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