiaca) l 23 drag Dickeya genomes sequenced by JHI (D.dadanGi, D,chrysanthemi, D.zeae, D.paradisiaca, D.dianthicola, D.“solani”, several unclassified) l Two drag genomes sequenced by ILVO (D.dianthicola, D.“solani”) l Several technologies: l 454 PE and single-‐end l Illumina PE and single-‐end
- 29. Bacteria: genomics: Introduc1on I ID Method Bases Con1gs ID Method Bases Con1gs Ddie_NCPPB_2976 dieffenbachiae Newbler de novo 4804891 76 Dunk_MK7 unknown Newbler de novo 4921532 57 Dze_NCPPB_3531 zeae Newbler de novo 4622472 27 Dch_NCPPB_516 chrysanthemi Minimus (meta) 4614776 33 Dze_NCPPB_2538 zeae Newbler de novo 4556975 41 Dda_NCPPB_898 dadanGi Minimus (meta) 4933637 45 Dze_CSL_RW192 zeae Newbler de novo 4692116 49 Dda_NCPPB_3537 dadanGi Minimus (meta) 4805222 38 Dunk_CSL_RW240 unknown Newbler de novo 4375181 73 Dunk_NCPPB_3274 unknown Minimus (meta) 5110316 54 Dch_NCPPB_3533 chrysanthemi Minimus (meta) 4769655 49 Dunk_DW0440 unknown Newbler de novo 4330262 147 Dso_IPO2222 solani Newbler de novo 4863391 96 (8) Dunk_NCPPB_569 unknown Minimus (meta) 4215441 58 Dch_NCPPB_402 chrysanthemi Newbler de novo 4718933 93 (12) Dze_NCPPB_3532 zeae Minimus (meta) 4555162 19 Dpa_NCPPB_2511 paradisiaca Newbler reference 4627470 43 Ddi_GBBC2039 dianthicola Minimus (meta) 4776142 237 Dso_MK16 solani Minimus (meta) 4868282 24 Dso_GBBC2040 solani CLCBio reference 4832847 224 Dze_MK19 zeae Newbler de novo 4668103 33 Ddi_NCPPB_3534 dianthicola MIRA (hybrid dn) 4831142 45 Ddi_IPO980 dianthicola Newbler de novo 4825313 62 Ddi_NCPPB_453 dianthicola Newbler de novo 4668151 46 Dso_MK10 solani Minimus (meta) 4931437 41
- 30. Bacteria: genomics: Introduc1on I ID Prodigal Excess tRNA ID Prodigal Excess tRNA Ddie_NCPPB_2976 4269 63 + 2 63 Dunk_MK7 4251 48 + 1 64 dieffenbachiae unknown Dze_NCPPB_3531 zeae 4072 59 62 Dch_NCPPB_516 chrysanthemi 4190 60 + 1 69 Dze_NCPPB_2538 zeae 4048 46 64 Dda_NCPPB_898 dadanGi 4330 43 62 Dze_CSL_RW192 zeae 4205 64 + 1 60 Dda_NCPPB_3537 dadanGi 4163 47 64 Dunk_CSL_RW240 unknown 3953 31 64 Dunk_NCPPB_3274 unknown 4439 63 69 Dch_NCPPB_3533 chrysanthemi 4211 51 + 1 65 Dunk_DW0440 unknown 3962 31 59 Dso_IPO2222 solani 4226 62 + 2 63 Dunk_NCPPB_569 unknown 3878 59 72 Dch_NCPPB_402 chrysanthemi 4204 58 61 Dze_NCPPB_3532 zeae 4062 55 64 Dpa_NCPPB_2511 paradisiaca 4094 75 + 2 64 Ddi_GBBC2039 dianthicola 4286 67 + 2 57 Dso_MK16 solani 4168 58 + 2 64 Dso_GBBC2040 solani 4231 57 + 4 63 Dze_MK19 zeae 4179 47 65 Ddi_NCPPB_3534 dianthicola 4333 64 + 2 77 Ddi_IPO980 dianthicola 4254 71 + 1 63 Ddi_NCPPB_453 dianthicola 4263 60 + 2 64 Dso_MK10 solani 4118 54 + 2 62
- 31. Bacteria: genomics: Introduc1on I l 25 BioProjects online l Genome assembly submissions in progress
- 32. Bacteria: genomics l Pairwise drag assembly comparisons (MegaBLAST, MUMmer) Dickeya zeae pairwise comparisons
- 33. Bacteria: genomics l Pairwise drag assembly comparisons (MegaBLAST, MUMmer) l Average Nucleo5de Iden5ty (ANI), unique regions Dickeya pairwise unique regions GBBC2039 NCPPB_3534 NCPPB_453 IPO_980 MK7 NCPPB_3274 NCPPB_2976 NC_014500 NCPPB_898 NCPPB_3537 GBBC2040 IPO_2222 MK10 MK16 CSL_RW240 DW_0440 NCPPB_2511 NC_012880 NCPPB_569 NCPPB_402 NCPPB_516 NCPPB_3533 NC_012912 NCPPB_3531 CSL_RW192 NC_013592 MK19 NCPPB_3532 NCPPB_2538 GBBC2039 NCPPB_3534 NCPPB_453 IPO_980 MK7 NCPPB_3274 NCPPB_2976 NC_014500 NCPPB_898 NCPPB_3537 GBBC2040 IPO_2222 MK10 MK16 CSL_RW240 DW_0440 NCPPB_2511 NC_012880 NCPPB_569 NCPPB_402 NCPPB_516 NCPPB_3533 NC_012912 NCPPB_3531 CSL_RW192 NC_013592 MK19 NCPPB_3532 NCPPB_2538 Dickeya average nucleo5de iden5ty (ANI)
- 34. Bacteria: genomics l Pairwise drag assembly comparisons (MegaBLAST, MUMmer) l Is D.“solani” a novel species? (95% ANI≈70% DNA-‐DNA hybridisa5on) GBBC2039 NCPPB_3534 NCPPB_453 IPO_980 MK7 NCPPB_3274 NCPPB_2976 NC_014500 NCPPB_898 NCPPB_3537 GBBC2040 IPO_2222 MK10 MK16 CSL_RW240 DW_0440 NCPPB_2511 NC_012880 NCPPB_569 NCPPB_402 NCPPB_516 NCPPB_3533 NC_012912 NCPPB_3531 CSL_RW192 NC_013592 MK19 NCPPB_3532 NCPPB_2538 GBBC2039 NCPPB_3534 NCPPB_453 IPO_980 MK7 NCPPB_3274 NCPPB_2976 NC_014500 NCPPB_898 NCPPB_3537 GBBC2040 IPO_2222 MK10 MK16 CSL_RW240 DW_0440 NCPPB_2511 NC_012880 NCPPB_569 NCPPB_402 NCPPB_516 NCPPB_3533 NC_012912 NCPPB_3531 CSL_RW192 NC_013592 MK19 NCPPB_3532 NCPPB_2538 Dickeya average nucleo5de iden5ty (ANI) GBBC2039 NCPPB_3534 IPO_980 NCPPB_453 NCPPB_2976 NCPPB_898 NCPPB_3537 NC_014500 GBBC2040 IPO_2222 MK10 MK16 GBBC2039 NCPPB_3534 IPO_980 NCPPB_453 NCPPB_2976 NCPPB_898 NCPPB_3537 NC_014500 GBBC2040 IPO_2222 MK10 MK16
- 35. Bacteria: genomics: Methods l Gene complement comparisons l Reciprocal best BLAST hits (RBBH) Organism 1 CDS: Organism 2 CDS: best BLAST hit: RBBH: not RBBH
- 36. Bacteria: genomics: Methods best BLAST hits reciprocal best BLAST hits
- 37. Bacteria: genomics D. solani IPO2222 70% iden5ty 100% iden5ty core accessory
- 38. Bacteria: genomics l Core and accessory genomes from RBBH Cliques: l Predicted Dickeya core genome: 2201 genes
- 39. Compara1ve Genomics l Predicted Dickeya species-‐specific accessory genome sizes Species Weak pruning Full pruning ‘core’ 2201 2201 chrysanthemi 32 36 dadanGi 11 14 dianthicola 102 127 paradisiaca 404 441 solani 120 157 zeae 33 40 l Accessory = RBBH with all other members of the species, but no other Dickeya l Weak pruning: remove all RBBH with <80% iden5ty, 40% coverage l Full pruning: remove all RBBH un5l minimal ‘clique’ found
- 40. Bacteria: genomics l Collinearity and conserva5on of synteny from RBBH l iADHoRe
- 41. Bacteria: genomics l Collinearity and conserva5on of synteny from RBBH l iADHoRe Dickeya Ddi/Dda Ddi/Dso Dze Dso
- 42. Bacteria: SysBio: Methods l 29 metabolic models (25 drag genomes, 4 published) l Presence/absence of metabolic pathways l Substrate dependence/survival l Flux Balance Analysis (FBA) l Steady-‐states/Elementary Modes l Associa5on with phenotype
- 43. Bacteria: SysBio: Results Compound presence/absence table Reac5on presence/absence table
- 44. Bacteria: SysBio: Results l Predicted substrate-‐dependent growth l experimental verifica5on Sonia Humphris/Anne-‐Laure Lucquet
- 45. Dickeya Virtual Machine l Aims: l A basis for collabora5on (joint compara5ve genomics paper) l Share sequencing and analysis data l Provide tools for analysis l Solu5on: l Ubuntu 12.04 as a virtual machine on USB s5ck
- 47. Bacteria: genomics: Introduc1on II l Campylobacter spp. l Collabora5on with University of Aberdeen (studentship available): >4200 genome sequences of outbreak isolates l Most prevalent (increasingly so) food-‐borne pathogen in Scotland: >6500 cases per annum; 60-‐80% of cases from chicken l Occasional water contamina5on l Mul5ple host-‐specific species (canle, pig, chicken) l No magic bullet – hygiene is not sufficient
- 48. Bacteria: genomics l Analysis underway (RBBH >34 day calcula5on on JHI cluster) 1.0e−05 7.5e−06 5.0e−06 2.5e−06 0.005 0.004 0.003 0.002 0.001 0.000 2000 4000 6000 8000 genecall count density Genecall count distribution (binsize=50) 0.0e+00 2e+06 3e+06 4e+06 assembly length density Total assembly length distribution (binsize=1e4)
- 49. Bacteria: genomics: Summary l 25 novel Dickeya drag genomes l Whole-‐genome differences (structure/similarity) l Gene complement differences l Core and accessory genomes l Metabolic reconstruc5on l Phenotypic differences l Dickeya VM for collaborators l 1034 novel Campylobacter genomes l Influence of gene complement on host associa5on
- 50. Acknowledgements l Bacteria: diagnos1cs l Ian Toth, Nicola Holden, Sonia Humphris (CMS) l Mar5na Bielaszewska, Helge Karch, Nadine Brandt (University of Münster) l John Elphinstone, Neil Parkinson, Valerie Bertrand (FERA, York) l Potato: NB-‐LRRs l Ingo Hein, Florian Jupe, Glenn Bryan, Sanjeev Sharma (CMS) l Peter Cock, Linda Milne (ICS) l Frank Wright, Katrin MacKenzie (BioSS) l Graham Etherington (TSL, Norwich) l Dan Bolser (University of Dundee/EBI) l Bacteria: genomics l Ian Toth, Sonia Humphris, Nicola Holden, Emma Douglas, Anne-‐Laure Lucquet (CMS) l Peter Cock, Iain Milne, Sue Jones (ICS) l Ken Forbes, Norval Strachan (University of Aberdeen) l Gerry Saddler (SASA, Edinburgh) l Steve Baeyen, Mar5ne Maes, Johan van Vaerenbergh (ILVO, Belgium) l John Elphinstone, Neil Parkinson (FERA, York) l Jan van der Wolf (PRI Wageningen) l Minna Pirhonen (University of Helsinki) l Potato: phylogene1cs l Paul Birch, Hazel McLellan (CMS) l Frank Wright (BioSS)