Resistance gene cloning in wheat

Burkhard Steuernagel2020-09-23

JIC Informatics – Burkhard Steuernagel 1

Wheat Pathogens

2

wikipediaAmber Hafeez

www.rusttracker.org

Hales et al.

Outline

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Annotation of NLR genes

Mutational Genomics

AgRenSeq

The NLR Gene Family

4

1617 1 6 4 5 10 3 12 2 9 11 19

Coiled Coil

8 7

NB-ARC LRR

P-loop RNBS-A Kin-2 RNBS-B RNBS-C GLPL RNBS-D

Linker

MHDV Motif1 LDLEDVID

0 100 200 300 400 500 600 700 800 900 1000

Coffee

Maize

Papaya

Cucumber

Soybean

Tomato

Potato

Brachypodium

NLRs per genome

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0 2000 4000 6000 8000 10000 12000 14000 16000 18000

IWGSC refseq v1.0

Zimin et al. 2017

Clavijo et al. 2017

Chapman et al. 2015

IWGSC 2014

NLRs per assembly

full-length fragmented

NLRs of Chinese Spring

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NLRs of Chinese Spring

NLR Annotation

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1617 1 6 4 5 10 3 12 2 9 11 19

Coiled Coil

8 7

NB-ARC LRR

P-loop RNBS-A Kin-2 RNBS-B RNBS-C GLPL RNBS-D

Linker

MHDV Motif1 LDLEDVID

20152020

NLR-Annotator

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• NLR loci (vs. NLR genes)• No gene structure• No difference between pseudo-genes and genes

https://github.com/steuernb/NLR-Annotator

NLR-Annotation in Chinese Spring

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Resistance Gene Enrichment Sequencing (RenSeq)

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arborbiosci.com

RenSeq Bait Libraries• Triticeae RenSeq v1 (60k baits)• Triticeae RenSeq v2 (60k baits; improved sensistivity)• Triticeae RenSeq v3 (220k baits; improved sensitivity; introns)• Ae. tauschii RenSeq (60k baits; Ae. tauschii specific)

https://github.com/steuernb/MutantHunterhttps://github.com/steuernb/AgRenSeq/releases/download/v1.0/

https://arborbiosci.com/applications/agrigenomics/resistance-gene-enrichment-sequencing-renseq/

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Mutational Genomics

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EMS

. . .

. . .

. . .

. . .

. . .

. . .

Mutant 2

Mutant 1

Mutant 3

Mutant 4

Mutant 5

Resistant parent

xx xx

x x

xxx

x xx

xxxxx

R1 R2 R3 R4 R5 R6 R7 R8 R9 R12R10 R13R11 R2500

MutRenSeq

• EMS mutagenesis

• RenSeq on wild type and mutants

• Assembly, annotation and mapping

• Mutational genomics

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RenSeq

Mapping De novo assembly

>candidate_contig_1wildtype: ATGCTTAGCGGCATAGCACGCCCTAGmutant1 : ATACTTAGCGGCATAGCACGCCCTAGmutant2 : ATGCTTAACGGCATAGCACGCCCTAGmutant3 : ---------deletion---------…mutantn : ATGCTTAGCGGCATAGCACGTCCTAG

Mutant Hunter

ContigAnnotation

https://github.com/steuernb/MutantHunter

MutRenSeq

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Sr22

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• Introgressed into wheat from T. boeoticum• Suppressed recombination

prevents map-based cloning• 6 EMS mutants

Sr22 sr22

Sr22

Sr22 Transgenics

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Fielder Transgenics

Sr45 (MutRenSeq)

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49S 52S 55S 57S 59S 61SSr45Mutants

*

CC NB LRR5’ UTR 3’ UTR

RenSeq contig1795_1

2,500

**

5,000

*

0 bp

FielderTransgenics

MutChromSeq

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historical mutantswild type

Mutational Genomics + Chromosome Flow Sorting

MutChromSeq Pm2

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wild type EMS mutants Chancellor

Forward genetics to clone single dominant genes

Pursue single genesEMS mutagenesis

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Mutational Genomics

Association Genetics using RenSeq (AgRenSeq)

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RenSeq

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. . . R1 R2 R3 R4 R5 R6 R7 R8 R9 R12R10 R13R11 R2500

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RKQQC

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87 k-mers

RKQQC

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964 bp658 bp

Candidate contigSr

33 C

DS

Novel Gene 1

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TRTTF

Sr46

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TRTTF

SrTA1662

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QTHJC

AgRenSeq

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AgRenSeq

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AgRenSeqDiversity panel

Phenotype

Gene candidate

Summary• NLR-Annotator

to annotate NLR loci in wheat

• Mutational Genomicsto pursue single dominant genes

• MutRenSeq• MutChromSeq

• AgRenSeqscreen diversity panels

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AcknowledgementsBrande WulffInma Hernandez-PinzonSanu Arora

Sambasivam PeriyannanEvans Lagudah

Kamil WitekFlorian JupeJonathan Jones

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Nikolai AdamskiJavi SanchezBeat Keller

Marie KubalákováJan VránaJaroslav Doležel

Jacob Enk

Brian SteffensonJesse Poland