Gene discovery and its applications in rice

Mathias Lorieux (IRD/CIAT)Rice 2010 Conference

September 2010September 2010

Plan

1. Using O. sativa related species to discover genes of g p gimportance- Oryza sativa x O. glaberrima introgressions

Wild species- Wild species

2. Sterility genes and interspecific bridges3. Gene discovery resources: mutant libraries and NAM y

populations; software4. Applying gene discovery to selection: RHBV5. Planned applications of new genomic tools

1. Using O. sativa related species to discover genes of importanceto discover genes of importance

b• Domestication allelic bottleneck• Wild species still have the “lost” alleles• Many traits of agronomical interest• Many traits of agronomical interest• Several examples of successful introgression• Transgressive effectsg

The A genome species of riceg p

O. glaberrimaO. glaberrima

O. sativa japonicaO. sativa japonica

O rufipogonO rufipogonO barthiiO barthiiO. sativa indicaO. sativa indica

O. longistaminataO. longistaminataO. O. glumaepatulaglumaepatulaO. rufipogonO. rufipogonO. barthiiO. barthii

OO meridionalismeridionalisO. O. meridionalismeridionalis

The triple domestication of ricep

japonicaj

O. glaberrima indicai

Chromosome Segment Substitution Lines

• CSSLs are specially useful for assessment of wild alleles p y– bypass sterility barriers– allow easier wild/cultivated phenotypic comparison

Q i k & l li i f /QTL f i• Quick & easy localization of genes/QTLs for traitsof interest

• Introduction in breeding programsg p g• Fixed lines• Derive NILs

Caiapo (japonica) x MG12 (IRGC103544)

312 lines scanned with 125 well distributed SSRs59 BC3DH lines cover the O. glaberrima genome

Residual background 3422 BC4F2 lines CSSLs C.P. Martinez

Mapping of a major resistance gene to Rice stripe necrosis virusto Rice stripe necrosis virus

Gutierrez et al, BMC Plant Biol. 2010

Yield components

Gutierrez et al, BMC Plant Biol. 2010

Striga resistance

Collab. J. Scholes, Sheffield Boisnard et al, 2010

A widely used population Trait Partner Name

Striga resistance U. Sheffield J. Scholes

D ht t l Af i Ri E b p B M hDrought tolerance AfricaRice, Embrapa, Fedearroz

B. MannehC. GuimaraesM. Diago

Osmotic adjustment IVIC T. Ghneim

Panicle architecture Cornell, CIAT S. McCouch

Root development Cirad N. Ahmadi

A i i CIAT ICAR C P M iAgronomic traits CIAT, ICAR, CIMMYT-India

C.P. MartinezR. Gupta

Bacterial blight R IRD - RPB V. Verdier

Nematode resistance IRD - RPB G ReversatNematode resistance IRD - RPB G. ReversatS. Bellafiore

Breeding (recurrent) Cirad M. Châtel

Bradyrhizobium LSTM-IRD B. Dreyfusy z y

Iron toxicityNitrogen UE

U. LouvainCIAT

P. BertinJ. Rane

IR64 (indica) x TOG5681

BC3F3 and BC2F4 population. Genotyping of 363 lines with 143 SSRs selected from the Core Map. 61 lines covering 95% O. glaberrima genome. Two gaps on Chr. 4 and 10

Performance of ILs under drought

B. Manneh (AfricaRice)

Cultivated x wild CSSLs

• Curinga x O. meridionalis acc. W2112/OR44 Laura Moreno – CIAT• Curinga x O. barthii acc. IRGC101937 Mamadou Cissoko – AfricaRice• Curinga x O. rufipogon acc. IRGC105491 J. David Arbelaez – Fedearroz• Curinga x O. glumaepatula acc. GEN1233 Priscila Rangel – CNPAF

Capacity builing at Cornell Uty (S. • Same genetic background: Acc. Curinga, tropical japonica elite line• Same SSR genetic map (Universal Core Genetic Map)

McCouch)

• BC1F1s genetic map; selection of target chr. Segments Fedearroz• BC2F1s 600 plants / pop. produced;

foreground check; background checkBC3F1 f d h k• BC3F1s foreground check

• BC3DH/F3 & BC4F1s• BC4F2/3

•BAC libraries & RefSeq (Rod Wing, AGI)Curinga x O. meridionalis BC3DH introgression lines

Tool: Universal Core Genetic MapO. meridionalis O. rufipogon O. barthii O. glumaepatula

85 - 91% polymorphism

Orjuela et al, TAG 2010

2. Genetic bases of the interspecific sterility

RM1900 9 RM190 RM19349 RM19350 RM19353

0,90,80,8

3,5

RM19357 RM19361 RM5199 RM19363 RM19367 RM19369 Os05260Int

0,90,00,00,80,80,00,8 RM19377

RM_S1_34 CG14 38E01

5,1

0,9

• Maternal allelic transmission depends onrecombination around S1CG14 38E01

RM19391 RM19398 RM3805 RM19414 RM19420

2,0

0,90,00,80,80 8

1

• Epistatic interaction between the three loci(BDM model)

• Sequencing of the region 2 candidate genesRM204 0,8 • Sequencing of the region 2 candidate genes

Garavito et al, Genetics 2010

Duplications at the S1A locus

Application: Opening the African rice diversityLinks

The O. sativa x O. glaberrima sterility barrier hampers full use of interspecific lines in breeding programsinterspecific lines in breeding programs

• Although O. sativa x O. glaberrima introgression lines (like CSSLs) can be fertile they generally produceCSSLs) can be fertile, they generally produce +/- sterile hybrids with O. sativa

• Sterility hampers full use of African rice for breedingSterility hampers full use of African rice for breeding

interspecific bridges

iBridges: specifications

• Lines with significant content of donor (O. glaberrima) genome• iBridges x O. sativa F1 hybrids are fertile (sativa-homozygous for S1)

direct use in breeding schemes (either MAS or classical; MARS)

• From many donor accessions broad access to the diversity available in donor/wild species for plant breeding

• A Generation Challenge Program competitive grant (starting July 2007)g g p g ( g J y )A. Ghesquière & M. Lorieux (IRD-LGDP/CIAT), D. Galbraith (AGI - Tucson), J. Tohme & C. P. Martinez (CIAT), M-N Ndjiondjop (AfricaRice) + selected NARs and Uties from Africa, Asia and South America

iBridges development scheme

3 O. sativa accessions X

F1 Hybrids

25-30 accessions of O. glaberrima

yb ds

BackcrossBackcross

• SAM for S1s allele (5%)

• Selection for fertility (50%)

BILs (BC1F4)• SSR – SNP genotyping• Evaluation for traits of interest Selection for S1

s leads to significant increase of plant fertility

What the iBridges will offer

• 25 pools of fertile BC1F3-4 lines, compatible to O. sativa40% of the lines are fertile vs < 5%!40% of the lines are fertile vs < 5%!

• A DNA microarray capable of revealing O. sativa x O. glaberrimapolymorphisms (high throughput, high resolution genome scanning)G ti k d th S t ilit• Genetic markers around the S1 sterility gene

allow to screen quickly interspecific lines for the presence of O. sativa compatible allele of S1

• A well-described technology for developing additional iBridges betweenA well described technology for developing additional iBridges between O. sativa and its other AA-genome (wild) relatives, to provide a broad access of the genetic diversity in the AA species complex

• A physical map of the O sativa x O glaberrima sterility “genes”A physical map of the O. sativa x O. glaberrima sterility genesallow to develop even more efficient strategies for future selection of materials

The approach could improve significantly the access to andThe approach could improve significantly the access to and the use of the genetic diversity available in African rice

3. Genomic resources for gene discoveryg y

• T-DNA and Tos17 mutants• Nested Association Mapping

Massive gene discovery platformNew Generation Sequencing technologies will make these

resources even more valuable

Gene discovery: T-DNA mutants

Gene2traits search

Gene2traits search

Gene2traits search

Gene discovery:Nested Association Mapping (NAM)Nested Association Mapping (NAM)

Tools: Software for geneticsmapdisto.free.fr

4. Applying gene discovery to selection• Marker-Assisted Selection can fasten (not always) the breeding

process• Particulary valuable for traits that are difficult or expensive or

time-consuming to evaluateN d i ti b i t i• Now used in routine by private companies

• Example: Rice hoja blanca virus (MADR, Fedearroz) (2007-2011)

RHBV resistance QTLs MADRQ

Fedearroz 2000 x WC366

MADR

Chr 4

Chr 5

RHBV incidence vs QTL presence

40

45

25

30

35

40

ED

IO V

HB

A AA_Fedearroz 2000

10

15

20

% P

RO

ME AB_Heterocigoto

BB_WC366

0

5

RM518RM16368

RM16416RM16416

LOCUSEl efecto fenotípico significativo es una característica de importancia en

el mejoramiento asistido por marcadores

Introgression of resistance genes in elite lines

Mapeo Fino: evaluaciónD ll d d Mapeo Fino: evaluaciónen invernadero y genotipificación

Desarrollo de marcadoresasociados con la resistencia

Definición de un marcadorespecífico para el gen de

i t i

Introgresión de QTLs y usode SNPs : evaluación en

ti ifi ió resistencia

Evaluación con el nuevo

campo y genotipificación

Comparación de la nueva Evaluación con el nuevomarcador sin evaluación

fenotípica

Comparación de la nuevametodología con el método

clásico

Identificación y optimización de una metodología optimizadapara la selección por RHBV

5. New genomic tools: How we will use themg

• SNP platform (Constanza Quintero)p ( Q )– Genetic diversity– Genetic mapping (Genes, QTLs)

MAB MAS MARS– MAB, MAS, MARS

• High throughput NGS-based SNP technologies– 1,000s of samples x 10,000s-100,000s of SNPs– Decipher genetic bases of interspecific sterility using advanced

backcross lines– Fine mapping and cloning of QTLspp g g Q– NAM

• Bioinformatics (key)Platform for MAS

Diversity of LAC germplasm

Graphical genotypes versus Indica

Graphical genotypes versus Tropical Japonica

Expected Indica x Japonica genetic map (IR64 x Azucena, NAM)( , )

Rice Association Analysis Initiative Upland Rice Breeding

Marker-Assisted Recurrent Selection and Genomic Selection

Cécile GrenierRice Association Analysis Initiative

Association Panels

Upland Rice Breeding

Synthetic Populations

Temperate Japonica

Tropical Japonica Nucleus

PCT-4A PCT-4B PCT-4C PCT‐11

Indicas(200)

Agronomic traits

p(200)

p(200)

Nucleus Oryza SNP

(24)

Evaluation under drought (leaf T°, WUE)Root traits Diseases (blast)

Leaf T°AgronomyDiseases

Agronomic traitsEvaluation under drought (leaf T°, WUE)Diseases (blast)

Tropical Japonica AP(200), low LD

SPn(400), medium LD

SPn+1(400), medium LD

Association mappingPan-genomic approach

600,000 SNP

3000 SNP

3000 SNP

Validation on phenotypic breeding

Pan genomic approach

Genomic estimated br din

Genomic estimated br din

Validation on phenotypic breedingbreeding

valuesbreeding

values

MARS & GS

breeding values

breeding values

MARS & GS