3rd Africa Rice Congress Theme 1: Climate resilient rice Mini symposium: Towards improved resistance to biotic stresses

1. Towards the fine mapping of the major QTL
conferring resistance to African Rice Gall
Midge (AfRGM)
Presenter: Marie Noelle Ndjiondjop

2. Outline
1. Introduction
2. Genetic basis of resistance to AfRGM
3. Identification of QTLs controlling resistance to
AfRGM using SSR and SNP markers
4. Validation and fine mapping
5. Conclusion

3. Introduction
Current status of AfRGM in Africa
What is African Rice Gall Midge (AfRGM)?
Adults are mosquito-like
and nocturnal
Females have robust,
reddish brown abdomens
Males have slender,
brown abdomens and
long antenna
Severe outbreak have
led to 100% yield loss
(Ethel, 1993)
•
•
45-80% yield loss
Most affected countries: Burkina Faso,
Mali and Nigeria
Occurs in lowland and upland ecologies
• 3 African Orseolia species- O. oryzivora Harris & Gagné,
- O.bonzii Harris
- O. nwanzei Harris & Nwilene
- O. oryzivora and O. nwanzei directly harmful
- O. bonzii causes PGM an alternative host of
the main parasitoids of AfRGM
- O. oryzivora and O. bonzii closely related and
O. nwanzei is distinct (Francis et al., 2006)
Objective of this study- To fine map major QTL associated with AfRGM
resistance in O. sativa

4. Genetic basis of resistance to AfRGM
1- Insect rearing
AfRGM insect culture was maintained on the susceptible variety, ITA306
Planting done in seed boxes and timed to coincide the plant age for infestation with emergence of adult midges from the
culture plants
Culture plants of 5 weeks bearing 3-week old adult midges transplanted to the screenhouse just prior to the transplanting of
test entry
2- Paddy greenhouse screening
Entries previously sown on nursery bed
Entries transplanted, 14 DAS in a paddy
screenhouse, in a 2-m row with a space of
0.18 m within and between rows
Flanking Infestation band is constituted of
highly susceptible variety around plots of plants
being screened
SES and Resistance Index based Assessment
recorded at 45 and 70 DAT
Galls were counted on all the 20 hills in each
row, 45 and 70 days after transplanting
Percentage tiller infestation was computed

5. Genetic basis of resistance to AfRGM
Populations development
Populations screening
Generation
Segregation
ITA306xTOS14519 ITA306xTOG7106 inheritance
0:20
0:1
34:445
4:86
Ratio
1:15
1:15
0.66ns
0.48ns
R:Seg:S
F3
0:20
0:1
Khi2
F2/BC1F2
R:S
Ratio
R:S
F1
35:358:253
-
Ratio
1:8:7
-
Khi2
6.89ns
-
ns = Not significant deviation from expected ratio at p= 0.001
recessive
2 genes

6. Identification of markers (SSRs and SNPs) link to
AfRGM resistance
Selection of a set
of 303 SSR markers
Selection of a set
of 500 SNP markers
Selection of the parental lines
Crossing
Genotyping with marker set
Development of a mapping population of
649 F3 families
Polymorphic markers between Parents
Whole population genotyping
with the SNPs markers
Phenotyping against
AfRGM
10 highly resistant lines
10 highly susceptible lines
Resistant bulk
Susceptible bulk
Genotyping with
polymorphic markers
Identification of potential markers for
the AfRGM
SSR Genotyping
SSR Genotyping
Pooling DNA
Linkage analysis (F test)
Identification of markers linked to
AfRGM resistance
- F test very significant
- High LOD score

7. Identification of SNP markers link to AfRGM
resistance in TOG7106
1
2
3
5
4
6
8
7
9
10
12
1
1

8. Identification of SNP markers link to
AfRGM resistance in TOS14519
2
3
5
4
6
AfRGM1
1
8
7
9
12
10
11
Epistatic interactions among QTLs were studied
• There is no significant epistasis, although the two largest QTLs may show
marginal interaction.

9. Large effect QTL for resistance to AfRGM
Fine mapping & QTL cloning
40
35
30
LOD
25
20
15
10
5
0
100
0
105
5
110
10
115
15
centiMorgans
120
20
125
25
25 SNP
markers
in ~3 Mb

10. Independent verification experiment:
Two linked QTLs for AfRGM resistance
P < 1010 with 2 QTLs
-Log10P
2 QTLs increase
resistance by 20%
Single QTL
Model
0
10
20
30
40
50
60
centiMorgans
AfRGM QTL verified
Positional cloning in progress
70
Significance
Threshold

11. Genomics to accelerate gene
identification
Understanding mechanism accelerates gene identification
ITA
• Comparison of chemicals in
resistant, susceptible, infecte
d and control plant using a
reversed-phase-HPLC
coupled to an
Electrospray(ESI)- IonTrap
mass spectrometer (Bruker
Esquire 6000 instrument)
• Metabolite detection and
difference search using
Bruker software
TOS
ITA
TOS
I
I
II
II
III
III
I
C
Insect tissue
I
C
First harvest date of leaf and insect tissue
I
: Infested plant growing in the cage
C : Control plant growing in the cage
I
C
I
Second harvest date of leaf and insect tissue

12. Chemical differences between
genotypes
• ANOVA for LC-MS of TOS and ITA leaves infested or uninfested with AfRGM
• Significant differences for the
MW316, MW5801, MW564, MW372, and MW446
compounds, with higher concentrations
in the resistant TOS genotypes
• Result consistent with a possible
defensive role for these compounds

13. Conclusion
1. Inheritance of AfRGM in both landraces TOG7106 (O. glaberrima).and
TOS14519 (O. sativa) is controlled by several recessive genes
2. BSA with SSR and whole-genome genotyping with SNP markers allowed
the identification of a major QTL, and 4 additional QTLs on other
chromosomes
3. The whole genome genotyping with SNP markers allowed the
identification of 12 QTLs in TOG7106. They are located on 7
chromosomes
4. We have verified the position of the QTL, and identified two linked
QTLs, which together cause a 20% increase in resistance to AfRGM
5. Fine scale mapping is now underway

14. Conclusion
6. We identified several novel flavonoid compounds related to known insect
defense metabolites, which occur at higher concentration in the resistant parent
TOS.
7. We are working to verify whether the QTL controlling this difference maps in
the AfRGM QTL region.
8. We also examined the rice genome annotation near the AfRGM QTL, and
find a tightly linked candidate gene corresponding to the expected biosynthetic
enzyme that may explain the chemical difference.

15. Collaborators
Center of Excellence for Rice Research