Solanaceous crops and bacterial wilt interactions ... · 23-25 February 2017, Bangkok Solanaceous...
Transcript of Solanaceous crops and bacterial wilt interactions ... · 23-25 February 2017, Bangkok Solanaceous...
ASRT 223-25 February 2017, Bangkok
Solanaceous crops and bacterial wilt interactions: overview &
focus on genetics of eggplant resistance
Jacques DINTINGER
Marie-Christine DAUNAY
2 PhD theses
A. Lebeau (2010), S. Salgon (2017)
Solanaceous crops and bacterial wilt interactions: overview &
focus on genetics of eggplant resistance
1. Bacterial wilt diversity
2. Interactions Bacterial Wilt x Solanaceous crops resistances
3. Genetics of eggplant resistance(s) to bacterial wilt
Genetic diversity of Bacterial wilt: Ralstonia solanacearum
very large host range (including Solanaceous crops), persistence in fields
extensive genetic diversity: Ralstonia solanacearum species complex (RSSC)
Clonal lines identified (rep-PCR, PFGE, AFLP, …)
structured in 4 phylotypes (ITS, egl, hrpB, …): I, II (A & B), III et IV (Fegan and Prior, 2005)
Phylotype IIA
Phylotype IIB
Phylotype III
Phylotype IV
Phylotype I
Subdivided in 10 clades (sequence variants (<1,8%) with ecological traits51 sequevars (highly conserved variants (< 1%)
: 2 clades, 11 sequevars
: 3 clades, 8 sequevars
: 1 clade, 16 sequevars
: 1 clade, 11 sequevars
: 3 clades, 4 sequevars
R. sol.Moko
R. pseudo sol.BDB
R. syzygii
Recently classified as 3 species
Phylotype
Mutation rate
Reprod.mode
Demogr.history
I 137.6Highlyrecombin.
Highlyexpanding
III 68.8Highlyrecombin.
Highlyexpanding
IIA 49.4 Recombin. Expanding
IIB 43.3 ClonalWeaklyexpanding
IV 108.2Highlyrecombin.
No expansion
Genetic diversity of bacterial wilt : a worldwide prevalence
Prevalence
-Strains from all phylotypes can wilt eggplant, tomato and pepper-Phylotype I : highest evolutionary potential, worldwide prevalence, highestvirulence diversity
=> Highest ability to adapt to new hosts/resistant cultivars?
Tomato
T1. CRA 66
T2. Okitsu Sozai n°1T3. NC 72 TR 4-4
T4. IRAT L3
T5. Hawaii 7996
T6. TML 46
T7.CLN 1463
T8. R3034
T9. L 285 S. lycop. escul. var. cerasiforme
T10. L 390 (susceptible control)
eggplant
E1. MM853, Dingras multiple purple
E2. MM643, SM6
E3. MM152, Ceylan SM164
E4. MM1752, EG203, Surya
E5. AG91-01, MM931
E6. AG91-25, MM960
E7. MM195 S. linnaeanum (parent of 1rst mappi.pop)
E8. MM738 (parent of 1rst mapping population)
E9. S56B, Terong Bulat Hijau
E10. Florida Market, MM 136 (suscept. control)
Pepper
P1. Narval, PM 1143
P2. PI 322719, PM 687
P3. Cristal Blanco, PM 1022, Pen 79 (C.baccatum)
P4. CM 334, PM 702
P5. 0209-4, BC3F5 (BC3F5[C. annuum x C.chinense])
P6. CA8, PBC631A
P7. MC4, PBC66
P8. Perennial, PM 659
P9. PBC384
P10. Yolo Wonder (susceptible control)
Nominated by crop breeding experts
Tomato: H. Laterrot (INRA), P. Hanson (AVRDC),
J. Scott (Univ. Florida), M.C. Daunay (INRA)
Eggplant: P. Hanson & J.-F. Wang (AVRDC) ,
MC. Daunay (INRA)
Pepper: P. Gniffke (AVRDC), A. Palloix (INRA)
A core-collection (Core-TEP) of worldwide genetic resources resistant to R. solanacearum
12 virulent strains, phylogenetically differentiated
A core-collection (Core-Rs) of R. solanacearum strains
Isolated from: Solanum lycopersicum, Vaccinium sp.Heliconia caribea
Pathogenic to
Phenotype ClusterPercentage of
wilted plant
Colonization
index
Highly resistant 1 ≤ 13,3% ≤ 16,67%
Moderately
resistant 2 ≤ 33,33%
16,67% ≤ … ≤
50%
Intermediate 3 ≤ 43,33% ≥ 43,33%
Susceptible 4 40% ≤ … ≤ 80% ≥ 46,67%
Highly
susceptible 5 ≥ 80% ≥ 86,67%
phenotype statistical partitioning (based on interactions Core-TEP x Core-Rs)
• partially resistant
• latently infected
Colonisation index (%)
% wilted
plants
veryrésistant
Moderately susceptible
Moderatelyrésistant
Very susceptible
Partiallyresistant
Latent infection
6 phenotypes defined
Latent
infection
very
resistant
Moderately
susceptible
Moderately
resistant
Very
susceptible
Partially
résistant
Interactions BW diversity X solanaceous crops resistance diversityStrains selected for genetic studies
Strain Alternative name Country AG91-25 MM738
CMR134 RUN215, CFBP7058 Cameroon 2 4
PSS366 RUN155 Taiwan 1 4
PSS4 RUN157, CIP410 Taiwan 5 5
GMI1000 RUN54, JS753 French Guyana 3.1 5
CFBP2957 RUN36, MT5 Martinique 2 4
CMR34 RUN147, CFBP7029 Cameroon 5 5
CFBP3059 RUN39, JS904 Burkina Faso 4 5
TO10 RUN969 Thaïland na na
1
2
3.1
4
5
highly resistant
moderately resistant
partially resistant
moderately susceptible
highly susceptible
Dissection of the resistance in eggplant AG91-25(breeding line of complex pedigree, involving S. aethiopicum)
Resistance evaluation: protocol
- 180 RILs population Inoculated through irrigation system- 2 greenhouses = 2 replications- 2-season repeat tests for each strain
- Scoring plants according a scale 0 to 4- Evaluation of plants 4 to 5 weeks
post-inoculation- Latent infection tested at the end of
trials
Variables analyzed :
Disease score (SCO): Rating means for each line
Wilting rate (W): % plants with rating ≥ 1
Colonization index (CI): % colonized plants
AUDPC: Area under disease progress curve
AG91-25 (0,038)
AG91-25 (0,041)
MM738 (0,56)
MM738 (0,45)
AG91-25 (0,35)
AG91-25 (0,40)
MM738 (0,68)
MM738 (0,73)
AG91-25 (0,12)
MM738 (0,16)
MM738 (0,30)
MM738 (0,54)
MM738 (0,28)
AG91-25 (0)AG91-25 (0)AG91-25 (0)
Resistance evaluation: results
Three different results with Waudpc variable:
Continuous distributions of RILs population. High differences of resistance between AG91-25 (R) and MM738 (S) (CFBP3059, CFBP2957,
PSS4, TO10)
Quantitative variable controlled by QTLs
Kernel density plots of the variable Waudpc
Continuous distribution but phenotype of parental lines are very close (CMR34)
Distribution due to environmental effects?
Discontinuous distribution with 2 discrete classes : Resistant /susceptible (GMI1000,
PSS3666, CMR134).
Qualitative variable controlled by major gene(s)
Genotyping by sequencing (GBS)
DNA extraction from eggplant population
51 2
DNA digestion with ApeKIenzyme
3
Multiplexing 96 samples with unique barcode
4
PCR amplification to obtain GBS “library”
Sequencing with illuminatechnology
Generation of SNPs
1 Quality control of raw data
2 Clean and demultiplex by barcode
3
Reference eggplant genome
“Denovo”
Stack 1 Stack 2 Stack 3 Stack X
Pipeline Stacks Stacks from parental lines assembled in loci in a catalog
Stacks from population compared to catalog and SNP calling
4 5
Results: A new densified genetic map
Genetic map with 14 LG corresponding to the 12 chromosomes of eggplant
Total map length of 1518 cM (Reference genetic map = 1281 cM) with 1036 markers (867 SNPs)
LG1= E01_ALG2= E02 LG3= E03LG4= E04 LG5= E05_A LG6= E06 LG7= E07 LG8= E08 LG9= E09LG10= E10 LG11= E11 LG12= E12LG13= E01_BLG14= E05_B
LG
Linkage map of the RILs [MM738 x AG91-25]population
Some regions very rich in distorted markers mean probable chromosome segments from S.aethiopicumancestor ( in AG91-25)
Sequences from GBS were aligned onto the tomato genomic sequence Orthology can be used to identify candidate genes thank to physical map of tomato.
Detection of major gene ERs1 on chromosome 9
ref_26947
novo_30512
novo_77150
novo_32243
novo_4278
novo_41086
novo_61997
novo_5749
novo_1505
emd03C01
CSO475b
COX190a
ref_17276
ref_67868
novo_89055
novo_51578
CMI065a
novo_7810
novo_58873
ref_34168
novo_72335
novo_59329
novo_5688
ref_8676
ref_54787
COX067a
novo_95361
novo_80111
ref_8690
novo_71272
CRO432b
EM134
emf01K21
0
5
10
15
20
9
ERs1
0
5
10
15
20
25
30
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ref_26947
novo_30512
novo_77150
novo_32243
novo_4278
novo_41086
novo_61997
novo_5749
novo_1505
emd03C01
CSO475b
COX190a
ref_17276
ref_67868
novo_89055
novo_51578
CMI065a
novo_7810
novo_58873
ref_34168
novo_72335
novo_59329
novo_5688
ref_8676
ref_54787
COX067a
novo_95361
novo_80111
ref_8690
novo_71272
CRO432b
EM134
emf01K21
0
5
10
15
20
9
ERs1
0
5
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15
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Strain LG QTL (cM)length of
CIeggplant
chr.synteny
blockTomato
chr.CMR134 9 106.36 0.360 9 sb35 9GMI1000 9 106.5 0.114 9 sb35 9PSS366 9 106.5 0.114 9 sb35 93strains 9 106.5 0.567 9 sb35 9
Graphic of LOD score according to marker position on LG9
Results of QTL analysis with strains CMR34, GMI1000 and PSS366
ERs1 positioned on the long arm of chromosome 9: Maximum estimated interval= 0,57 cM
Anchor markers with known position on tomato and eggplant reference genomes
Physical position estimated thanks to anchor markers: interval of 1.7 Mbp
ERs1 only detected with strains of phylotype I
ERs1 Phylotype I - specific?
QTL analysis results with strains CFBP3059, CFBP2957, PSS4, TO10
E02
QTL on chromosome 2 detected with 4 strains: PSS4 and TO10 (I), CFBP3059 (III) and CFBP2957 (IIA) Broad-spectrum QTL explaining 12 to 38 % of phenotypic variance QTL on chromosome 5 detected with 2 strains: CFBP2957 and CFBP3059 Major QTL explaining 17 to 50 % of phenotypic variance
E05_b novo_430290,0CDX125a1,9ref_590745,0novo_152146,2novo_324517,8CRX184b8,4CMC184b8,7novo_95239,3novo_700139,6novo_147561_110,2novo_122979_110,8novo_9828913,2novo_49324_114,3CAU275b16,2CAU273a17,4novo_5079127,9novo_6401630,9novo_2339131,9novo_2425632,7novo_4258433,8novo_6931534,6novo_1728437,7
CFB
P2957
CFB
P3059
0
5
10
15
20
25
30
E05_B
novo_717770,0novo_908352,5novo_269683,3novo_703916,1novo_183738,0ref_3373721,7novo_2392747,7novo_9262750,4novo_10162153,3ecm00969,8COF324b70,6COI393a72,7novo_5333574,5CAU393a75,3D_emh02E0876,3novo_480679,7CRC098b80,6novo_7115681,8novo_3016984,7novo_273286,6novo_6679388,8novo_2911096,2ref_50397,6novo_8402103,3novo_86713106,5ref_2107108,6novo_92566109,4novo_76218112,4novo_88131113,3novo_43591114,7novo_95332115,4novo_71535116,7novo_22241120,8novo_33634121,5novo_88421124,3novo_82960125,1novo_35611128,7novo_145307138,7novo_83500143,3novo_25595147,7
CFB
P2957
CFB
P3059
PSS4
TO10
0
5
10
15
20
25
E02
novo_430290,0CDX125a1,9ref_590745,0novo_152146,2novo_324517,8CRX184b8,4CMC184b8,7novo_95239,3novo_700139,6novo_147561_110,2novo_122979_110,8novo_9828913,2novo_49324_114,3CAU275b16,2CAU273a17,4novo_5079127,9novo_6401630,9novo_2339131,9novo_2425632,7novo_4258433,8novo_6931534,6novo_1728437,7
CFB
P2957
CFB
P3059
0
5
10
15
20
25
30
E05_B
novo_717770,0novo_908352,5novo_269683,3novo_703916,1novo_183738,0ref_3373721,7novo_2392747,7novo_9262750,4novo_10162153,3ecm00969,8COF324b70,6COI393a72,7novo_5333574,5CAU393a75,3D_emh02E0876,3novo_480679,7CRC098b80,6novo_7115681,8novo_3016984,7novo_273286,6novo_6679388,8novo_2911096,2ref_50397,6novo_8402103,3novo_86713106,5ref_2107108,6novo_92566109,4novo_76218112,4novo_88131113,3novo_43591114,7novo_95332115,4novo_71535116,7novo_22241120,8novo_33634121,5novo_88421124,3novo_82960125,1novo_35611128,7novo_145307138,7novo_83500143,3novo_25595147,7
CFB
P2957
CFB
P3059
PSS4
TO10
0
5
10
15
20
25
E02
Graphics of LOD score
Straineggplant
chr.QTL position
(cM)length of CI
(cM)synteny
blockTomato
chr.
CFBP30592 71 5 sb06 25 8 3 sb20 12
CFBP29572 78 4 sb06 2
5 5 5 sb20 12
PSS4 2 70.6 4 sb06 2
TO10 2 71 4 sb06 2
Candidate genes for resistance to R. solanacearum
The example of major gene ERs1 on eggplant chr. 9 colocalizing with a cluster of R genes on tomato and potato
Comparison of the synteny block 35 between eggplant transcripts on chr 9 (EO9), tomato genes on chr 9 (T09) and potato genes on chr 9 (P09) (Salgon et al, submitted)
ERs1 interval on chr 9/T09 : 1 CC-NBS-LRR gene 2 RLK gene
ERs1
QTL interval on chr 5/T012 : 4 RLK genes 1 CC-NBS-LRR gene 5 genes coding for
resistance fragment protein
QTL interval on chr 2/T02 : Several clusters of R genes
Heterogeneous Inbred Family (HIF) Material for Near Isogenic Lines (NILs) production and fine mapping of ERs1
Finding SNPs closely linked to the gene ERs1 and defining a narrower interval
To target more precisely candidate genes in the corresponding zone
To use markers more closely linked for breeding
Ongoing: fine mapping of major ERs1 gene
ERs1
1,7 Mb
HIF-1HIF-2HIF-3HIF-4HIF-5
R RRRS
SRRS
R=resistantS=susceptible
b/b homozygous AG91-25a/a homozygous MM738a/b heterozygous
HIF-6HIF-7HIF-8HIF-9
QTL Chr.a Donor parent
Linked marker
Forward primer Reverse primerMarker type
Digestion Enzyme
EBWR2 E02 E6 ecm009ATCTAGTACCATCAAGTCTAAGCAGCA
GTTTAACAACAGCTGAGGCCATGAAA
SSR no -
EBWR9 E09 E6 s_903CCCATTTCACACACAAGCAA
CTCTATTGCCACCCCAAGTG
CAP yes NcoI
Transfer of resistance by SAM in susceptible cultivars BR and BS in Réunion island (strain I-31)
marker CAP s_903 linked to specific major QTL ERs1
marker SSR ecm009 linked to generalist QTL EBWR2
Transfer of resistance by SAM: example of backcross 4
s_903 marker (CAP) used for
transfering ERs1 major gene
ecm009 marker (SSR) used for
transfering EBWR2 QTL
11 individuals carrying both ERs1 and EBWR2 alleles
0
10
20
30
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50
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100
0 6 13 20 27 34 41
W (
%)
Days after inoculation
BC4F1(BR*E1)
BC4F1(BR*E2)
BC4F1(BR*E3)
BC4F1(BR*E6)
BC4F1(BS*E1)
BC4F1(BS*E2)
BC4F1(BS*E3)
BC4F1(BS*E6)
E8
E6
Observed % of resistant plants in BC4F1 > theoretical 1:1 ratio
Severe distortion of segregation of marker s_903 in favor of donnor or resistance E6
Selective advantage of genotypes carrying the segments from E6, particularly that one with
major gene ERs1 probably originating from S. aethiopicum
Transfer of resistance by SAM: example of backcross 4
Take Home messages
Resistance to R. solanacearum in eggplant is a complex system with a specific major gene and non-specific QTL,
ERs1 is (at least) phylotype I strains - specific : deployment of resistant breeding lines in areas where phylotype I is predominant,
Transfer of major gene ERs1 in susceptible lines by MAS is effective , but more breeder-friendly markers must be developed from closely linked SNPs sequences,
Cloning ERs1 is a priority for the years to come
ERs1 does not control virulent strains of phylotype I (PSS4, TO10) nor strains of other phylotypes:
need to discover other major genes / QTLs usable by breeders for future creation of broad-spectrum resistance by pyramiding
eggplant
E1. MM853, Dingras multiple purple
E2. MM643, SM6
E3. MM152, Ceylan SM164
E4. MM1752, EG203, Surya
E5. AG91-01, MM931
E6. AG91-25, MM960
E7. MM195 S. linnaeanum (parent of 1rst mapping pop)
E8. MM738 (parent of 1rst mapping population)
E9. S56B, Terong Bulat Hijau
E10. Florida Market, MM 136 (susceptible control)
E1 E3 E4 E9
Mining for new major resistance factors
E8 (susceptible)
Ongoing studies at CIRAD- INRA
X
DH
Aknowledgment
Ralsto team in Réunion Island Main partners
On a local level
On a national level
International level
Financial support
Thank you for your
attention