GENESALB : Genetic analysis of resistance to South American leaf blight - SALB (Microcyclus ulei) in rubber tree (Hevea spp.)

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® Thematic: Other Species

ANR n°: ANR-07-GPLA-017 Acronym: GENESALB

Total Cost: 1 710 Keuros Total Grant: 442 Keuros

P059

_{Date of beginning: 01.01.2008 – End: 31.12.2010}

Edition 2007

Genetic analysis of resistance to South American Leaf Blight

(SALB) in rubber tree (

Hevea

spp.)

Marc SEGUIN1_{(coordinator), Françoise GRANET}4_{, Xavier ARGOUT}1_{, Angélique BERGER}1_{, Eric} CAVALOC4_{, Philippe CUBRY}1,4_{, Fabien DOARE}2_{, Sandra ESPEOUT}1,4_{, Fernando FONSECA}5_{, Dominique} GARCIA1,3_{, Vincent LE GUEN}1_{, Carlos MATTOS}6_{, Valérie PUJADE-RENAUD}1_{, Christelle WEBER}1

(1) Cirad, UMR-1098 - DAP, TA A96/03, Av. Agropolis, 34398 Montpellier, France (2) Cirad, UR-31, Campus Agronomique, BP 701, 97387 Kourou, French Guiana

(3) Universidade Estadual de Santa Cruz, Rod. Ilhéus-Itabuna BR415, km 16 Ilhéus – BA CEP 45.662-000. Brazil (4) Michelin - CPN, 23 Place des Carmes Déchaux, 63040 Clermont-Ferrand, France

(5) Plantações E. Michelin Ltda, BR 163, km16,5, Distrito de Ouro Branco do Sul, Itiquira, MT. Caixa Postal 080 CEP 78.700-090, Brazil

(6) Plantações Michelin da Bahia Ltda, Km 05 Rod. Ituberá - Camamu, Igrapiúna, BA. CEP 45.443-000, Brazil

Aims

Hevea brasiliensis cultivation is nearly the only source of production of natural rubber, an irreplaceable strategic commodity upon which are dependent various industrial sectors. The worldwide area of rubber tree cultivation has continuously increased still the beginning of the 20th century, except in South and Central America, where an endemic disease has prevented its development. This foliar disease, called South American Leaf Blight (SALB), is due to the Ascomycota fungus Microcyclus ulei. It causes severe foliar decay and can end in the death of trees of the most susceptible cultivars, such as high yielding cultivars from Asia or Africa.

The objective of the present project is to enforce and develop the research investment in molecular genetics of SALB tolerance initiated with the CMB project. It is proposed to combine complementary actions in genetic mapping and in candidate genes identification for speeding the characterization of genetics factors of natural resistance to SALB and ultimately setting a marker-aided selection program.

Results

QTL analysis of a durable resistance (MDF180) – [subproject 1]

A new resistance to SALB has recently been described in the cultivar MDF180 originated from Peru (Le Guen et al. 2008, Crop Prot, 27:1498-1503), lasting for more than 30 years in areas under high inoculum pressure. Resistance of MDF180 to SALB is characterized by the complete absence of sexual phase of the fungus (teleomorph), a limited asexual sporulation, and no foliar decay. Pseudo-testcross QTL mapping was performed on 298 F1 descendants from a Susceptible(PB260)xMDF180 cross, located in French Guiana. On this progeny, we built a genetic map encompassing 256 markers (95 AFLPs and 177 SSRs) and leading to a sufficient coverage of the 18 Hevea chromosomes. Surprisingly, the genetic determinism of this durable resistance is relatively simple, being genetically supported mainly by one major gene (linkage group g15), governing resistance to Guianese strains of the fungus, and a strong QTL (linkage group g13), efficient against Brazilian strains.

Fine mapping of a major resistance gene from FX2784 – [subproject 1]

We previously identified and mapped an other major resistance gene, governing the complete resistance of the FX2784 cultivar. Using SSR mapping of 125 F1 individuals located in French Guiana, we mapped this major gene on the linkage group g2. In the present project, we localized more accurately this major gene by SSR mapping on a population of 295 additional F1 individuals from FX2784, evaluated for SALB resistance in Brazil (Michelin PEM estate, Mato Grosso). The results confirm that the same locus explain the complete resistance in the both locations (French Guiana and Brazil) and the mapping results are highly congruent. Two SSR markers flank the resistance locus at 5 cM.

Candidate genes identification – [subproject 3]

Candidate genes (ESTs) were first identified and cloned using SSH approach based on “Susceptible vs Resistance” or “Inoculated vs Non-inoculated” subtraction. A sub-set of candidate genes was then defined among the 6992 cloned SSH-unigenes using expression analysis by macroarrays hybridizations. Three hundred eighty two ESTs, over- or under-expressed during infection, were retained for further map integration.

EST-SSRs identification and mapping – [subproject 2]

We apply several genotyping methods for polymorphic genetic marker development and candidate ESTs mapping. First of all, we search for SSR repeats (di- to hexa-nucleotide repeats) in the whole set of unigenes using the ESTtik software. We found 287 ESTs with at least 1 SSR motif. Genetic polymorphism and mapping were carried out on 54 EST-SSRs using the bin-mapping method.

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Perspectives

During the third and last year of the project (2010), the following research activities will be carried out:

a) Genetic mapping – [subproject

1

]: SSR mapping of 2 additional progenies located at the Michelin PMB

estate (Bahia, Brazil: PB260xMDF180 & WxFDR5597).

b) QTL mapping and diversity of genetic determinisms – [subproject

1

]: field evaluation of SALB resistance

for 3 progenies in Brazil (PB260xFX2784; PB260xMDF180; WxFDR5597); SALB resistance evaluation in controlled conditions of 1 fourth progeny in French Guiana (PB260xRO38); QTL mapping finalized at the end of 2010, when complete phenotypic data available

c) Candidate genes characterization – [subproject

3

]: expression analysis by Q-PCR of a subset of candidate

ESTs

d) Genetic markers development and candidate genes mapping in 4 progenies – [subproject

2

]: achievement

of EST-SSRs mapping; EST-SNPs identification and mapping using HRM genotyping method; development of genetic markers from BAC clone sequences, corresponding to candidate genes and/or to chromosome regions containing major genes/QTLs.

Publications / Congress

Publications (in prep.)

1. Le Guen V., Garcia D., Doaré F., Condina V., Couturier C., Weber C., Espéout S., Seguin M. Genetic mapping for major gene and QTLs of resistance to South American Leaf Blight due to Microcyclus ulei, in a highly resistant rubber tree cultivar.

2. Garcia D., Carels N., de Sousa L.A, Sizenando Andrade S.J., Pujade-Renaud V., Mattos C.R.R., Cascardo J.C.M. Profiling of genes from Hevea brasiliensis involved in the resistance to Microcyclus ulei.

Congress

1. Berger A, Déon M, Doaré F, Goujon E, Garcia D, Seguin M, Pujade-Renaud V (2010). Identification de gènes candidats impliqués dans les processus de résistance de l’hévéa à Microcyclus ulei par la technique des macroarrays. In: Journées Jean Chevaugeon, Aussois, 25-29 Janv 2010. Poster

2. Le Guen V, Garcia D, Doaré F, Weber C, Chambon A, Seguin M (2009) Natural pyramiding provides rubber tree with durable resistance to South American Leaf Blight. In: Plant-GEM 8, Lisbon 2009, 7-10 October 2009, Lisbon, Portugal. Poster n° S2.P.16, abstract p.94.

3. Koop D.M., Conceição L., Cardoso S.E.A., de Sousa L.A., Silva D. da C., Garcia D. (2009) Estudo histológico e molecular da morte celular programada (PCD) na interação Hevea – Microcyclus ulei. In: XLII Congresso Brasileiro de Fitopatologia. Rio de Janeiro (Brasil). 3 a 7 agosto de 2009. Tropical Plant Pathology. Vol. 34. S257. Poster 868.

4. Argout X., Garcia D., Montoro P., Pujade-Renaud V., Ruiz M., Seguin M., Sidibé Bocs S. (2009). Statement of transcriptomics and bioinformatics analyses conducted at CIRAD in rubber tree: towards the genome analysis. In: Hevea genome and transcriptome. IRRDB Workshop on Hevea Genome and Transcriptome. 2009/06/03-05, Montpellier, France. Book of abstracts. Montpellier, France: Cirad, IRRDB, IFC, p.49. 5. Garcia D., Carels N., Araújo L.d.S., Koop D.M., Pujade-Renaud V., Silva D.d.C., Mattos C.R.R., Cascardo

J.C.M. (2009). Transcriptome comparison of resistant and susceptible Hevea brasiliensis cultivars infected by Microcyclus ulei. In: Hevea genome and transcriptome. IRRDB Workshop on Hevea Genome and Transcriptome. Book of abstracts. Montpellier, France. Cirad, IRRDB, IFC, p. 30-48. 2009/06/03-05, Montpellier, France.

6. Seguin M, Argout X, Cavaloc E, Doaré F, Espéout-Fois S, Fonseca F, Garcia D, Granet F, Le Guen V, Mattos C, Pujade-Renaud V, Weber C (2008) GENESALB: Genetic analysis of resistance to South American Leaf Blight – SALB (Microcyclus ulei) in rubber tree (Hevea spp.). In: Séminaire Génoplante 2008. 1-3 Avril 2008, 1p. Arles, France. Poster

Academic training / reports 1. Masters :

- Déon Marine (2008) « Expression analysis by macro-arrays hybridization ». Montpellier University UM2, France - Bouchata Karima (2009) « EST-SSR mapping ». Blaise Pascal University, Clermont-Ferrand, France.

- Goujon Eric (2009) « Expression analysis using Q-PCR »Blaise Pascal University, Clermont-Ferrand, France. 2. PhD :

- Le Guen Vincent (2008) « Resistance QTL mapping » Montpellier Univ. UM2 & Montpellier SupAgro, France

Total permanent scientist

Montpellier: 2.6 EFTs ; Clermont-Ferrand: 1.6 EFTs ; French Guiana: 0.6 EFTs ; Brazil(researchers only): 1.5 EFTs

Temporary contracts

Mr Philippe CUBRY, Michelin post-doctoral fellow, 18 months, Montpellier from April 1, 2008, to September 30, 2009.

Mrs Sandra ESPEOUT, Michelin technician, 18 months, Montpellier

from July 1, 2009, to December 31, 2010

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GENESALB

Genetic analysis of resistance

(SALB Mi

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(SALB Microcyclus ulei)

(SALB, Microcyclus ulei)

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)

(photo: M. Seguin)

Introduction

Hevea brasiliensis cultivation is nearly the only source of

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production of natural rubber (latex), an irreplaceable strategic

bio-p

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),

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polymer for various industrial sectors

Worldwide production is

polymer for various industrial sectors. Worldwide production is

y

threaten by the South American Leaf Blight (SALB) due to the

fungus Microcyclus ulei (Ascomycota)

CIRAD and Michelin

fungus Microcyclus ulei (Ascomycota). CIRAD and Michelin

ll b

t

i

1992

(CMB Ci d Mi h li B é il)

collaborate, since 1992, on a program (CMB, Cirad-Michelin-Brésil)

collaborate, since 1992, on a program (CMB, Cirad Michelin Brésil)

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aiming at the creation of new varieties, combining high latex

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,

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yield and tolerance to SALB The ultimate objective is both to

yield and tolerance to SALB. The ultimate objective is both to

allow rubber farming development in the American inter-tropical

zone endemically affected by the disease, and to prevent the risk

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f th

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of accidental introduction of the pathogen in the current Asian and

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African producing regions

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African producing regions.

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The aim of the present project is to reinforce genetic mapping and

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The aim of the present project is to reinforce genetic mapping and

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candidate

genes

identification

in

order

to

speed

up

the

Re

o

candidate

genes

identification

in

order

to

speed

up

the

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characterization of genetics factors of natural resistance to

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characterization of genetics factors of natural resistance to

SALB

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SALB and, ultimately, to set up a marker-aided selection program.

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Figure 1: M

ulei symptoms on

(

Figure 1: M. ulei symptoms on

(

Hevea adult leaves: A) necrosis, B)

)

,

)

conidian sporulation C) stromata

conidian sporulation, C) stromata

(

ith

i

t i i

)

(perithecia containing ascospores)

(photos: M. Seguin)

(p

g

)

F

to

g15

g13

to

g15

g13

M574

0 EM9/108

4 a140

0 R

lt (2008 2009)

EM9/108

4 ₅

_EM12/214

Results (2008-2009)

EM9/362

16 A2394

14 (

)

Candidate gene identification: 6 EST

EM38/85

18 EM12/57

18 A2394

14 Candidate gene identification: 6 EST

EM12/57

18 EM12/41

19 produced and sequenced (Sanger) ->

19 a289

20 T2086

27 A2415

produced and sequenced (Sanger) ->

T2086

20 M15md

21 M15md

_{EM 4/304}

libraries (Fig. 2) -> identification of 394

21 EM3/252

23

36 ₃₈

EM54/304

_EM54/303

libraries (Fig. 2) > identification of 394

S

d R

lti

t 48

d 24 h i (F

EM3/252

23 EM1/344

24

38 EM54/303

S and R cultivars at 48 and 24 hpi (F

ssh69

29 EM3/419

35 p (

similar analyses of 5 EST SSH libr

EM3/419

35 A2483

38 EM54/114

6 similar analyses of 5 EST-SSH libr

A2483

38 EM7/153

40

56 EM54/114

ssh70

59 y

MDF180(R)

[7]

) > final identification of

EM12/276

46 TAs2709

54 ssh70

59 MDF180(R)

[7]

) -> final identification of

TAs2709

54 EM54/259

61 EM54/259

68 ssh28

A2685

67 ss

8

68 EM2/132

73 EM2/132

Candidate gene mapping: identificatio

73 A2523

79 Candidate gene mapping: identificatio

EST SSR

l

d b bi

i

A2523

79 A2508

83 ESTSSRs analyzed by binmapping

-EM54/275

89 EST SSRs analyzed by bin mapping

bi

d > 19

d

th

STS/EM36/13-14

92 bin-mapped -> 19 mapped on the com

pp

major QRL

_{QTL/mapping: 1) “fine” mapping of a m}

A2697

111 j

Q

of MDF180

QTL/mapping: 1) fine mapping of a m

A2697

111 cultivar: gSSR mapping and phenotyp

cultivar: gSSR mapping and phenotyp

M2f l

h

i

l id

a095

125 a095

121 A2757

same M2fx locus than previously iden

125 A2492

130 same M2fx locus than previously iden

(LG) 2 fl

k d b 2 SSR

A2492

130 group (LG) g2 flanked by 2 gSSRs a

TAs2456

137 g

p (

) g

y

g

2) QTL mapping of PB260xMDF180: 159

Fi

4 T

li k

f th

2) QTL mapping of PB260xMDF180: 159

Figure 4

: Two linkage groups of the

MDF180 parental map -> 18 LG (sat

MDF180

parental

map.

Genetic

MDF180 parental map -> 18 LG (sat

MDF180

parental

map.

Genetic

markers:

gSSRs

AFLPs

EST SSRs

Resistance QTL on LG g13 and of a

markers:

gSSRs,

AFLPs,

EST-SSRs,

SALB

i

l

i

Resistance QTL on LG g13 and of a

(Fi

4) Aft

M13 1b

LG 13

[2]

SALB resistance loci

(Fig. 4). After M13-1bn on LG g13

(

g

)

g

[2]

[ ]

a

Resistance gene identified in rubber tre

g

Prospects

QTL mapping of other resistance sources: 1) 171 additional [PB260

2) 269 [S x FDR5597(R)] F1 in Bahia; 3) 312 additional [PB260 x RO

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l

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f C

did t G

Candidate gene characterization: expression analysis of Candidate G

g

p

y

Candidate gene mapping: SNPs identification and genotyping by San

gSSR identification in BAC clone sequences

Cited

[1] L

Publications and communications

[1]. L

[2] L

Publications and communications

[2]. L

Academic courses: 3 MScD reports (2008-2009)

[3]. S

Academic courses: 3 MScD reports (2008 2009)

d 1 PhD

t (2008)

[4]. L

and 1 PhD report (2008)

[ ]

[5] A

p

(

)

Communications: 4 posters and 2 oral communi

[5]. A

[6] D

Communications: 4 posters and 2 oral

communi-[6]. D

[7] L

p

cations in 2 international and 2 national congress

[7]. L

cations in 2 international and 2 national congress

(photo M Seguin)

(photo M. Seguin)

Defoliation due to M.

(2008 2010):

ulei in Bahia

(2008-2010):

(2008 2010):

to South American Leaf Blight

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bb

t

(H

)

in rubber tree (Hevea spp )

in rubber tree (Hevea spp.)

(

pp )

( h t

M S

i )

(photo: M. Seguin)

1

1 1 3

Seguin Marc

1 , Argout Xavier

1 , Berger Angélique

1 , Bouchata Karima

1,3

, Déon

Segu

a c ,

gout

a e ,

e ge

gé que ,

ouc ata

a

,

éo

Marine

1 3

Doaré Fabien

2 Garcia Dominique

1 4

Goujon Eric

3 Le

Marine

1,3

, Doaré Fabien

2 , Garcia Dominique

1,4

, Goujon Eric

3 , Le

Guen Vincent

1 , Pujade-Renaud Valérie

1,3

, Weber Christelle

1 Guen Vincent , Pujade Renaud Valérie

, Weber Christelle

(1) Cirad-Bios, UMR-1098-DAP, TA A96/03, Av. Agropolis, 34398 Montpellier, France; (2)

Cirad-Bios, UR-31, Campus Agronomique, BP 701, 97387 Kourou, French Guiana; (3)

C ad

os, U

3 , Ca pus

g o o

que,

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ou ou,

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Gu a a; (3)

Université Blaise Pascal Bât Biologie Végétale Recherche 24 avenue des Landais BP 80026

Université Blaise Pascal, Bât. Biologie Végétale Recherche, 24 avenue des Landais BP 80026,

63177 Aubière; (4) Universidade Estadual de Santa Cruz Laboratório de Genômica e Biologia

63177 Aubière; (4) Universidade Estadual de Santa Cruz, Laboratório de Genômica e Biologia

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R d Ilhé

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BR 415 K

16 Ilhé

B hi CEP 45 662 000 B

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Molecular, Rod. Ilhéus-Itabuna BR 415, Km 16, Ilhéus, Bahia CEP 45.662-000, Brazil

Granet Françoise

5 Cavaloc Eric

7 Cubry Philippe

1,5

Espéout Sandra

1,5

Granet Françoise , Cavaloc Eric , Cubry Philippe

, Espéout Sandra ,

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7 S

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5 Fonseca Fernando

6 , Mattos Carlos

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7 , Scomparin Cassio

,

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5 (5) Michelin

CPN 23 Place des Carmes Déchaux 63040 Clermont Ferrand France;

(5) Michelin – CPN, 23 Place des Carmes Déchaux, 63040 Clermont-Ferrand, France;

(6) Pl

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õ

E Mi h li

Ltd

BR 163 k 16 5 Di t it

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(6) Plantações E. Michelin Ltda, BR 163, km16,5, Distrito de Ouro Branco do Sul, Itiquira,

Mato Grosso, Caixa Postal 080, CEP 78.700-090, Brazil; (7) Plantações Michelin da Bahia

; ( )

ç

Ltda, Km 05, Ituberá - Camamu, Bahia, CEP 45.443-000, Brazil

Ltda, Km 05, Ituberá Camamu, Bahia, CEP 45.443 000, Brazil

Material & Methods

i

l ti

[S R] F1

ti

l ti

351 [PB260 MDF180]

apping populations: [SxR] F1 segregating populations: 351 [PB260xMDF180]

pp g p p

[

]

g g

g p p

[

]

ndividuals located in French Guiana; 295 [PB260xFX2784] in Mato Grosso (Brazil)

ndividuals located in French Guiana; 295 [PB260xFX2784] in Mato Grosso (Brazil).

esistance evaluation: natural (field) or controlled infestations (with isolated strains

of M ulei) in French Guiana; Resistance parameters

[1][2]

: 3 parameters based on

of M. ulei) in French Guiana; Resistance parameters

[1][2]

: 3 parameters based on

ntensity of necrosis/chlorosis conidia sporulation and stromata formation (

Fig 1

)

ntensity of necrosis/chlorosis, conidia sporulation and stromata formation (

Fig. 1

)

l

k

f

i

i SSR

AFLP

[1]

[3][4]

olecular markers for mapping: genomic SSRs, AFLPs

pp g g

[1]

[ ]

[3][4]

[ ][ ]

apping strategy: bin-mapping and pseudo-testcross (H brasiliensis: 2n=2x=36)

apping strategy: bin-mapping and pseudo-testcross (H. brasiliensis: 2n=2x=36)

f

S

(S)

( )

S SS

andidate gene identification: Susceptible(S) vs Resistant(R) EST-SSH libraries:

g

p

( )

sequence analysis (ESTtik pipeline

[5]

); expression analyses at different hours after

sequence analysis (ESTtik pipeline

[5]

); expression analyses at different hours after

controlled infestations (hpi) through macroarrays hybridization; signal quantification

(ImageQ ant TL soft are) and non parametric normali ation

[6]

(ImageQuant-TL software) and non-parametric normalization

[6]

.

andidate gene genotyping for mapping: EST-SSRs from SSH clones

andidate gene genotyping for mapping: EST SSRs from SSH clones

S

i

ibl

R

i

S

inoc: susceptible

R

inoc: resistant

_SSH

_Subtractive

_{Expected expression of}

(PB260) inoculated

(RO38) inoculated

library

combination

p

the cloned ESTs during

(

)

(

)

y

(tester – driver)

g

infestation

(

)

A

S

inoc

R

inoc

S

>

R

A

S

inoc –

R

inoc

S

>

R

B

R

inoc –

S

inoc

R

>

S

E

R

inoc

R

control

Ò in

R

E

R

inoc –

R

control

Ò in

R

F

R

control –

R

inoc

Ô in

R

(photos: F Doaré)

Fi

2 F

SSH lib

i

tt d

filt

d h b idi

d

ith

33 P l b ll d

(photos: F. Doaré)

Figure 2

: Four SSH libraries spotted on macroarray filters and hybridized with

33 P labelled

otal leaf cDNAs from PB260(

S

) or RO38(

R

) cultivars inoculated or not with a M ulei isolate

otal leaf cDNAs from PB260(

S

) or RO38(

R

) cultivars inoculated or not with a M. ulei isolate.

T SSH libraries of PB260 vs RO38 (S vs R)

T-SSH libraries of PB260 vs RO38 (S vs R)

I1

5428 unigenes Macroarrays analysis of 4/6

_I1

5428 unigenes. Macroarrays analysis of 4/6

4 candidate genes differentially expressed in

Fi

3) UESC (D G

i )

id d d t f

Fig. 3). UESC (D. Garcia) provided data from

g

)

(

) p

raries from 2 other cultivars (PB314(S) vs

I

(

( )

f 674 candidate genes from 6992 unigenes

I

f 674 candidate genes from 6992 unigenes.

I2

n of 282/6992 ESTs containing SSRs -> 142

_I2

n of 282/6992 ESTs containing SSRs > 142

0 ( 0%)

f EST SSR

l

hi

d

-> 70 (50%) of EST-SSRs polymorphic and

70 (50%) of EST SSRs polymorphic and

l t PB260 MDF180

(Fi

4)

mplete PB260xMDF180 progeny (Fig. 4).

p

p g

y (

g

)

major SALB resistance gene from the FX2784

I3

ping of the population from Mato Grosso ->

I3

ping of the population from Mato Grosso >

ifi d i F

h G i

d

li k

tified in French Guiana: mapped on linkage

t 5 M (d t

t h

)

at 5cM (data not shown);

(

);

9 SSRs 96 AFLPs 14 EST SSRs mapped in

II

9 SSRs, 96 AFLPs, 14 EST-SSRs mapped in

II

turated map) -> identification of one major

a major Resistance gene (M15md) on g15

d M2f ( 2) M15 d i th thi d

j

SALB

and M2fx (g2), M15md is the third major SALB

III

(g ),

j

ee

III

ee

.

x MDF180] F1 in Bahia;

Figure 3

: Cluster analysis of expression

O38] F1 in French Guiana

[2]

Figure 3

: Cluster analysis of expression

/macroarray

data:

classification

tree

O38] F1 in French Guiana

[2]

G

b Q PCR

/macroarray

data:

classification

tree

bt i

d

f

th

71 SSH EST

Genes by Q-PCR

obtained

for

the

71 SSH-ESTs

y

ger sequencing and HRM;

_{differentially}

_expressed

_between

ger sequencing and HRM;

_{differentially}

_expressed

_between

PB260/S and RO38/R at 24 hpi

PB260/S and RO38/R at 24 hpi.

d references:

Lespinasse D Grivet L Troispoux V Rodier Goud M Pinard F Seguin M (2000) Theor Appl Genet 100: 975 984

Lespinasse D, Grivet L, Troispoux V, Rodier-Goud M, Pinard F, Seguin M (2000) Theor. Appl. Genet. 100: 975-984.

L G

V G

i D M tt

C R R D

é F L

i

D S

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