adventures in bioinformatics (2012)
DESCRIPTION
An end-of-year personal update from 2012, given at JHI's Cellular and Molecular Sciences seminar seriesTRANSCRIPT
Adventures in Bioinforma1cs (2012)
Leighton Pritchard
Summary l Potato: NB-‐LRRs
l Bacteria: diagnos5cs
l Potato: phylogene5cs
l Bacteria: genomics
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
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
NB-‐LRR:
Not NB-‐LRR:
No false posi5ves No false nega5ves
Potato: NB-‐LRRs: Method l Mo5f iden5fica5on/composi5on
l Train MEME (psp-‐gen) on posi5ve and nega5ve examples to build model
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
NB-‐LRR:
Not NB-‐LRR:
‘good’ score
‘bad’ score
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
Potato: NB-‐LRRs: Results l NB-‐LRR model used to build gene enrichment bead ‘array’
l Iden5fied 338 addi5onal candidate NB-‐LRRs
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.
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.
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
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
Amplifica5on of nega5ve samples: discard
Group (species) IV
Group (species) V
Simultaneous design of primers specific to all subgroups of the input sequence set
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.
Validate in vitro: unseen data (performance es5mates)
Bacteria: diagnos1cs: Method
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.
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
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.
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.
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
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
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.
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)
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
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.
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
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…)
0.3
0.00.5
1.01.5
2.02.5
3.0
Glycine m
ax - gi|356535507|ref|XP_003536286.1|/33-161
Populus trichocarpa - gi|224066587|ref|XP_002302150.1|/16-144
StNac1_5/28-157
Populus trichocarpa - gi|224068212|ref|XP_002302682.1|/11-142
Arabidopsis lyrata subsp. lyrata - gi|297829590|ref|XP_002882677.1|/9-137
Hordeum vulgare subsp. vulgare - gi|188593543|dbj|BAG
32519.1|/6-134
Solyc12g056790.1.1/22-150
Brachypodium distachyon - gi|357120932|ref|XP_003562178.1|/6-134
Oryza sativa Indica G
roup - gi|125550359|gb|EAY96181.1|/6-134
Populus trichocarpa - gi|224061527|ref|XP_002300524.1|/8-141
Arabidopsis lyrata subsp. lyrata - gi|297807857|ref|XP_002871812.1|/13-130
Ricinus comm
unis - gi|255548119|ref|XP_002515116.1|/20-150
Petunia x hybrida - gi|21358787|gb|AAM47025.1|/27-156
Arabidopsis thaliana - gi|18399166|ref|NP_564439.1|/16-144
Arabidopsis thaliana - gi|15229161|ref|NP_190522.1|/13-142
Oryza sativa Indica G
roup - gi|125562576|gb|EAZ08024.1|/21-149
Malus x dom
estica - gi|302398995|gb|ADL36792.1|/8-118
Petunia x hybrida - gi|21105736|gb|AAM34767.1|AF509867_1/35-163
Populus trichocarpa - gi|224130704|ref|XP_002320907.1|/2-130
Arabidopsis lyrata subsp. lyrata - gi|297792793|ref|XP_002864281.1|/3-134
PGSC0003DMP400034078/40-167
Ricinus comm
unis - gi|255558043|ref|XP_002520050.1|/5-124
Populus trichocarpa - gi|224068220|ref|XP_002302683.1|/30-151
Oryza sativa Indica G
roup - gi|218202386|gb|EEC84813.1|/10-144
Sorghum bicolor - gi|242063552|ref|XP_002453065.1|/19-156
Arabidopsis lyrata subsp. lyrata - gi|297819354|ref|XP_002877560.1|/223-364
Populus trichocarpa - gi|224126509|ref|XP_002329572.1|/6-134
Solyc02g036430.1.1/6-134
Glycine m
ax - gi|356576366|ref|XP_003556303.1|/34-162
Populus trichocarpa - gi|224082532|ref|XP_002306731.1|/16-144
Vitis vinifera - gi|147832851|emb|CAN63961.1|/30-157
Medicago truncatula - gi|357490459|ref|XP_003615517.1|/25-153
Populus trichocarpa - gi|224068206|ref|XP_002302681.1|/2-128
Glycine m
ax - gi|356542037|ref|XP_003539478.1|/17-145
Medicago truncatula - gi|357464261|ref|XP_003602412.1|/22-150
Populus trichocarpa - gi|224098826|ref|XP_002311281.1|/6-134
Physcomitrella patens subsp. patens - gi|168001860|ref|XP_001753632.1|/6-133
Ricinus comm
unis - gi|255548101|ref|XP_002515107.1|/14-139
Zea mays - gi|293333166|ref|NP_001169690.1|/11-142
Arabidopsis lyrata subsp. lyrata - gi|297848426|ref|XP_002892094.1|/3-133
PGSC0003DMP400030569/6-134
Arabidopsis lyrata subsp. lyrata - gi|297830402|ref|XP_002883083.1|/6-134
Arabidopsis lyrata subsp. lyrata - gi|297848422|ref|XP_002892092.1|/3-133
Petunia x hybrida - gi|21105746|gb|AAM34772.1|AF509872_1/10-138
Medicago truncatula - gi|357519943|ref|XP_003630260.1|/6-134
Arabidopsis thaliana - gi|186509710|ref|NP_186970.2|/6-134
Glycine m
ax - gi|356562269|ref|XP_003549394.1|/9-144
Vitis vinifera - gi|225453680|ref|XP_002268892.1|/14-144
Populus trichocarpa - gi|224063971|ref|XP_002301327.1|/11-142
Populus trichocarpa - gi|224130136|ref|XP_002320761.1|/16-140
Malus x dom
estica - gi|302399015|gb|ADL36802.1|/8-136
Arabidopsis lyrata subsp. lyrata - gi|297848416|ref|XP_002892089.1|/3-139
Arabidopsis thaliana - gi|18398893|ref|NP_566375.1|/27-155
Medicago truncatula - gi|357451351|ref|XP_003595952.1|/6-134
Physcomitrella patens subsp. patens - gi|168001016|ref|XP_001753211.1|/1-128
Arabidopsis lyrata subsp. lyrata - gi|297851744|ref|XP_002893753.1|/24-152
Arabidopsis lyrata subsp. lyrata - gi|297797411|ref|XP_002866590.1|/6-134
Arabidopsis thaliana - gi|15229292|ref|NP_187093.1|/2-138
Arabidopsis thaliana - gi|15237698|ref|NP_196060.1|/28-170
Hordeum vulgare subsp. vulgare - gi|371925001|tpe|CBZ41162.1|/9-137
Populus trichocarpa - gi|224127202|ref|XP_002329425.1|/10-136
Arabidopsis lyrata subsp. lyrata - gi|297795805|ref|XP_002865787.1|/4-145
PGSC0003DMP400054092/6-134
Oryza sativa Japonica G
roup - gi|222641843|gb|EEE69975.1|/10-134
Populus trichocarpa - gi|224109872|ref|XP_002315338.1|/5-133
Triticum aestivum
- gi|289551912|gb|ADD10614.1|/9-137
Arabidopsis thaliana - gi|238479717|ref|NP_001154602.1|/27-188
Arabidopsis lyrata subsp. lyrata - gi|297810007|ref|XP_002872887.1|/5-138
Solyc06g061080.2.1/6-134
Oryza sativa Japonica G
roup - gi|115479907|ref|NP_001063547.1|/10-144
Arabidopsis thaliana - gi|15217677|ref|NP_171725.1|/3-130
Populus trichocarpa - gi|224120980|ref|XP_002318467.1|/13-137
Oryza sativa Indica G
roup - gi|125541671|gb|EAY88066.1|/16-153
Populus trichocarpa - gi|224100709|ref|XP_002311983.1|/10-138
Vitis vinifera - gi|147843196|emb|CAN80540.1|/1150-1280
Brachypodium distachyon - gi|357144217|ref|XP_003573214.1|/29-157
Arabidopsis thaliana - gi|145332955|ref|NP_001078343.1|/4-137
Arabidopsis lyrata subsp. lyrata - gi|297819600|ref|XP_002877683.1|/13-142
Medicago truncatula - gi|358346553|ref|XP_003637331.1|/12-142
Sorghum bicolor - gi|242091638|ref|XP_002436309.1|/30-161
Arabidopsis thaliana - gi|15229511|ref|NP_188400.1|/6-134
Arabidopsis thaliana - gi|18420448|ref|NP_568414.1|/21-148
Populus trichocarpa - gi|224127206|ref|XP_002329426.1|/6-134
Glycine m
ax - gi|356560207|ref|XP_003548385.1|/11-141
Ricinus comm
unis - gi|255548117|ref|XP_002515115.1|/12-128
Arabidopsis thaliana - gi|4585977|gb|AAD25613.1|AC005287_15/3-131
Vitis vinifera - gi|147780480|emb|CAN73478.1|/9-139
Arabidopsis lyrata subsp. lyrata - gi|297819354|ref|XP_002877560.1|/5-145
Triticum aestivum
- gi|292659258|gb|ADE34584.1|/22-150
Arabidopsis thaliana - gi|15233215|ref|NP_191081.1|/11-151
Populus trichocarpa - gi|224134993|ref|XP_002321956.1|/11-139
Zea mays - gi|293332711|ref|NP_001169185.1|/6-140
Glycine m
ax - gi|356535729|ref|XP_003536396.1|/19-147
Medicago truncatula - gi|357482369|ref|XP_003611470.1|/6-148
Vitis vinifera - gi|147843195|emb|CAN80539.1|/9-137
Arabidopsis thaliana - gi|145334985|ref|NP_171726.2|/3-133
Arabidopsis lyrata subsp. lyrata - gi|297794579|ref|XP_002865174.1|/6-135
Populus trichocarpa - gi|224063969|ref|XP_002301326.1|/14-145
Sorghum bicolor - gi|242079615|ref|XP_002444576.1|/32-160
Ricinus comm
unis - gi|255573304|ref|XP_002527580.1|/6-134
Oryza sativa Japonica G
roup - gi|115464001|ref|NP_001055600.1|/6-134
Solyc06g074170.2.1/32-159
Sorghum bicolor - gi|242045528|ref|XP_002460635.1|/6-135
Arabidopsis lyrata subsp. lyrata - gi|297832960|ref|XP_002884362.1|/6-134
Solyc06g073050.2.1/19-148
Arabidopsis thaliana - gi|12322791|gb|AAG51388.1|AC011560_20/9-137
Populus trichocarpa - gi|224118488|ref|XP_002317831.1|/11-139
Medicago truncatula - gi|357491761|ref|XP_003616168.1|/6-134
Arabidopsis lyrata subsp. lyrata - gi|297829588|ref|XP_002882676.1|/27-155
Medicago truncatula - gi|358346543|ref|XP_003637326.1|/12-142
Solyc11g008010.1.1/97-225
Arabidopsis lyrata subsp. lyrata - gi|297846266|ref|XP_002891014.1|/10-136
Picea sitchensis - gi|116793533|gb|ABK26780.1|/6-134
Populus trichocarpa - gi|224104873|ref|XP_002313601.1|/19-146
Arabidopsis thaliana - gi|334182236|ref|NP_171727.2|/3-133
Oryza sativa Japonica G
roup - gi|115435780|ref|NP_001042648.1|/9-137
Brachypodium distachyon - gi|357129971|ref|XP_003566632.1|/9-137
NbNac2_1/29-157
Arabidopsis thaliana - gi|15225866|ref|NP_180298.1|/14-141
Vitis vinifera - gi|296089033|emb|CBI38736.3|/9-139
Hordeum vulgare subsp. vulgare - gi|326509519|dbj|BAJ91676.1|/22-150
Brachypodium distachyon - gi|357133590|ref|XP_003568407.1|/6-134
Solyc08g077110.2.1/6-134
Arabidopsis lyrata subsp. lyrata - gi|297846226|ref|XP_002890994.1|/6-139
Ricinus comm
unis - gi|255555833|ref|XP_002518952.1|/9-142
Arabidopsis lyrata subsp. lyrata - gi|297819350|ref|XP_002877558.1|/46-187
Arabidopsis thaliana - gi|15237609|ref|NP_201211.1|/6-134
Jatropha curcas - gi|337743347|gb|AEI73170.1|/3-124
Arabidopsis thaliana - gi|15234225|ref|NP_192064.1|/4-136
Hordeum vulgare subsp. vulgare - gi|326496190|dbj|BAJ90716.1|/6-145
Arabidopsis lyrata subsp. lyrata - gi|297806417|ref|XP_002871092.1|/9-137
Populus trichocarpa - gi|224120762|ref|XP_002330945.1|/237-361
Ricinus comm
unis - gi|255552430|ref|XP_002517259.1|/5-133
Arabidopsis lyrata subsp. lyrata - gi|297829586|ref|XP_002882675.1|/27-155
Populus trichocarpa - gi|224130574|ref|XP_002328323.1|/7-135
Vitis vinifera - gi|147854237|emb|CAN83436.1|/6-134
Selaginella moellendorffii - gi|302756881|ref|XP_002961864.1|/1-126
PGSC0003DMP400029635/22-150
Vitis vinifera - gi|296088554|emb|CBI37545.3|/6-134
Arabidopsis lyrata subsp. lyrata - gi|297811871|ref|XP_002873819.1|/6-134
Glycine m
ax - gi|356560211|ref|XP_003548387.1|/4-136
Zea mays - gi|223949493|gb|ACN28830.1|/24-161
Populus trichocarpa - gi|224112333|ref|XP_002316156.1|/6-134
Populus trichocarpa - gi|224130708|ref|XP_002320908.1|/6-138
PGSC0003DMP400037231/6-134
Glycine m
ax - gi|356551985|ref|XP_003544352.1|/5-133
Medicago truncatula - gi|7716952|gb|AAF68626.1|AF254124_1/6-134
Arabidopsis thaliana - gi|21593389|gb|AAM65338.1|/9-137
Sorghum bicolor - gi|242056921|ref|XP_002457606.1|/9-137
PGSC0003DMP400040416/22-150
Zea mays - gi|293336322|ref|NP_001169920.1|/6-134
Glycine m
ax - gi|356512837|ref|XP_003525122.1|/3-131
Setaria italica - gi|326369339|gb|ADZ55681.1|/6-134
Petunia x hybrida - gi|21105740|gb|AAM34769.1|AF509869_1/12-140
Arabidopsis thaliana - gi|186516558|ref|NP_001119122.1|/9-137
Hordeum vulgare subsp. vulgare - gi|326508056|dbj|BAJ86771.1|/12-139
Medicago truncatula - gi|358346547|ref|XP_003637328.1|/7-149
Brachypodium distachyon - gi|357159131|ref|XP_003578349.1|/8-135
NbNac1_1/28-157
Medicago truncatula - gi|358346553|ref|XP_003637331.1|/193-323
Glycine m
ax - gi|356557052|ref|XP_003546832.1|/6-134
Vitis vinifera - gi|147843196|emb|CAN80540.1|/669-799
Petunia x hybrida - gi|21105751|gb|AAM34774.1|AF509874_1/6-134
Solyc02g081270.2.1/6-134
Hordeum vulgare subsp. vulgare - gi|326526251|dbj|BAJ97142.1|/9-143
Arabidopsis thaliana - gi|15234216|ref|NP_192061.1|/4-137
Oryza sativa Japonica G
roup - gi|115449815|ref|NP_001048559.1|/16-153
Vitis vinifera - gi|147787054|emb|CAN62333.1|/14-159
Medicago truncatula - gi|357476695|ref|XP_003608633.1|/6-137
Medicago truncatula - gi|357453161|ref|XP_003596857.1|/18-145
Ricinus comm
unis - gi|255558041|ref|XP_002520049.1|/13-134
Sorghum bicolor - gi|242042457|ref|XP_002468623.1|/6-134
Oryza sativa Japonica G
roup - gi|110289615|gb|AAP55107.2|/6-134
Populus trichocarpa - gi|224080239|ref|XP_002306067.1|/7-131
Glycine m
ax - gi|356576364|ref|XP_003556302.1|/4-132
Arabidopsis thaliana - gi|14334572|gb|AAK59465.1|/10-136
Arabidopsis thaliana - gi|42562467|ref|NP_174529.2|/6-141
Populus trichocarpa - gi|224109864|ref|XP_002315336.1|/39-167
Populus trichocarpa - gi|224127506|ref|XP_002320091.1|/16-140
Solyc03g080090.2.1/27-156
Zea mays - gi|212274312|ref|NP_001130458.1|/9-136
Vitis vinifera - gi|225463127|ref|XP_002265611.1|/20-148
Arabidopsis lyrata subsp. lyrata - gi|297848424|ref|XP_002892093.1|/3-133
Medicago truncatula - gi|358346870|ref|XP_003637487.1|/12-128
Solyc05g055470.2.1/10-138
Populus trichocarpa - gi|224145744|ref|XP_002336258.1|/2-133
Oryza sativa Japonica G
roup - gi|115474875|ref|NP_001061034.1|/9-143
Ricinus comm
unis - gi|255557623|ref|XP_002519841.1|/6-134
Ricinus comm
unis - gi|255571259|ref|XP_002526579.1|/6-134
Oryza sativa Indica G
roup - gi|218187925|gb|EEC70352.1|/9-137
Populus trichocarpa - gi|224127510|ref|XP_002320092.1|/11-142
Arabidopsis thaliana - gi|15218843|ref|NP_176766.1|/6-134
Picea sitchensis - gi|116786496|gb|ABK24128.1|/6-136
Medicago truncatula - gi|357464259|ref|XP_003602411.1|/230-358
Glycine m
ax - gi|356576117|ref|XP_003556180.1|/19-147
Arabidopsis lyrata subsp. lyrata - gi|297853370|ref|XP_002894566.1|/6-134
Picea sitchensis - gi|148909054|gb|ABR17630.1|/7-147
Zea mays - gi|226493114|ref|NP_001147448.1|/9-137
Solyc11g068750.1.1/82-209
Arabidopsis thaliana - gi|15223276|ref|NP_171609.1|/3-139
Medicago truncatula - gi|357460717|ref|XP_003600640.1|/6-134
Glycine m
ax - gi|356537835|ref|XP_003537430.1|/6-134
Populus trichocarpa - gi|224130712|ref|XP_002320909.1|/12-143
Ricinus comm
unis - gi|255548097|ref|XP_002515105.1|/3-141Ricinus com
munis - gi|255548099|ref|XP_002515106.1|/3-128
Populus trichocarpa - gi|224120988|ref|XP_002318469.1|/13-137
Populus trichocarpa - gi|224115462|ref|XP_002317040.1|/6-139
Vitis vinifera - gi|296088017|emb|CBI35300.3|/6-134
Oryza sativa Japonica G
roup - gi|115465864|ref|NP_001056531.1|/18-149
Ricinus comm
unis - gi|255569031|ref|XP_002525485.1|/6-134
Glycine m
ax - gi|356516356|ref|XP_003526861.1|/5-133
Brachypodium distachyon - gi|357154515|ref|XP_003576809.1|/6-142
Arabidopsis thaliana - gi|15237699|ref|NP_196061.1|/9-137
Solyc04g072220.2.1/32-160
Ricinus comm
unis - gi|255583748|ref|XP_002532627.1|/11-139
Oryza sativa Indica G
roup - gi|125542167|gb|EAY88306.1|/6-134
Glycine m
ax - gi|255646322|gb|ACU23644.1|/22-150
Physcomitrella patens subsp. patens - gi|168016362|ref|XP_001760718.1|/6-131
Vitis vinifera - gi|147787053|emb|CAN62332.1|/14-144
Populus trichocarpa - gi|224120448|ref|XP_002318332.1|/6-134
Arabidopsis lyrata subsp. lyrata - gi|297811023|ref|XP_002873395.1|/6-134
Petunia x hybrida - gi|21105742|gb|AAM34770.1|AF509870_1/19-147
Populus trichocarpa - gi|224132570|ref|XP_002321355.1|/8-141
Arabidopsis thaliana - gi|30689531|ref|NP_197847.3|/13-142
Populus trichocarpa - gi|224125708|ref|XP_002329698.1|/6-134
Populus trichocarpa - gi|224120984|ref|XP_002318468.1|/13-137
Ricinus comm
unis - gi|255565737|ref|XP_002523858.1|/17-145
Glycine m
ax - gi|356561847|ref|XP_003549188.1|/6-134
Arabidopsis thaliana - gi|110742030|dbj|BAE98952.1|/3-139
Petunia x hybrida - gi|21105744|gb|AAM34771.1|AF509871_1/23-151
Oryza sativa Japonica G
roup - gi|115481670|ref|NP_001064428.1|/18-129
Oryza sativa Indica G
roup - gi|125564636|gb|EAZ10016.1|/116-246
Arabidopsis thaliana - gi|6456751|gb|AAF09254.1|AF201456_1/9-137
Ricinus comm
unis - gi|255560596|ref|XP_002521312.1|/9-137
Sorghum bicolor - gi|242049706|ref|XP_002462597.1|/9-136
Vitis vinifera - gi|296089034|emb|CBI38737.3|/14-144
Populus trichocarpa - gi|224079646|ref|XP_002305903.1|/6-134
Arabidopsis lyrata subsp. lyrata - gi|297818896|ref|XP_002877331.1|/10-137
Oryza sativa Japonica G
roup - gi|115480567|ref|NP_001063877.1|/6-134
Vitis vinifera - gi|147802301|emb|CAN70406.1|/9-137
Glycine m
ax - gi|356520206|ref|XP_003528755.1|/5-139
Vitis vinifera - gi|359474569|ref|XP_002280894.2|/6-135
Solyc03g098190.2.1/6-134
PGSC0003DMP400040418/1-129
Arabidopsis lyrata subsp. lyrata - gi|297848420|ref|XP_002892091.1|/11-146
Glycine m
ax - gi|356525677|ref|XP_003531450.1|/3-131
Malus x dom
estica - gi|302398999|gb|ADL36794.1|/9-137
Populus trichocarpa - gi|224138076|ref|XP_002326512.1|/6-134
Zea mays - gi|226531876|ref|NP_001146336.1|/6-134
Picea sitchensis - gi|148907008|gb|ABR16648.1|/6-136
Sorghum bicolor - gi|242077812|ref|XP_002448842.1|/6-134
Arabidopsis thaliana - gi|15237939|ref|NP_197228.1|/6-134
Solyc11g008000.1.1/10-139
Brachypodium distachyon - gi|357119028|ref|XP_003561248.1|/14-150
Vitis vinifera - gi|225466227|ref|XP_002267333.1|/6-134
StNac2_5/32-160
Brachypodium distachyon - gi|357144944|ref|XP_003573468.1|/9-143
Sorghum bicolor - gi|242045530|ref|XP_002460636.1|/6-134
Glycine m
ax - gi|356499060|ref|XP_003518362.1|/5-133
Solyc11g005920.1.1/4-132
PGSC0003DMP400046923/18-147
Vitis vinifera - gi|225464868|ref|XP_002272914.1|/17-144
PGSC0003DMP400054118/10-139
PGSC0003DMP400054265/31-159
Glycine m
ax - gi|356520204|ref|XP_003528754.1|/5-139
Arabidopsis thaliana - gi|15237469|ref|NP_199471.1|/6-135
Sorghum bicolor - gi|242040125|ref|XP_002467457.1|/6-134
Arabidopsis lyrata subsp. lyrata - gi|297810005|ref|XP_002872886.1|/4-136
Zea mays - gi|293336942|ref|NP_001168544.1|/26-154
Vitis vinifera - gi|147837829|emb|CAN73792.1|/84-212
Vitis vinifera - gi|147843196|emb|CAN80540.1|/9-139
Populus trichocarpa - gi|224095914|ref|XP_002310505.1|/7-135
Arabidopsis lyrata subsp. lyrata - gi|297833156|ref|XP_002884460.1|/3-135
Glycine m
ax - gi|356512014|ref|XP_003524716.1|/6-136
Vitis vinifera - gi|147802300|emb|CAN70405.1|/13-141
Populus trichocarpa - gi|224135105|ref|XP_002327567.1|/5-133
Arabidopsis thaliana - gi|15229931|ref|NP_190015.1|/14-141
Hordeum vulgare subsp. vulgare - gi|326492385|dbj|BAK01976.1|/6-134
Oryza sativa Japonica G
roup - gi|115477845|ref|NP_001062518.1|/21-149
Brachypodium distachyon - gi|357141157|ref|XP_003572109.1|/6-135
Brachypodium distachyon - gi|357168397|ref|XP_003581627.1|/5-133
Hordeum vulgare subsp. vulgare - gi|295881154|gb|ADG
56507.1|/3-134
Arabidopsis thaliana - gi|15242390|ref|NP_196495.1|/6-134
Glycine m
ax - gi|356508975|ref|XP_003523228.1|/5-133
Arabidopsis lyrata subsp. lyrata - gi|297812351|ref|XP_002874059.1|/13-140
Arabidopsis thaliana - gi|18399168|ref|NP_564440.1|/16-144
PGSC0003DMP400001112/27-156
Ricinus comm
unis - gi|255548095|ref|XP_002515104.1|/4-138
Glycine m
ax - gi|356528635|ref|XP_003532905.1|/6-134
Glycine m
ax - gi|356522462|ref|XP_003529865.1|/12-142
Vitis vinifera - gi|147765514|emb|CAN78113.1|/6-135
Arabidopsis lyrata subsp. lyrata - gi|297841167|ref|XP_002888465.1|/6-134
Glycine m
ax - gi|356547116|ref|XP_003541963.1|/17-144
Arabidopsis lyrata subsp. lyrata - gi|297800286|ref|XP_002868027.1|/6-136
Sorghum bicolor - gi|242080633|ref|XP_002445085.1|/12-146
Glycine m
ax - gi|356516174|ref|XP_003526771.1|/22-150
Glycine m
ax - gi|356560215|ref|XP_003548389.1|/5-139
Vitis vinifera - gi|225448908|ref|XP_002265550.1|/13-141
Arabidopsis lyrata subsp. lyrata - gi|297812653|ref|XP_002874210.1|/13-142
Medicago truncatula - gi|357516809|ref|XP_003628693.1|/6-134
Arabidopsis lyrata subsp. lyrata - gi|297798362|ref|XP_002867065.1|/9-137
Vitis vinifera - gi|296089035|emb|CBI38738.3|/64-192
Arabidopsis lyrata subsp. lyrata - gi|297843024|ref|XP_002889393.1|/2-138
Malus x dom
estica - gi|302399003|gb|ADL36796.1|/6-134
Arabidopsis thaliana - gi|145324118|ref|NP_001077648.1|/16-156
Arabidopsis thaliana - gi|18398891|ref|NP_566374.1|/27-155
Arabidopsis lyrata subsp. lyrata - gi|297846400|ref|XP_002891081.1|/16-144
Arabidopsis lyrata subsp. lyrata - gi|297826043|ref|XP_002880904.1|/14-141
Solyc05g055480.2.1/17-145
Glycine m
ax - gi|356531066|ref|XP_003534099.1|/6-134
Medicago truncatula - gi|357464643|ref|XP_003602603.1|/7-135
Medicago truncatula - gi|358346559|ref|XP_003637334.1|/11-141
Brachypodium distachyon - gi|357137711|ref|XP_003570443.1|/14-151
Arabidopsis thaliana - gi|21618244|gb|AAM67294.1|/16-144
Oryza sativa Indica G
roup - gi|218184374|gb|EEC66801.1|/31-142
Sorghum bicolor - gi|242032581|ref|XP_002463685.1|/4-155
Glycine m
ax - gi|356569398|ref|XP_003552888.1|/6-134
Malus x dom
estica - gi|302399033|gb|ADL36811.1|/9-137
Arabidopsis lyrata subsp. lyrata - gi|297846402|ref|XP_002891082.1|/16-144
Eutrema halophilum
- gi|312282343|dbj|BAJ34037.1|/24-151
Populus trichocarpa - gi|224135051|ref|XP_002321971.1|/10-134
PGSC0003DMP400049938/4-132
Arabidopsis thaliana - gi|6714418|gb|AAF26106.1|AC012328_9/6-126
Medicago truncatula - gi|358347538|ref|XP_003637813.1|/53-186
Petunia x hybrida - gi|21105732|gb|AAM34765.1|AF509865_1/4-132
Malus x dom
estica - gi|302399031|gb|ADL36810.1|/9-145
Glycine m
ax - gi|356563453|ref|XP_003549977.1|/6-136
PGSC0003DMP400054120/30-158
Hordeum vulgare subsp. vulgare - gi|326530494|dbj|BAJ97673.1|/13-150
Arabidopsis thaliana - gi|42562475|ref|NP_174582.3|/24-152
PGSC0003DMP400010437/31-158
Solyc03g078120.2.1/4-132
Populus trichocarpa - gi|224063973|ref|XP_002301328.1|/11-142
Arabidopsis thaliana - gi|15236721|ref|NP_193532.1|/6-136
Physcomitrella patens subsp. patens - gi|168025227|ref|XP_001765136.1|/5-132
Populus trichocarpa - gi|224116242|ref|XP_002331996.1|/2-129
Vitis vinifera - gi|296089035|emb|CBI38738.3|/682-812
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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)
Potato: phylogene1cs: Results
0.3
0.0 0.5 1.0 1.5 2.0 2.5
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NAC1
NAC2
NTL1
NTL13NTL12
NTL11
NTL10
NTL9NTL8
NTL7
NTL6NTL5
NTL4
NTL3
NTL2
ArabidopsisSolanaceaeCereals
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
NbNac1_1/28-157PGSC0003DMP400001112/27-156
Solyc03g080090.2.1/27-156
Solyc06g073050.2.1/19-148PGSC0003DMP400046923/18-147
gi|115474875|ref|NP_001061034.1|/9-143gi|326526251|dbj|BAJ97142.1|/9-143
gi|357144944|ref|XP_003573468.1|/9-143
gi|242080633|ref|XP_002445085.1|/12-146gi|293332711|ref|NP_001169185.1|/6-140
gi|110289615|gb|AAP55107.2|/6-134gi|242040125|ref|XP_002467457.1|/6-134
gi|357141157|ref|XP_003572109.1|/6-135gi|326496190|dbj|BAJ90716.1|/6-145
gi|242045528|ref|XP_002460635.1|/6-135
gi|357154515|ref|XP_003576809.1|/6-142gi|125564636|gb|EAZ10016.1|/116-246
gi|357137711|ref|XP_003570443.1|/14-151gi|242063552|ref|XP_002453065.1|/19-156
gi|223949493|gb|ACN28830.1|/24-161gi|115449815|ref|NP_001048559.1|/16-153
gi|125541671|gb|EAY88066.1|/16-153
gi|326530494|dbj|BAJ97673.1|/13-150
gi|292659258|gb|ADE34584.1|/22-150
gi|326509519|dbj|BAJ91676.1|/22-150
gi|357144217|ref|XP_003573214.1|/29-157
gi|242079615|ref|XP_002444576.1|/32-160
gi|293336942|ref|NP_001168544.1|/26-154
gi|115477845|ref|NP_001062518.1|/21-149
gi|357119028|ref|XP_003561248.1|/14-150
gi|115465864|ref|NP_001056531.1|/18-149
gi|242091638|ref|XP_002436309.1|/30-161gi|293333166|ref|NP_001169690.1|/11-142
gi|212274312|ref|NP_001130458.1|/9-136gi|242049706|ref|XP_002462597.1|/9-136gi|357159131|ref|XP_003578349.1|/8-135
gi|326508056|dbj|BAJ86771.1|/12-139gi|115479907|ref|NP_001063547.1|/10-144
gi|218202386|gb|EEC84813.1|/10-144
gi|222641843|gb|EEE69975.1|/10-134gi|295881154|gb|ADG56507.1|/3-134
gi|242032581|ref|XP_002463685.1|/4-155
gi|302756881|ref|XP_002961864.1|/1-126
gi|168001860|ref|XP_001753632.1|/6-133
gi|168016362|ref|XP_001760718.1|/6-131
gi|168025227|ref|XP_001765136.1|/5-132
gi|168001016|ref|XP_001753211.1|/1-128
gi|148909054|gb|ABR17630.1|/7-147
gi|116793533|gb|ABK26780.1|/6-134
gi|224120762|ref|XP_002330945.1|/237-361
gi|224116242|ref|XP_002331996.1|/2-129
gi|224082532|ref|XP_002306731.1|/16-144
gi|224066587|ref|XP_002302150.1|/16-144
Solyc12g056790.1.1/22-150
PGSC0003DMP400029635/22-150
gi|21105740|gb|AAM34769.1|AF509869_1/12-140
gi|21105742|gb|AAM34770.1|AF509870_1/19-147PGSC0003DMP400054265/31-159
StNac2_5/32-160
Solyc04g072220.2.1/32-160NbNac2_1/29-157
gi|225463127|ref|XP_002265611.1|/20-148
gi|356576117|ref|XP_003556180.1|/19-147
gi|356535729|ref|XP_003536396.1|/19-147
gi|18399168|ref|NP_564440.1|/16-144
gi|297846402|ref|XP_002891082.1|/16-144
gi|297846400|ref|XP_002891081.1|/16-144
gi|14334572|gb|AAK59465.1|/10-136
gi|297846266|ref|XP_002891014.1|/10-136
gi|15237609|ref|NP_201211.1|/6-134
gi|297797411|ref|XP_002866590.1|/6-134
gi|297811023|ref|XP_002873395.1|/6-134
gi|15242390|ref|NP_196495.1|/6-134
gi|357519943|ref|XP_003630260.1|/6-134
gi|356525677|ref|XP_003531450.1|/3-131
gi|356512837|ref|XP_003525122.1|/3-131
gi|356508975|ref|XP_003523228.1|/5-133gi|356516356|ref|XP_003526861.1|/5-133
gi|357464643|ref|XP_003602603.1|/7-135
gi|255552430|ref|XP_002517259.1|/5-133
gi|224095914|ref|XP_002310505.1|/7-135
gi|224130574|ref|XP_002328323.1|/7-135
gi|302399015|gb|ADL36802.1|/8-136
gi|302399003|gb|ADL36796.1|/6-134
gi|296088554|emb|CBI37545.3|/6-134
Solyc03g078120.2.1/4-132
PGSC0003DMP400049938/4-132
Solyc11g005920.1.1/4-132
gi|21105732|gb|AAM34765.1|AF509865_1/4-132
gi|357133590|ref|XP_003568407.1|/6-134
gi|188593543|dbj|BAG32519.1|/6-134gi|115464001|ref|NP_001055600.1|/6-134
gi|326369339|gb|ADZ55681.1|/6-134
gi|242077812|ref|XP_002448842.1|/6-134
gi|293336322|ref|NP_001169920.1|/6-134
gi|125550359|gb|EAY96181.1|/6-134
gi|357168397|ref|XP_003581627.1|/5-133
gi|326492385|dbj|BAK01976.1|/6-134
PGSC0003DMP400054118/10-139
Solyc11g008000.1.1/10-139
PGSC0003DMP400040418/1-129
Solyc05g055470.2.1/10-138
gi|21105746|gb|AAM34772.1|AF509872_1/10-138
Solyc05g055480.2.1/17-145
PGSC0003DMP400040416/22-150
gi|21105744|gb|AAM34771.1|AF509871_1/23-151
gi|21105736|gb|AAM34767.1|AF509867_1/35-163
PGSC0003DMP400054120/30-158
Solyc11g008010.1.1/97-225gi|224109864|ref|XP_002315336.1|/39-167
gi|356535507|ref|XP_003536286.1|/33-161
gi|356576366|ref|XP_003556303.1|/34-162
gi|147802300|emb|CAN70405.1|/13-141
gi|356576364|ref|XP_003556302.1|/4-132
gi|147802301|emb|CAN70406.1|/9-137
gi|302399033|gb|ADL36811.1|/9-137
gi|255583748|ref|XP_002532627.1|/11-139gi|224100709|ref|XP_002311983.1|/10-138
gi|224109872|ref|XP_002315338.1|/5-133
gi|6456751|gb|AAF09254.1|AF201456_1/9-137
gi|15237699|ref|NP_196061.1|/9-137
gi|297806417|ref|XP_002871092.1|/9-137
gi|21593389|gb|AAM65338.1|/9-137gi|297829590|ref|XP_002882677.1|/9-137
gi|238479717|ref|NP_001154602.1|/27-188gi|18398891|ref|NP_566374.1|/27-155
gi|297829586|ref|XP_002882675.1|/27-155
gi|297829588|ref|XP_002882676.1|/27-155
gi|18398893|ref|NP_566375.1|/27-155gi|15237698|ref|NP_196060.1|/28-170
gi|357482369|ref|XP_003611470.1|/6-148
gi|357476695|ref|XP_003608633.1|/6-137
gi|356563453|ref|XP_003549977.1|/6-136
gi|356512014|ref|XP_003524716.1|/6-136gi|224138076|ref|XP_002326512.1|/6-134
gi|224126509|ref|XP_002329572.1|/6-134
gi|255557623|ref|XP_002519841.1|/6-134
Solyc08g077110.2.1/6-134
PGSC0003DMP400030569/6-134
gi|21105751|gb|AAM34774.1|AF509874_1/6-134
Solyc06g061080.2.1/6-134gi|42562467|ref|NP_174529.2|/6-141
gi|297846226|ref|XP_002890994.1|/6-139
gi|297800286|ref|XP_002868027.1|/6-136
gi|15236721|ref|NP_193532.1|/6-136gi|297794579|ref|XP_002865174.1|/6-135
gi|15237469|ref|NP_199471.1|/6-135
gi|297830402|ref|XP_002883083.1|/6-134
gi|15229511|ref|NP_188400.1|/6-134
gi|224125708|ref|XP_002329698.1|/6-134gi|224120448|ref|XP_002318332.1|/6-134gi|255569031|ref|XP_002525485.1|/6-134gi|356531066|ref|XP_003534099.1|/6-134
gi|356561847|ref|XP_003549188.1|/6-134
PGSC0003DMP400054092/6-134
Solyc03g098190.2.1/6-134
gi|296088017|emb|CBI35300.3|/6-134
gi|242045530|ref|XP_002460636.1|/6-134
gi|115480567|ref|NP_001063877.1|/6-134
gi|4585977|gb|AAD25613.1|AC005287_15/3-131
gi|297853370|ref|XP_002894566.1|/6-134
gi|255573304|ref|XP_002527580.1|/6-134gi|224098826|ref|XP_002311281.1|/6-134
gi|224112333|ref|XP_002316156.1|/6-134
gi|147854237|emb|CAN83436.1|/6-134
gi|356557052|ref|XP_003546832.1|/6-134
gi|356528635|ref|XP_003532905.1|/6-134
gi|357451351|ref|XP_003595952.1|/6-134
Solyc02g036430.1.1/6-134
PGSC0003DMP400037231/6-134
Solyc02g081270.2.1/6-134
gi|225466227|ref|XP_002267333.1|/6-134
gi|255571259|ref|XP_002526579.1|/6-134
gi|224135105|ref|XP_002327567.1|/5-133
gi|224079646|ref|XP_002305903.1|/6-134
gi|356551985|ref|XP_003544352.1|/5-133
gi|356499060|ref|XP_003518362.1|/5-133
gi|357491761|ref|XP_003616168.1|/6-134gi|357460717|ref|XP_003600640.1|/6-134
gi|356537835|ref|XP_003537430.1|/6-134
gi|356569398|ref|XP_003552888.1|/6-134
gi|242042457|ref|XP_002468623.1|/6-134
gi|226531876|ref|NP_001146336.1|/6-134
gi|125542167|gb|EAY88306.1|/6-134
gi|357120932|ref|XP_003562178.1|/6-134
gi|297841167|ref|XP_002888465.1|/6-134
gi|15218843|ref|NP_176766.1|/6-134
gi|15237939|ref|NP_197228.1|/6-134
gi|297811871|ref|XP_002873819.1|/6-134gi|297832960|ref|XP_002884362.1|/6-134
gi|186509710|ref|NP_186970.2|/6-134
gi|116786496|gb|ABK24128.1|/6-136
gi|148907008|gb|ABR16648.1|/6-136
gi|357453161|ref|XP_003596857.1|/18-145
gi|356542037|ref|XP_003539478.1|/17-145gi|356547116|ref|XP_003541963.1|/17-144
gi|225464868|ref|XP_002272914.1|/17-144
gi|224104873|ref|XP_002313601.1|/19-146
gi|255565737|ref|XP_002523858.1|/17-145
Solyc06g074170.2.1/32-159
PGSC0003DMP400010437/31-158
Solyc11g068750.1.1/82-209
PGSC0003DMP400034078/40-167
gi|297818896|ref|XP_002877331.1|/10-137
gi|15229931|ref|NP_190015.1|/14-141gi|312282343|dbj|BAJ34037.1|/24-151
gi|18420448|ref|NP_568414.1|/21-148gi|297812351|ref|XP_002874059.1|/13-140
gi|15225866|ref|NP_180298.1|/14-141
gi|297826043|ref|XP_002880904.1|/14-141
gi|226493114|ref|NP_001147448.1|/9-137gi|242056921|ref|XP_002457606.1|/9-137
gi|289551912|gb|ADD10614.1|/9-137gi|371925001|tpe|CBZ41162.1|/9-137
gi|357129971|ref|XP_003566632.1|/9-137
gi|218187925|gb|EEC70352.1|/9-137gi|115435780|ref|NP_001042648.1|/9-137
gi|224080239|ref|XP_002306067.1|/7-131gi|224120988|ref|XP_002318469.1|/13-137
gi|224120984|ref|XP_002318468.1|/13-137
gi|224120980|ref|XP_002318467.1|/13-137
gi|224135051|ref|XP_002321971.1|/10-134
gi|15233215|ref|NP_191081.1|/11-151
gi|297819350|ref|XP_002877558.1|/46-187gi|297819354|ref|XP_002877560.1|/5-145
gi|297819354|ref|XP_002877560.1|/223-364
gi|224132570|ref|XP_002321355.1|/8-141
gi|297807857|ref|XP_002871812.1|/13-130
gi|356562269|ref|XP_003549394.1|/9-144
gi|218184374|gb|EEC66801.1|/31-142
gi|302398995|gb|ADL36792.1|/8-118
gi|297795805|ref|XP_002865787.1|/4-145
gi|297843024|ref|XP_002889393.1|/2-138gi|15229292|ref|NP_187093.1|/2-138
gi|297833156|ref|XP_002884460.1|/3-135
gi|15234225|ref|NP_192064.1|/4-136gi|297810005|ref|XP_002872886.1|/4-136
gi|297810007|ref|XP_002872887.1|/5-138
gi|145332955|ref|NP_001078343.1|/4-137gi|15234216|ref|NP_192061.1|/4-137
gi|297792793|ref|XP_002864281.1|/3-134
gi|297848416|ref|XP_002892089.1|/3-139
gi|15223276|ref|NP_171609.1|/3-139gi|110742030|dbj|BAE98952.1|/3-139
gi|15217677|ref|NP_171725.1|/3-130
gi|297848422|ref|XP_002892092.1|/3-133
gi|334182236|ref|NP_171727.2|/3-133
gi|297848426|ref|XP_002892094.1|/3-133
gi|297848424|ref|XP_002892093.1|/3-133
gi|145334985|ref|NP_171726.2|/3-133
gi|297848420|ref|XP_002892091.1|/11-146
gi|224127206|ref|XP_002329426.1|/6-134
gi|224127202|ref|XP_002329425.1|/10-136
gi|255558041|ref|XP_002520049.1|/13-134
gi|255558043|ref|XP_002520050.1|/5-124
gi|224130708|ref|XP_002320908.1|/6-138
gi|224068212|ref|XP_002302682.1|/11-142
gi|224063973|ref|XP_002301328.1|/11-142
gi|224127510|ref|XP_002320092.1|/11-142
gi|255548097|ref|XP_002515105.1|/3-141gi|255548099|ref|XP_002515106.1|/3-128
gi|337743347|gb|AEI73170.1|/3-124
gi|255548101|ref|XP_002515107.1|/14-139
gi|255548117|ref|XP_002515115.1|/12-128
gi|255548119|ref|XP_002515116.1|/20-150
gi|224130712|ref|XP_002320909.1|/12-143
gi|224068220|ref|XP_002302683.1|/30-151
gi|224063969|ref|XP_002301326.1|/14-145
gi|224063971|ref|XP_002301327.1|/11-142
gi|224127506|ref|XP_002320091.1|/16-140gi|224130136|ref|XP_002320761.1|/16-140
gi|255548095|ref|XP_002515104.1|/4-138
gi|224130704|ref|XP_002320907.1|/2-130gi|224068206|ref|XP_002302681.1|/2-128
gi|358346547|ref|XP_003637328.1|/7-149
gi|356560211|ref|XP_003548387.1|/4-136
gi|356560215|ref|XP_003548389.1|/5-139
gi|356520206|ref|XP_003528755.1|/5-139
gi|356520204|ref|XP_003528754.1|/5-139
gi|356560207|ref|XP_003548385.1|/11-141gi|356522462|ref|XP_003529865.1|/12-142
gi|358346559|ref|XP_003637334.1|/11-141
gi|358346870|ref|XP_003637487.1|/12-128
gi|358346553|ref|XP_003637331.1|/193-323
gi|358347538|ref|XP_003637813.1|/53-186
gi|358346553|ref|XP_003637331.1|/12-142
gi|225453680|ref|XP_002268892.1|/14-144
gi|296089034|emb|CBI38737.3|/14-144
gi|147787053|emb|CAN62332.1|/14-144
gi|147843196|emb|CAN80540.1|/9-139
gi|296089035|emb|CBI38738.3|/682-812
gi|296089033|emb|CBI38736.3|/9-139
gi|296089035|emb|CBI38738.3|/64-192gi|147787054|emb|CAN62333.1|/14-159
gi|147780480|emb|CAN73478.1|/9-139gi|255555833|ref|XP_002518952.1|/9-142
gi|302399031|gb|ADL36810.1|/9-145
gi|224061527|ref|XP_002300524.1|/8-141
gi|224115462|ref|XP_002317040.1|/6-139
gi|297798362|ref|XP_002867065.1|/9-137
gi|186516558|ref|NP_001119122.1|/9-137
gi|42562475|ref|NP_174582.3|/24-152
gi|297851744|ref|XP_002893753.1|/24-152gi|30689531|ref|NP_197847.3|/13-142
gi|297812653|ref|XP_002874210.1|/13-142gi|15229161|ref|NP_190522.1|/13-142
gi|297819600|ref|XP_002877683.1|/13-142
gi|224134993|ref|XP_002321956.1|/11-139
gi|224118488|ref|XP_002317831.1|/11-139gi|357464259|ref|XP_003602411.1|/230-358
gi|357464261|ref|XP_003602412.1|/22-150gi|255646322|gb|ACU23644.1|/22-150gi|356516174|ref|XP_003526771.1|/22-150
gi|357490459|ref|XP_003615517.1|/25-153
gi|302398999|gb|ADL36794.1|/9-137gi|255560596|ref|XP_002521312.1|/9-137
gi|147837829|emb|CAN73792.1|/84-212gi|21358787|gb|AAM47025.1|/27-156
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|7716952|gb|AAF68626.1|AF254124_1/6-134
gi|357516809|ref|XP_003628693.1|/6-134
gi|225448908|ref|XP_002265550.1|/13-141
gi|12322791|gb|AAG51388.1|AC011560_20/9-137
gi|147765514|emb|CAN78113.1|/6-135gi|359474569|ref|XP_002280894.2|/6-135
gi|6714418|gb|AAF26106.1|AC012328_9/6-126
gi|147832851|emb|CAN63961.1|/30-157
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|/669-799gi|147843196|emb|CAN80540.1|/1150-1280
gi|147843195|emb|CAN80539.1|/9-137
0.01
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…
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
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
Bacteria: genomics: Introduc1on I ID Prodigal Excess tRNA ID Prodigal Excess tRNA
Ddie_NCPPB_2976 dieffenbachiae
4269 63 + 2 63 Dunk_MK7 unknown
4251 48 + 1 64
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
Bacteria: genomics: Introduc1on I l 25 BioProjects online
l Genome assembly submissions in progress
Bacteria: genomics l Pairwise drag assembly comparisons (MegaBLAST, MUMmer)
Dickeya zeae pairwise comparisons
Bacteria: genomics l Pairwise drag assembly comparisons (MegaBLAST, MUMmer)
l Average Nucleo5de Iden5ty (ANI), unique regions
Dickeya pairwise unique regions
GBB
C2039
NCPP
B_3534
NCPP
B_453
IPO_980
MK7
NCPP
B_3274
NCPP
B_2976
NC_014500
NCPP
B_898
NCPP
B_3537
GBB
C2040
IPO_2222
MK10
MK16
CSL_RW240
DW_0440
NCPP
B_2511
NC_012880
NCPP
B_569
NCPP
B_402
NCPP
B_516
NCPP
B_3533
NC_012912
NCPP
B_3531
CSL_RW192
NC_013592
MK19
NCPP
B_3532
NCPP
B_2538
NCPPB_2538NCPPB_3532MK19NC_013592CSL_RW192NCPPB_3531NC_012912NCPPB_3533NCPPB_516NCPPB_402NCPPB_569NC_012880NCPPB_2511DW_0440CSL_RW240MK16MK10IPO_2222GBBC2040NCPPB_3537NCPPB_898NC_014500NCPPB_2976NCPPB_3274MK7IPO_980NCPPB_453NCPPB_3534GBBC2039
Dickeya average nucleo5de iden5ty (ANI)
Bacteria: genomics l Pairwise drag assembly comparisons (MegaBLAST, MUMmer)
l Is D.“solani” a novel species? (95% ANI≈70% DNA-‐DNA hybridisa5on) GBB
C2039
NCPP
B_3534
NCPP
B_453
IPO_980
MK7
NCPP
B_3274
NCPP
B_2976
NC_014500
NCPP
B_898
NCPP
B_3537
GBB
C2040
IPO_2222
MK10
MK16
CSL_RW240
DW_0440
NCPP
B_2511
NC_012880
NCPP
B_569
NCPP
B_402
NCPP
B_516
NCPP
B_3533
NC_012912
NCPP
B_3531
CSL_RW192
NC_013592
MK19
NCPP
B_3532
NCPP
B_2538
NCPPB_2538NCPPB_3532MK19NC_013592CSL_RW192NCPPB_3531NC_012912NCPPB_3533NCPPB_516NCPPB_402NCPPB_569NC_012880NCPPB_2511DW_0440CSL_RW240MK16MK10IPO_2222GBBC2040NCPPB_3537NCPPB_898NC_014500NCPPB_2976NCPPB_3274MK7IPO_980NCPPB_453NCPPB_3534GBBC2039
Dickeya average nucleo5de iden5ty (ANI) GBB
C2039
NCPP
B_3534
IPO_980
NCPP
B_453
NCPP
B_2976
NCPP
B_898
NCPP
B_3537
NC_014500
GBB
C2040
IPO_2222
MK10
MK16
MK16
MK10
IPO_2222
GBBC2040
NC_014500
NCPPB_3537
NCPPB_898
NCPPB_2976
NCPPB_453
IPO_980
NCPPB_3534
GBBC2039
Bacteria: genomics: Methods l Gene complement comparisons
l Reciprocal best BLAST hits (RBBH)
Organism 2
Organism 1
CDS:
CDS:
not RBBH RBBH: best BLAST hit:
Bacteria: genomics: Methods
best BLAST hits reciprocal best BLAST hits
Bacteria: genomics
D. solani IPO2222
70% iden5ty 100% iden5ty core accessory
Bacteria: genomics Cliques: l Core and accessory genomes from RBBH
l Predicted Dickeya core genome: 2201 genes
Compara1ve Genomics l Predicted Dickeya species-‐specific accessory genome sizes
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
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
Bacteria: genomics l Collinearity and conserva5on of synteny from RBBH
l iADHoRe
Bacteria: genomics l Collinearity and conserva5on of synteny from RBBH
l iADHoRe
Ddi/Dda
Dze
Dso
Ddi/Dso
Dickeya
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
Bacteria: SysBio: Results
Compound presence/absence table
Reac5on presence/absence table
Bacteria: SysBio: Results l Predicted substrate-‐dependent growth
l experimental verifica5on
Sonia Humphris/Anne-‐Laure Lucquet
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
Dickeya Virtual Machine
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
Bacteria: genomics l Analysis underway (RBBH >34 day calcula5on on JHI cluster)
0.000
0.001
0.002
0.003
0.004
0.005
2000 4000 6000 8000genecall count
dens
ity
Genecall count distribution (binsize=50)
0.0e+00
2.5e−06
5.0e−06
7.5e−06
1.0e−05
2e+06 3e+06 4e+06assembly length
dens
ity
Total assembly length distribution (binsize=1e4)
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
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)