Th1_Towards the fine mapping of the major QTL conferring resistance to African Rice Gall Midge (AfRGM)
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Th1_Towards the fine mapping of the major QTL conferring resistance to African Rice Gall Midge (AfRGM)

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3rd Africa Rice Congress ...

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

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  • 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