Cloning, Mapping and Identification of the Rpp1-Rpp5 Asian ... · Cloning, Mapping and Identification of the Rpp1-Rpp5 Asian Soybean Rust Resistance Genes Jenelle Meyer11, Danielle

Cloning, Mapping and Identification Cloning, Mapping and Identification of the of the Rpp1Rpp1--Rpp5 Rpp5 Asian Soybean Asian Soybean

Rust Resistance GenesRust Resistance Genes

JenelleJenelle MeyerMeyer11, Danielle Silva, Danielle Silva22, , MartijnMartijn van de Mortelvan de Mortel33, , ChunlingChunlingYangYang33, Chris Zhang, Chris Zhang33, Kerry Pedley, Kerry Pedley44,, John HillJohn Hill33,, Randy Randy

ShoemakerShoemaker1,31,3, Ricardo Abdelnoor, Ricardo Abdelnoor22, Steven Whitham, Steven Whitham33 and and Michelle GrahamMichelle Graham1,31,3

11USDAUSDA--ARS, Corn Insects and Crop Genetics Research Unit, ARS, Corn Insects and Crop Genetics Research Unit, 22Embrapa Embrapa SojaSoja, , 33Dept. of Plant Pathology, Iowa State UniversityDept. of Plant Pathology, Iowa State University

44USDAUSDA--ARS Foreign DiseaseARS Foreign Disease--Weed Science Research Unit,Weed Science Research Unit,

www.swicofil.com, cropwatch.unl.edu/ archives/2005/crop05-6.htm, http://www.uoguelph.ca/research/news/articles/2005/June/aphid_biocontrol.shtml, Soybean plant and flower. Credit: Jamie O'Rourke, ISU,

Outline

• Introduction to Asian Soybean Rust

• Analyses of the Rpp4 locus in susceptible genotype Wm82 and the resistant genotype PI459025B

• Status of Rpp1-3 and Rpp5

OutlineOutline

•• Introduction to Asian Soybean RustIntroduction to Asian Soybean Rust

•• Analyses of the Rpp4 locus in susceptible Analyses of the Rpp4 locus in susceptible genotype Wm82 and the resistant genotype genotype Wm82 and the resistant genotype PI459025B PI459025B

•• Status of Rpp1Status of Rpp1--3 and Rpp53 and Rpp5

Images: Unknown source, DTNImages: Unknown source, DTN

Asian Soybean Rust (ASR)Asian Soybean Rust (ASR)Asian Soybean Rust (ASR)

• Caused by the fungus Phakopsora pachyrhizi• Infects many legume species (soybean, Phaseolus, kudzu)• First identified in the Eastern Hemisphere early 1900’s• Now in all major soybean producing countries• Yield losses range from 10-80%• U.S. commercial cultivars lack resistance

•• Caused by the fungus Caused by the fungus PhakopsoraPhakopsora pachyrhizipachyrhizi•• Infects many legume species (soybean, Phaseolus, kudzu)Infects many legume species (soybean, Phaseolus, kudzu)•• First identified in the Eastern Hemisphere early 1900First identified in the Eastern Hemisphere early 1900’’ss•• Now in all major soybean producing countriesNow in all major soybean producing countries•• Yield losses range from 10Yield losses range from 10--80%80%•• U.S. commercial cultivars lack resistanceU.S. commercial cultivars lack resistance

Asian Soybean Rust ResistanceAsian Soybean Rust Resistance

•• Resistance is very rare, Resistance is very rare, over 16,000 germplasm over 16,000 germplasm accessions screenedaccessions screened

•• Breeding efforts rely on Breeding efforts rely on germplasm with partial germplasm with partial resistance or toleranceresistance or tolerance

•• Five resistance loci have Five resistance loci have been identified: been identified: Rpp1, Rpp2, Rpp1, Rpp2, Rpp3, Rpp4, Rpp5Rpp3, Rpp4, Rpp5

Our objective: To clone the Our objective: To clone the Rpp4Rpp4 ASR resistance gene.ASR resistance gene.

Microscopic Studies of Microscopic Studies of Rpp2, Rpp3Rpp2, Rpp3 and and Rpp4Rpp4Susceptible InteractionSusceptible InteractionResistant InteractionResistant Interaction

Images: D. da Silva, M. van de MortelImages: D. da Silva, M. van de Mortel

Resistance to ASR mediated by Resistance to ASR mediated by Rpp4 Rpp4 leads to the formation of red/brown leads to the formation of red/brown lesions and localized cell death, indicative of a hypersensitivelesions and localized cell death, indicative of a hypersensitive response.response.

Microarray Studies of Microarray Studies of Rpp2Rpp2

van de Mortel et al. (2007) used microarray van de Mortel et al. (2007) used microarray analyses to examine changes in gene expression analyses to examine changes in gene expression in response to ASRin response to ASR

•• Susceptible and resistant genotypes both had:Susceptible and resistant genotypes both had:-- early induction of basal defense genes early induction of basal defense genes

followed by return to normal levels.followed by return to normal levels.-- a second defense response a second defense response

•• However, the 2nd defense response occurred However, the 2nd defense response occurred 24 hours earlier in the resistant line24 hours earlier in the resistant line

•• A similar response was seen for A similar response was seen for Rpp3Rpp3 and and Rpp4Rpp4 (van de Mortel et al., in preparation).(van de Mortel et al., in preparation).

Chorismate mutase0

1

2

Chalcone isomerase

0

1

2

Isoflavone 2'-hydroxylase

2Δ

2

4

6

2'-hydroxydihydro-daidzein reductase

0

1

2

3

Isoflavone-7-O-methytransferase

Time (hai)0 48 96 144 192 240 288

0

1

2

3

We hypothesize We hypothesize Rpp4Rpp4 is a classical disease is a classical disease resistance genes.resistance genes.

van de Mortel et al. 2007. Molecular Plant-Microbe Interactionsvan de Mortel et al. 2007. Molecular Plant-Microbe Interactions

Research Objectives:Research Objectives:

1.1. Identify markers linked to Identify markers linked to Rpp4Rpp4..

2.2. Develop BAC contig corresponding to the Develop BAC contig corresponding to the Rpp4 Rpp4 locus.locus.

3. Sequence candidate BACs.3. Sequence candidate BACs.

4. Identify candidate resistance genes.4. Identify candidate resistance genes.

5.5. Silence candidate genes using VIGS (virus induced Silence candidate genes using VIGS (virus induced gene silencing).gene silencing).

PI459025 × BRS184F2 population (80)

76.76 cM

89.94 cM

Soybean Consensus Map (linkage group G)

Silva et al. 2008. Theoretical and Applied Genetics

I45C04

79L

04F

79L

04R

I13L

24R

I13L

24F

I16L09

I40P

17R

I40P

17F

I51A11

I16I22

Satt

288

I16K21

Sat_

143

I40P17

I13L24

73.4 cM 76.8 cM

I79L04

M18K13

M90

G19

R

M17

1H04

R

M171H04

M17

1H04

F

M158G13

M15

8G13

F

I45O

14F

I45O14

I63J17

M3L17

M4E17

M33J17

M27E18

M92N02

I42K08

I75D11

I75D

11R

I10C05

I52J05I5

2J05

R

I75D

11F

I57P16

I57P

16F

I57P

16R

M70A12

M176I01

A88

5_1

M90G19

79.2 cM

TheThe Wm82 Wm82 Rpp4Rpp4 BAC ContigBAC Contig

‘‘Williams82Williams82’’ is susceptible to ASR.is susceptible to ASR.

Rpp4Rpp4 Candidate Genes Encode NBS/LRR ProteinsCandidate Genes Encode NBS/LRR Proteins

55 kb 75 kb 100 kb 125 kb 150 kb 175 kb 200 kb

1 3 4 5 8 9 13 1514

1. Q9ZT68 Resistance protein candidate RGC2K 1E-113

2. Q01HF9 uncharacterized protein 5E-53

3. A5BCQ9 uncharacterized protein 1E-93

4. Unknown5. Q9ZT68 Resistance protein candidate RGC2K 1E-110

6. Unknown7. Q6UUN7 Putative gag-pol polyprotein* 1E-14

8. Q43105 Cellulase 1E-05

9. Q9LFC1 Uncharacterized protein 2E-46

10. Q9M2D1 Copia-type polyprotein* 4E-47

11. Q9M2D1 Copia-type polyprotein 0.012. Q2R3T4 Retrotranposon protein 1E-105

13. Q9ZR3T4 Retrotranposon Tto1 1E-22

14. Unknown15. Q9ZT68 Resistance protein candidate RGC2K 1E-111

126 7 10 11

Rpp4 candidate R-genes

2Rpp4C1Rpp4C1 Rpp4C2Rpp4C2 Rpp4C3Rpp4C3

TheThe Rpp4Rpp4 Contig Can be Linked to the Soybean Contig Can be Linked to the Soybean Whole Genome Assembly.Whole Genome Assembly.

I45C04

I16L09

I51A11

I16I22

Satt

288

I16K21Sa

t_14

3

I40P17

I13L24

I79L04

M18K13

M171H04

M158G13

I45O14

I63J17

M3L17

M4E17

M33J17

M27E18

M92N02

I42K08

I75D11

I10C05

I52J05

I57P16

M70A12

M176I01

A88

5_1

M90G19

73.4 cM 76.8 cM 79.2 cM

Scaffold_21Scaffold_21

No other RNo other R--genes candidates can be identified in this region.genes candidates can be identified in this region.www.phytozome.netwww.phytozome.net

SoybeanSoybean Rpp4Rpp4 Candidate GenesCandidate Genes

•• Three Three Rpp4Rpp4 candidate genes identified, belonging to candidate genes identified, belonging to the CCthe CC--NBSNBS--LRR family of resistance genes.LRR family of resistance genes.

•• Most closely related to the lettuce Most closely related to the lettuce RGC2RGC2 genes genes which confer resistance to downy mildew and root aphidwhich confer resistance to downy mildew and root aphid11..

•• One additional homolog (RLG) of the One additional homolog (RLG) of the Rpp4Rpp4 candidate candidate genes was located in the genome on LG D1A.genes was located in the genome on LG D1A.

•• Genes are large, spanning 15Genes are large, spanning 15--25kb.25kb.

•• Genes are not represented by ESTs nor any public Genes are not represented by ESTs nor any public microarray platform.microarray platform.

11Meyers et al. 1998. Plant Cell.Meyers et al. 1998. Plant Cell.

Structure of the Structure of the Rpp4 Rpp4 Candidate GenesCandidate Genes

NBSNBS-- required for ATP binding, signaling moleculerequired for ATP binding, signaling molecule

LRRLRR-- provide binding surface for pathogen avr protein or plant targeprovide binding surface for pathogen avr protein or plant targett

Rpp4C1Rpp4C1

Rpp4C2Rpp4C2

Rpp4C3Rpp4C3

RLG RLG (LGD1A)(LGD1A)

Can we use the Wm82Can we use the Wm82 Rpp4 Rpp4 candidate genes to candidate genes to silence silence Rpp4Rpp4 in the resistant genotype?in the resistant genotype?

Rpp4C1Rpp4C1Rpp4C2Rpp4C2Rpp4C3Rpp4C3








Assumption 1: Assumption 1: Rpp4Rpp4 is a member of this gene cluster.is a member of this gene cluster.Assumption 2: Assumption 2: Rpp4Rpp4 shares >85% nucleotide identity with the shares >85% nucleotide identity with the

genes in this cluster.genes in this cluster.

VirusVirus--Induced Gene Silencing (VIGS)Induced Gene Silencing (VIGS)

dsRNA forms during virus replication & triggers soybean dsRNA forms during virus replication & triggers soybean defenses leading to postdefenses leading to post--transcriptional gene silencingtranscriptional gene silencing

Bean pod mottle virus (BPMV) genome organization:Bean pod mottle virus (BPMV) genome organization:

Virus engineered by and image provided by Chunquan Chris Zhang (Virus engineered by and image provided by Chunquan Chris Zhang (Iowa State University)Iowa State University)

RNARNA--22

VPgVPgPoly(A)Poly(A)468 (775) – 3522*CR / MPCR / MP SS--CPCPLL--CPCP

insertinsert

VPgVPgPoly(A)Poly(A)CoCo--ProPro HelicaseHelicase ProPro RdRpRdRp

VPgVPg

RNARNA--11

Zhang et al. 2009. Molecular Zhang et al. 2009. Molecular PlantPlant--Microbe InteractionsMicrobe Interactions

Can we use the Wm82Can we use the Wm82 Rpp4 Rpp4 candidate genes to candidate genes to silence silence Rpp4Rpp4 in the resistant genotype?in the resistant genotype?









Assumption 1: Assumption 1: Rpp4Rpp4 is a member of this gene cluster.is a member of this gene cluster.Assumption 2: Assumption 2: Rpp4Rpp4 shares >85% nucleotide identity with the shares >85% nucleotide identity with the

genes in this cluster.genes in this cluster.

Silencing Silencing Rpp4 Rpp4 with VIGSwith VIGS

1. Infect1. Infect Rpp4 Rpp4 genotype with genotype with VIGS constructVIGS construct

2. Infect VIGS 2. Infect VIGS plants with plants with P. P. pachyrhizipachyrhizi

EmptyEmptyVIGSVIGSBPMVBPMV

No silencing ofNo silencing ofRpp4Rpp4

RR genotypegenotypeRR phenotypephenotype

Control: Mock VIGSControl: Mock VIGS Control: Empty VIGSControl: Empty VIGS

Rpp4Rpp4

Rpp4Rpp4

Rpp4Rpp4VIGSVIGSBPMVBPMV

Silencing ofSilencing of Rpp4Rpp4

R R genotypegenotypeS S phenotypephenotype

Exp: NBS VIGSExp: NBS VIGS Exp: LRR VIGSExp: LRR VIGS






Gene2Gene2Gene3Gene3Gene1Gene1



Are there differences in gene number or gene Are there differences in gene number or gene expression between the R and S parents? expression between the R and S parents?

•• Rpp4_Rpp4_NBD_F/R: Amplify and differentiate the NBD of all NBD_F/R: Amplify and differentiate the NBD of all Rpp4Rpp4candidate genescandidate genes

Wm82 (S): Wm82 (S): Rpp4Rpp4C1, Rpp4C2 and C1, Rpp4C2 and Rpp4Rpp4C3 (Wm82)C3 (Wm82)PI459025B (R): PI459025B (R): Rpp4Rpp4C1, C1, Rpp4Rpp4C2, C2, Rpp4Rpp4C3, C3, Rpp4Rpp4C4C4

and and Rpp4Rpp4C5 (PI459025B)C5 (PI459025B)

•• Rpp4_Rpp4_F/R:F/R: Amplify the NBD of all Amplify the NBD of all Rpp4Rpp4 candidate genes.candidate genes.

Quantitative RTQuantitative RT--PCR of PCR of Rpp4Rpp4 Candidate GenesCandidate Genes

Rpp4_F/R Rpp4_F/R primers (amplify all genes) primers (amplify all genes)

ASR ASR Mock ControlMock ControlTime Point Time Point Fold Change (R:S)Fold Change (R:S) Fold Change (R:S)Fold Change (R:S)

12 hai12 hai 6.496.49 5.025.02

24 hai24 hai 3.923.92 2.842.84

72 hai72 hai 4.824.82 2.862.86

216 hai216 hai 5.085.08 3.013.01

12 hai mock12 hai ASR

216 hai ASR 216 hai mock

24 hai ASR

72 hai ASR

24 hai mock

72 hai mock

Gene1 CCGTGene1 CCGTCACAATAATAGGCCTTTTATAA ATAA 2020Gene2 CCGTGene2 CCGT----ATAGCTTATAAATAGCTTATAA 2020Gene3 CCGene3 CCGGTCAATAGCTCAATAGC----ATAAATAA 2020Gene4 CCGene4 CCAATCAATAGCTCAATAGC----ATAAATAA 2020Gene5 CCGTCAATAGene5 CCGTCAATACCCTTACTTAAAAAAA 2020

Sequences of expression products from Sequences of expression products from Rpp4_Rpp4_NBD_F/R primers:NBD_F/R primers:

Which of the Which of the Rpp4Rpp4 Candidate Genes Candidate Genes is Expressed ?is Expressed ?

Theoretical: Similar gene expression across all genesTheoretical: Similar gene expression across all genes

Primers:Primers: Rpp4_Rpp4_NBD_F/RNBD_F/R

SampleSampleWilliams82/ASR/12haiWilliams82/ASR/12haiWilliams82/ASR/72haiWilliams82/ASR/72haiWilliams82/Mock/12haiWilliams82/Mock/12haiWilliams82/Mock/72haiWilliams82/Mock/72haiWilliams82 TotalWilliams82 TotalWilliams82 Genomic DNAWilliams82 Genomic DNAExpected ExpressionExpected Expression

SampleSamplePI459025B/ASR/12haiPI459025B/ASR/12haiPI459025B/ASR/72haiPI459025B/ASR/72haiPI459025B/Mock/12haiPI459025B/Mock/12haiPI459025B/Mock/72haiPI459025B/Mock/72haiPI459025B TotalPI459025B TotalPI459025B Genomic DNAPI459025B Genomic DNA

Rpp4C1Rpp4C100000000003535

136136

Rpp4C1Rpp4C1000000000099

Rpp4C2Rpp4C266111100882727105105

Rpp4C2Rpp4C2002200002277

Rpp4C3Rpp4C380808888797990903373372727105105

Rpp4C3Rpp4C3000000000066

Sequencing of RTSequencing of RT--PCR productsPCR products

Rpp4C4Rpp4C493939090878795953653652727

Rpp4C5Rpp4C500000000002222

Primers:Primers: Rpp4_Rpp4_NBD_F/RNBD_F/R


SampleSamplePI459025B/ASR/12haiPI459025B/ASR/12haiPI459025B/ASR/72haiPI459025B/ASR/72haiPI459025B/Mock/12haiPI459025B/Mock/12haiPI459025B/Mock/72haiPI459025B/Mock/72haiPI459025B TotalPI459025B TotalPI459025B Genomic DNAPI459025B Genomic DNAExpected ExpressionExpected Expression

Rpp4C1Rpp4C100000000003535

136136

Rpp4C1Rpp4C10000000000994141

Rpp4C2Rpp4C266111100882727105105

Rpp4C2Rpp4C20022000022777777

Rpp4C3Rpp4C380808888797990903373372727105105

Rpp4C3Rpp4C30000000000662727


Rpp4C4Rpp4C493939090878795953653652727122122

Rpp4C5Rpp4C500000000002222

100100

Rpp4C4Rpp4C4 is the candidate gene for is the candidate gene for Rpp4Rpp4--mediated resistance.mediated resistance.


SampleSamplePI459025B/ASR/12haiPI459025B/ASR/12haiPI459025B/ASR/72haiPI459025B/ASR/72haiPI459025B/Mock/12haiPI459025B/Mock/12haiPI459025B/Mock/72haiPI459025B/Mock/72haiPI459025B TotalPI459025B TotalPI459025B Genomic DNAPI459025B Genomic DNAExpected ExpressionExpected Expression

Rpp4C1Rpp4C100000000003535

136136

Rpp4C1Rpp4C10000000000994141

Rpp4C2Rpp4C266111100882727105105

Rpp4C2Rpp4C20022000022777777

Rpp4C3Rpp4C380808888797990903373372727105105

Rpp4C3Rpp4C30000000000662727


Rpp4C4Rpp4C493939090878795953653652727122122

Rpp4C5Rpp4C500000000002222

100100

Sequencing of the Rpp4 locus in PI459025BSequencing of the Sequencing of the Rpp4Rpp4 locus in PI459025Blocus in PI459025B

Sequencing of the Rpp4 locus in PI459025BSequencing of the Sequencing of the Rpp4Rpp4 locus in PI459025Blocus in PI459025B

1. Develop BAC library from PI459025B (TOFU)

1. Screen library with primers designed from the Rpp4candidate genes from Wm82.

1. Sequence candidate BACs.

1. Identify candidate resistance genes.

1.1. Develop BAC library from PI459025B (TOFU)Develop BAC library from PI459025B (TOFU)

1.1. Screen library with primers designed from the Screen library with primers designed from the Rpp4Rpp4candidate genes from Wm82.candidate genes from Wm82.

1.1. Sequence candidate BACs.Sequence candidate BACs.

1.1. Identify candidate resistance genes.Identify candidate resistance genes.

Glyma18g: 55,747,053..55,914,731Glyma18g: 55,747,053..55,914,731

TOFU 98D16TOFU 98D16

TOFU 51G21TOFU 51G21

TOFU 2H03TOFU 2H03

TOFU 86D10TOFU 86D10TOFU 66N11TOFU 66N11

TOFU 3J15TOFU 3J15

TOFU 81O07TOFU 81O07

TOFU 4O11TOFU 4O11

The Rpp4 locus in PI459025BThe Rpp4The Rpp4 locus in PI459025Blocus in PI459025B

Rpp4 homologyRpp4 homology

Transposon or Retrotransposon homologyTransposon or Retrotransposon homology

167,678 bp?167,678 bpbp?

Glyma18g: 55,747,053..55,914,731Glyma18g: 55,747,053..55,914,731

TOFU 98D16TOFU 98D16

TOFU 51G21TOFU 51G21

TOFU 2H03TOFU 2H03

TOFU 86D10TOFU 86D10TOFU 66N11TOFU 66N11

TOFU 3J15TOFU 3J15

TOFU 81O07TOFU 81O07

4O114O11


167,678 bp?167,678 bpbp?

Initiated sequencing of eight Rpp4 candidate BACs.Initiated sequencing of eight Initiated sequencing of eight Rpp4Rpp4 candidate BACs.candidate BACs.

Rpp4 homologyRpp4 homology

Transposon or Retrotransposon homologyTransposon or Retrotransposon homology

81O0781O0798D1698D16

51G2151G212H032H03

86D1086D1066N1166N11

3J153J154O114O11

Rpp4R1Rpp4R1

Rpp4R2*

Rpp4R2*

Rpp4R3Rpp4R3

Rpp4R4*

Rpp4R4*

Rpp4R5Rpp4R5

Rpp4R6Rpp4R6

Rpp4R7Rpp4R7

Rpp4R8Rpp4R8

00 50K50K 100K100K 150K150K 200K200K 250K250K 300K300K 350K350K 400K400K 450K450K 469K469K


Rpp4C1Rpp4C1Rpp4C2Rpp4C2 Rpp4C5Rpp4C5 Rpp4C3Rpp4C3 Rpp4C4Rpp4C4

The good...• Rpp4C4 is still our candidate for Rpp4.• Other genes in this cluster could also provide ASR resistance.

The bad...• The genes in the cluster range from 25 to 45 kb.

The ugly…• The transformation vector is only 9 kb.

The good.The good.....•• Rpp4C4 is still our candidate for Rpp4C4 is still our candidate for Rpp4Rpp4..•• Other genes in this cluster could also provide ASR resistance.Other genes in this cluster could also provide ASR resistance.

The bad...The bad...•• The genes in the cluster range from 25 to 45 kb.The genes in the cluster range from 25 to 45 kb.

The uglyThe ugly……•• The transformation vector is only 9 kb.The transformation vector is only 9 kb.

• The The Rpp4Rpp4 candidate genes are members of the most candidate genes are members of the most common class of Rcommon class of R--genes.genes.

•• There are >300 NBS/LRR RThere are >300 NBS/LRR R--genes in Arabidopsis and genes in Arabidopsis and >500 in Rice>500 in Rice11..

•• NBS/LRR RNBS/LRR R--genes are often clustered in the genome genes are often clustered in the genome leading to duplication, recombination, and the evolution of leading to duplication, recombination, and the evolution of new pathogen specificitiesnew pathogen specificities11..

Why is ASR Resistance Rare?Why is ASR Resistance Rare?

However, BLASTN searches (E<10However, BLASTN searches (E<10--44) failed to identify ) failed to identify additional genes with homology to the additional genes with homology to the Rpp4 Rpp4 candidate candidate genes.genes.

11Meyers et al., 2005Meyers et al., 2005

Is the rarity of Is the rarity of Rpp4Rpp4 homologs linked to cost?homologs linked to cost?

•• Its possible that the cost of maintaining the Its possible that the cost of maintaining the Rpp4Rpp4homologs genes in the absence of ASR is greater homologs genes in the absence of ASR is greater that the benefit derived from resistance during that the benefit derived from resistance during pathogen attack.pathogen attack.

•• In the absence of In the absence of P. syringae, RPM1P. syringae, RPM1 reduces reduces seed number in Arabidopsis by 9% (seed number in Arabidopsis by 9% (TianTian et al. et al. 2003).2003).

•• Given the agronomic importance of soybean, a 9% Given the agronomic importance of soybean, a 9% reduction in yield would have been immediately reduction in yield would have been immediately selected against.selected against.

If so, wild legumes may be a better source of If so, wild legumes may be a better source of resistance genes.resistance genes.

Status of Status of Rpp1Rpp1, , Rpp2Rpp2, , Rpp3Rpp3 and and Rpp5Rpp5

Rpp1Rpp1-bRpp1Rpp1-b

LG G (18)LG G (18)

Status of Rpp1 ResearchStatus of Status of Rpp1Rpp1 ResearchResearch• Rpp1 (immune response) and Rpp1-b (hypersensitive response) map to the same region on soybean LG G (Hyten et al. 2007, Chakraborty et al. 2009).

• Rpp1 and Rpp1-b are either tightly linked genes or are alleles of the same gene.

• Microarray analyses of Rpp1 complete (Fredrick, USDA/ARS)

• Rpp1 and Rpp1-b are located between SSR markers Sct_187 and Sat_064.

• Marker information can be used to browse genome sequence for candidate genes.

•• Rpp1 (immune response) and Rpp1-b (hypersensitive response) map to the same region on soybean LG G (Hyten et al. 2007, Chakraborty et al. 2009).

• Rpp1 and Rpp1-b are either tightly linked genes or are alleles of the same gene.

•• Microarray analyses of Microarray analyses of Rpp1 Rpp1 complete complete (Fredrick, USDA/ARS)(Fredrick, USDA/ARS)

•• Rpp1 Rpp1 and and Rpp1Rpp1--b b are located between SSR are located between SSR markers Sct_187 and Sat_064.markers Sct_187 and Sat_064.

•• Marker information can be used to browse Marker information can be used to browse genome sequence for candidate genesgenome sequence for candidate genes..

http://soybase.org/gbrowse/cgi-bin/gbrowse/gmax1.01/http://soybase.org/gbrowse/cgi-bin/gbrowse/gmax1.01/

Position on C18Position on C18 SSR markers SSR markers

Gene models: 7 NBS/LRR resistance genes, 3 protein kinasesGene models: 7 NBS/LRR resistance genes, 3 protein kinases

LG JLG J

Rpp2rpp2?Rpp2rpp2?

• Rpp2 mapped to LG J by Silva et al.and Garcia et al.(2007). rpp2? mapped to

same region by Garcia et al.

• Comparison with genome sequence identified 19 NBS/LRRs, 15 protein kinases and 3 Mlo-like genes.

• Fine mapping under way (Diers and Hudson)

• Sequencing underway in PI 230970 (Rpp2, Graham)

• VIGS of candidate genes underway (Graham, Whitham and Pedley)

• Microarray analyses of Rpp2 complete.

• Rpp2 mapped to LG J by Silva et al.and Garcia et al.(2007). rpp2? mapped to

same region by Garcia et al.

•• Comparison with genome sequence Comparison with genome sequence identified 19 NBS/identified 19 NBS/LRRsLRRs, 15 protein kinases , 15 protein kinases and 3 and 3 MloMlo--like genes.like genes.

•• Fine mapping under way (Fine mapping under way (DiersDiers and Hudson)and Hudson)

•• Sequencing underway in PI 230970 (Sequencing underway in PI 230970 (Rpp2, Rpp2, Graham)Graham)

•• VIGS of candidate genes underway (Graham, VIGS of candidate genes underway (Graham, WhithamWhitham and and PedleyPedley))

•• Microarray analyses of Microarray analyses of Rpp2Rpp2 complete.complete.

Status of Status of Rpp2 Rpp2 ResearchResearch

LG C2 (6)LG C2 (6)

Rpp3Rpp?Rpp3Rpp?

Status of Status of Rpp3 Rpp3 and and RppRpp? (? (HyuugaHyuuga) Research) Research• Rpp?(Hyuuga) mapped to LG C2 by Monteros et al. 2007 (Hyuuga x Dillon).

• Rpp3 mapped to same region by Hyten et al. 2009 (PI 462312 x Wm82).

• Rpp? and Rpp3 are either allelic or tightly linked genes.

• Comparison with genome sequence identified 6 NBS/LRR genes and 26 receptor-like kinases in a 900 kb region.

• Microarray analyses of Rpp3 complete (Fredrick and Whitham)

• Rpp?(Hyuuga) mapped to LG C2 by Monteros et al. 2007 (Hyuuga x Dillon).

• Rpp3 mapped to same region by Hyten et al. 2009 (PI 462312 x Wm82).

•• Rpp? Rpp? and and Rpp3 Rpp3 are either allelic or tightly are either allelic or tightly linked genes.linked genes.

•• Comparison with genome sequence Comparison with genome sequence identified 6 NBS/LRR genes and 26 identified 6 NBS/LRR genes and 26 receptorreceptor--like kinases in a 900 kb region.like kinases in a 900 kb region.

•• Microarray analyses of Microarray analyses of Rpp3 Rpp3 complete complete (Fredrick and Whitham)(Fredrick and Whitham)

LG N (3)LG N (3)

Rpp5rpp5Rpp5rpp5

Status of Status of Rpp5 Rpp5 ResearchResearch• Rpp5 (D, PI 200526), Rpp5 (ID, PI 471904), rpp5 (R, PI200456) mapped to LG N by Garcia et al. 2008.

• Genes are either allelic or tightly linked.

• Comparison with genome sequence identified no obvious candidate resistance genes.

• Fine mapping currently underway (Diers)

• Rpp5 (D, PI 200526), Rpp5 (ID, PI 471904), rpp5 (R, PI200456) mapped to LG N by Garcia et al. 2008.

•• Genes are either allelic or tightly linked.Genes are either allelic or tightly linked.

•• Comparison with genome sequence Comparison with genome sequence identified no obvious candidate resistance identified no obvious candidate resistance genes.genes.

•• Fine mapping currently underway (Diers)Fine mapping currently underway (Diers)

SummarySummary

•• Release of the soybean genome sequence and Release of the soybean genome sequence and improvements in genotyping have facilitated the improvements in genotyping have facilitated the mapping of ASR resistance genes.mapping of ASR resistance genes.

•• In all cases but one (In all cases but one (Rpp5)Rpp5) these efforts have these efforts have identified a cluster of candidate resistance genes.identified a cluster of candidate resistance genes.

•• Microarray analyses have been essential in Microarray analyses have been essential in understanding the mechanisms behind resistance.understanding the mechanisms behind resistance.

•• However, identifying a single candidate gene However, identifying a single candidate gene remains difficult.remains difficult.

SummarySummary

•• Our analyses ofOur analyses of Rpp4Rpp4 suggest that resistance loci suggest that resistance loci are much more complex then expected.are much more complex then expected.

•• By combining molecular approaches, we have By combining molecular approaches, we have been able to identify a single candidate resistance been able to identify a single candidate resistance gene.gene.

•• Continued analyses of the Continued analyses of the Rpp4 Rpp4 locus in soybean locus in soybean and and Phaseolus Phaseolus will help us understand how novel will help us understand how novel resistance specificities are generated and may resistance specificities are generated and may shed light on ASRshed light on ASR’’s ability to overcome resistance s ability to overcome resistance so quickly.so quickly.

AcknowledgementsAcknowledgements

Ricardo AbdelnoorRicardo AbdelnoorDanielle SilvaDanielle Silva

Michelle GrahamMichelle GrahamJenelle MeyerJenelle MeyerLori LincolnLori LincolnCatherine FordCatherine Ford

Randy ShoemakerRandy ShoemakerJamie OJamie O’’RourkeRourkeGreg PeifferGreg PeifferJohanna DobbsJohanna Dobbs

Kerry PedleyKerry Pedley

Rex NelsonRex NelsonSteven Cannon Steven Cannon David GrantDavid Grant

Steve WhithamSteve WhithamMartijn van de MortelMartijn van de MortelChunling YangChunling Yang

John HillJohn HillChris ZhangChris ZhangAl EggenbergerAl Eggenberger

Thanks toThanks to……David David HytenHyten, USDA, USDA--ARS, ARS, Rpp1, Rpp1Rpp1, Rpp1--bbBrian Brian DiersDiers, Univ. of Illinois, , Univ. of Illinois, Rpp2, Rpp5Rpp2, Rpp5Roger Roger BoermaBoerma, Univ. of Georgia, , Univ. of Georgia, Rpp3Rpp3

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