Towards the fine mapping of the major QTL conferring resistance to African Rice Gall Midge
(AfRGM)
Presenter: Marie Noelle Ndjiondjop
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
Outline
Current status of AfRGM in Africa
Introduction
Severe outbreak have led to 100% yield loss
(Ethel, 1993) • Most affected countries: Burkina Faso,
Mali and Nigeria• Occurs in lowland and upland ecologies
45-80% yield loss
Objective of this study- To fine map major QTL associated with AfRGM resistance in O. sativa
What is African Rice Gall Midge (AfRGM)?
Adults are mosquito-like and nocturnalFemales have robust, reddish brown abdomensMales have slender, brown abdomens and long antenna
• 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)
2- Paddy greenhouse screening
Flanking Infestation band is constituted of highly susceptible variety around plots of plants being screened
Galls were counted on all the 20 hills in each row, 45 and 70 days after transplanting
Percentage tiller infestation was computed
SES and Resistance Index based Assessment recorded at 45 and 70 DAT
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
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
Genetic basis of resistance to AfRGM
Generation Segregation ITA306xTOS14519 ITA306xTOG7106 inheritance
F1
R:S 0:20 0:20
Ratio 0:1 0:1 recessive
F2/BC1F2
R:S 34:445 4:86
Ratio 1:15 1:15 2 genes
Khi2 0.66ns 0.48ns
F3
R:Seg:S 35:358:253 -
Ratio 1:8:7 -
Khi2 6.89ns -
ns = Not significant deviation from expected ratio at p= 0.001
Genetic basis of resistance to AfRGM
Populations development
Populations screening
Identification of markers (SSRs and SNPs) link to AfRGM resistance
Development of a mapping population of 649 F3 families
Polymorphic markers between Parents
Resistant bulk Susceptible bulk
Identification of potential markers for the AfRGM
10 highly susceptible lines
Selection of the parental linesSelection of a set
of 303 SSR markers
Genotyping with polymorphic markers
10 highly resistant lines
Phenotyping against AfRGM
Genotyping with marker setCrossing
SSR
Gen
otyp
ing
SSR
Gen
otyp
ing
Whole population genotyping with the SNPs markers
Identification of markers linked to AfRGM resistance
- F test very significant- High LOD score
Linkage analysis (F test)
Pooling DNA
Selection of a setof 500 SNP markers
9 10
3
11
5421 6
8
712
Identification of SNP markers link to AfRGM resistance in TOG7106
9 10
3
11
54
AfRGM1
21 6
8
712
Identification of SNP markers link to AfRGM resistance in TOS14519
Epistatic interactions among QTLs were studied • There is no significant epistasis, although the two largest QTLs may show
marginal interaction.
100 105 110 115 120 1250
5
10
15
20
25
30
35
25 SNP markersin ~3 Mb
LOD
centiMorgans
Large effect QTL for resistance to AfRGMFine mapping & QTL cloning
0 5 10 15 20 25
centiMorgans
-Log
10P
Independent verification experiment: Two linked QTLs for AfRGM resistance
AfRGM QTL verified
Positional cloning in progress
SignificanceThreshold
P < 1010 with 2 QTLs
Single QTLModel
2 QTLs increase resistance by 20%
0 10 20 30 40 50 60 70
Genomics to accelerate gene identification
Understanding mechanism accelerates gene identification• Comparison of chemicals in
resistant, susceptible, infected 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
Insect tissue I C I First harvest date of leaf and insect tissue Second harvest date of leaf and insect tissue : Infested plant growing in the cage : Control plant growing in the cage
ITA ITA TOS
I
I
C
I C I C
ITA TOS ITA
I
II
III III
II
• ANOVA for LC-MS of TOS and ITA leaves infested or uninfested with AfRGM
• Significant differences for the MW316, MW580-1, MW564, MW372, and MW446 compounds, with higher concentrations in the resistant TOS genotypes
• Result consistent with a possible
defensive role for these compounds
Chemical differences between genotypes
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
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.
Collaborators
Center of Excellence for Rice Research