rapid gene cloning in plants
TRANSCRIPT
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BrandeWulffJohnInnesCentreNorwich,UK
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Edgar McFadden 1891-1956
“Itrequiresaboutthesamelengthof4metodevelopahybridstrainofwheatasitdoestoraisea[child]”
A.W.Erickson,1945
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R1.X
.R1
Introducing resistance genes by wide crossing
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R1.
Stacking cloned resistance genes
R2.
R3.R1R2R3
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The three challenges of wild grasses
Agronomy
MutaGonalgenomics GenomecomplexityreducGonAlienintrogressions
.R1
Giantgenomes
Arabidopsis0.12GB
Wildgrass~7Gb
Wheat17Gb
GenesRgenes
SuppressedrecombinaGon
Recombina
Gon
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Mutagenesis & sequencing
EMS
R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 R14 R2500Resistantparent ...
x x x xMutant1 ...
xx x x xxxxxx
xx
xx
xx
Mutant2
Mutant3
Mutant4
Mutant5
...
...
...
...
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MutRenSeq:resistancegeneenrichmentsequencingofmutants
Baits
GenesRgenes
GenomicDNA
MutantHunter
1.Assemblewildtype
2.Mapmutants
3.DetectmutaGons
4.DeterminemutaGonoverlap
Candidategene
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Wheatstemrust
1999
Wheatgrowingarea
2015 2008
2006
2013
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IsolaGonofNB-LRRsbyMutRenSeq
1S 2S 3S1 5S1 6S 7S1Sr22Mutants
**
7,5005,0002,500
CC NB LRR5’UTR 3’UTR
RenSeqcon7g10555_1
RenSeqcon7g3841_1
bp0
FielderTransgenics
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InancientRomanreligion,theRobigaliawasafes7valheldApril25.Itsmainritualwasadogsacrificetoprotectgrainfieldsfromdisease.Thesacrificialofferingwasthebloodandentrailsofanunweanedpuppy.
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EnrichedDNATotalgDNA
2a Targetedenrichment
1 Mutagenesisandscreening
EMS
3 Bioinforma7csSequencing
R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 R14 R2500Resistantparent ...
x x x xMutant1
xx x x xxxxxx
xx
xx
xx
Mutant2
Mutant3
Mutant4
Mutant5 ..
...
...
...
...
1 2 3 4 5 6 7
12
345
6
7
Rela7veDNAcontentGAA-FITC
fluo
rescen
ce
2b Chromosomeflowsor7ngandamplifica7on
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MutChromSeq
αβ-hydrolasefold5’UTR 3’UTR
ChromSeqcon7g_946_1
10 110 kb
2 **
Barleyeceriferum-q
ChromSeqcon7g_7526_1
010
**
NB-ARC LRRsCC
7
2
689
***
5’UTR 3’UTR
5kb
Wheatpowderymildewresistance2
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Mutate RenSeq BioinformaGcs TransgenicScreenSr22
Sr45
Sr1644
Sr43
Sr44
Sr2020
Sr40
Sr25
Sr51
Sr53
Clone Stack Deploy
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TradiGonalBreedingWildwheat
Breadwheat
Introgression
MutSeq
Clonedgene
ClonedgenesWildwheatdiversitypanel
AgRenSeq
Genepyramid
Engineering
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OpenWildWheat
AliMehrabi,IlamUniversity,IranAlisonBentley,Na7onalIns7tuteofAgriculturalBotany,UKAmirSharon,Ins7tuteofCerealCropImprovement,IsraelBrandeWulff,JohnInnesCentre,UK BrianSteffenson,UniversityofMinnesota,USA BurkhardSteuernagel,JohnInnesCentre,UK CarolinaPaolaSansaloni,CIMMYT,MexicoEvansLagudah,CSIRO,AustraliaFiruzaNasyrova,AcademyofSciences,TajikistanHananSela,Ins7tuteofCerealCropImprovement,IsraelJanDvorak,UCDavis,USAJessePoland,KansasStateUniversity,USAKlausMayer,HelmholtzZentrumMünchen,GermanyKseniaKrasileva,EarlhamIns7tute,UKMarioCaccamo,Na7onalIns7tuteofAgricultualBotany,UKMarGnMascher,IPKGatersleben,GermanyMikeAmbrose,GermplasmResourcesUnit,UKMinghengLuo,UCDavis,USAParveenChhuneja,PunjabAgriculturalUniversity,India
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AsyrafHacaBurkhardSteuernagelDeepikaNarangGuotaiYu LucyMcCannNgoniKangaraSanuAroraSreyaGhoshYajuanYue
Acknowledgements