harnessing interdisciplinary approaches for germplasm development
DESCRIPTION
Tools to accelerate efficiency of germplasm improvement,how these tools support breedersTRANSCRIPT
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
“IITA - the appliance of science”
Harnessing interdisciplinary approaches for
germplasm development
Sarah Hearne
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Development of varieties that are:
High yield potential
High yielding under farmer
conditions
Pest and disease resistant
Resistant to abiotic stress
Effective use of nutrients
Market preferred characteristics
Nutritious
Desired duration
The bottom line
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Breeding
Agronomy
IPM
Pathology
Weed science
Entomology
Biometrics
Bioinformatics
Nematology
Physiology
Biotechnology
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Tools to
accelerate /
better target /
improve
efficiency of
germplasm
improvement
Molecular tools -markers
Statistical methodologies
Data management, analysis and
decision support tools
Phenotyping
Methodologies
Benchmark sites
Breeding and selection
methodologies
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
How these tools
support
breeders?
Rapid targeted selection of segregants
Molecular breeding
Rapid integrated data analysis
Stress interactions / host-pest interactions
Target breeding for specific stress
complexes / pest genotypes
Selection of parents
Heterotic grouping
Diversity assessment – allelic / general
Predictive breeding
Variety production and seed systems
DUS (distinct, uniform, stable)& VCU (value for cultivation and use)
Seed purity
Impact assessment
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Three years-
outputs and
outcomes
Maize molecular breeding
projects, methodologies
applications
Plans for the future
Maize
Cowpea
Cassava
Musa
Striga
Some highlights
Maize genetic diversity
global diversity
Striga
distribution, diversity
pathogenicity
Resource development and use
cowpea, cassava and musa
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
DTMA
Interdisciplinary team with many talents working
across centers!
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
DTMA
Polygenic with high degree of epistasis
GxE issues
Phenotyping complexity – precision issues
Can’t apply markers across populations for MAS
Drought
Multiple interconnected themes within the initiative
Genomics
Breeding
Seed systems
Impact assessment
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Phenotyping Increase throughput
Enhance precision
Benchmark sites with precision irrigation
-Nigeria, Kenya, Zimbabwe, Zambia, Mexico
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Phenotyping
New tools
implemented
Collaboration with
breeders and
physiologists
Biomass/ senescence
Spectroradiometry – NDVI – rapid biomass
assessment
TranspirationLeaf / canopy temperature
NIRS & ash contentWater use
efficiency /
effectiveness
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Molecular
breeding
DTMA- largest public sector molecular breeding
project for maize in the world
Monitor and coordinate phenotyping, genotyping,
data analysis and breeding turnaround across
projects
Work with physiologists on phenotyping methods
and site characterization
Work with molecular team on genotyping
technologies and bottlenecks
Work with biometricians on issues of pop size
and marker number and on simulations of new
approaches using real data
Work with bioinformaticians on issues of data
handling and processing
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Molecular
breeding
What strategy to use for molecular
breeding?
What markers to use?
How to do the genotyping?
What traits to look at using markers?:
Make adapted materials more
drought tolerant
Make drought tolerant materials
more disease tolerant
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Molecular
breeding
What strategy to use for molecular
breeding?
What markers to use?
How to do the genotyping?
What traits to look at using markers?:
Classic QTL
ABQTL
MARS
GWS
For breeding - SNP
Both drought and disease
-list of diseases and populations
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Molecular
breeding
Molecular breeding training course 2008
DTMA scientists
NARS and private sector partners
Side meetings
Breeding strategy?
Evaluated our own QTL data and looked at
necessary population sizes and marker
numbers – NO classic QTL
Private sector present advising
ABQTL and MARS breeding strategies
Defined a template for population planning
and molecular breeding implementation
Worked with breeders to define possible
populations – 35 potentials
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Select
parents
Make
populationsPhenotype test cross
populations
in 3 or more
multilocation trialsGenotype parents
Using ~1500 SNP markers
Find marker trait
associations for
drought tolerance
Recombine the best materials
using marker data to stack or
pyramid favorable markers
for two or more cycles
(no phenotyping)
Isolate new lines
and phenotype
under drought
Use lines to
create new
drought tolerant
varieties
Marker assisted recurrent selection: MARS
-new lines
Genotype populations
Using 200-300 polymorphic
SNP markers
Seed based DNA extraction
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Y1
Y2
Y3
mid p
Sel C0
Breeder lines
MARS C2S1 lines
If you have 20 regions under selection, freq of optimum
genotype goes from 1 per trillion in cycle 0 to 1 in 5 in
cycle 3
Molecular
breeding
Power of MARS
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Select
parents
Make
populationsPhenotype test cross
populations
in 3 or more
multilocation trialsGenotype parents
Using ~1500 SNP markers
Find marker trait
associations for
drought tolerance
Recombine the best materials
using marker data to stack or
pyramid favorable markers
for two or more cycles
(no phenotyping)
Isolate new lines
and phenotype
under drought
Use lines to
create new
drought tolerant
varieties
Marker assisted recurrent selection: MARS
-new lines
Genotype populations
Using 200-300 polymorphic
SNP markers
Seed based DNA extraction
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Molecular
breeding
Annual meeting 2008
Scheduling of phenotyping and genotyping
and cost of genotyping
Some seasons ~6500 individuals to
genotype
3 weeks to extract DNA and genotype
Genotyping option
Illumina golden gate – 1536 SNP markers
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Molecular
breeding
Illumina golden gate – 1536 SNP markers
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Molecular
breeding
Workshop Feb 2009
SNPlex no longer an option
BeadXpress
Illumina system 384 markers
Single plex assays – KBiosciences
DTMA – buy BeadXpress
Analysis of throughput and cost
Discussion with private sector
Visit to KBioscience
Presented data at 2009 DTMA annual
meeting in Zim - use KBiosciences
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Molecular
breeding
DTMA – 20 molecular breeding projects. 18
MARS/GWS (explained in next slide), 2 ABQTL (line
conversion) – WA had 5 MARS/GWS populations
Two genotyping platforms-
Illumina golden gate-
One diverse 1536 illumina OPA, 1330 good
SNP
KASPar – single plex primer extension based
assays designed for 1111 SNP
Will be using new 60k infinium assay in 2010
Genotyping by sequencing under evaluation
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Building on MARS
populations
Some of the hit list:-
MSV
Striga hermonthica
GLS
Nematodes
We have high
breeding value
segregating
populations
We have
genotype data
What other high
value traits we
can phenotype
for…
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Molecular
breedingTolerance to MSV
Breeder, Virologist, Biometrician
and Mol Geneticist
Abebe + – populations, field phenotyping
Lava – Screenhouse phenotyping and indexing,
field phenotyping
Sarah – Candidate marker identification,
population genotyping, tracking, data curation
Sarah and Jose – Data analysis and marker
identification
Contribute knowledge and markers for simple
traits to incorporate into breeding work
Guy – Data handling and manipulation
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Molecular
breedingResistance and tolerance to Striga
Breeders, Physiologist, Biometrician
and Mol Geneticist
Abebe and Baffour – populations, field
phenotyping
Sarah – Screenhouse phenotyping
Sarah – Candidate marker identification,
population genotyping, tracking, data
curation
Sarah and Jose – Data analysis and
marker identification
Contribute knowledge and markers for
identified traits to incorporate into breeding
work
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
GLS Field phenotyping issues - Reliability
Not a simple trait – or a single species (Lava)
Development of improved phenotyping
e.g. HTP detached leaf assay for GLS
evaluations with pathologist - Ranajit
Enable controlled infestation with different
genotypes of pathogen (pathotypes / species
with agroecological niches)
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
CML 488 maize inbred line
Un-infected Meloidogyne infested
Black
Lesions
Reduced root
mass
“Root galling”
Nematodes
Pratylenchus
Meloidogyne
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Nematodes
Interaction
between drought
and nematode –
flowering date
and ASI
Nematode and drought interaction
Breeders, Nematologist, Physiologist,
Biometrician and Mol Geneticist
Abebe and Baffour – lines – parents of MARS
populations
Danny – Screenhouse phenotyping-
nematodes
Sarah and Jorge – Data analysis
Contribute knowledge and markers for
identified traits to incorporate into breeding
work
Sarah – Screenhouse phenotyping-
drought
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
What other
applications to
support
breeders?
Selection of parents
Heterotic grouping
Diversity assessment – allelic / general
Predictive breeding
Variety production and seed systems
DUS (distinct, uniform, stable)& VCU (value for cultivation and use)
Seed purity
Impact assessment
Genotyping of potential parents
Testing the quality of outgrower produced
hybrid seed in Zimbabwe
Genotyping IITA released maize lines
using 60k SNP chip to facilitate tracking of
IITA germplasm for impact assessment
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Numbers,
numbers
everywhere…
Molecular breeding is a balancing
act
$How do we balance competing
demands for funds and optimize
genetic gain?
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Analysis of pop
size and marker
density on QTL
detection
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PVE=1% PVE=2% PVE=3% PVE=4% PVE=5% PVE=10% PVE=20% PVE=30%
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Population size
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MD= 5 cM
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Population size
PVE=1%
PVE=2%
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MD= 10 cM
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MD= 20 cM
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0.20
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Stan
dar
d e
rro
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f e
stim
ate
d e
ffe
ct
Population size
PVE=1%
PVE=2%
PVE=3%
PVE=4%
PVE=5%
PVE=10%
PVE=20%
PVE=30%
MD= 20 cM
0.100.150.200.250.300.350.400.450.500.550.600.650.70
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Esti
mat
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eff
ect
Population size
MD= 40 cM
0.00
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0.20
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0.30
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05
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58
06
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Stan
dar
d e
rro
r o
f e
stim
ate
d e
ffe
ct
Population size
PVE=1%
PVE=2%
PVE=3%
PVE=4%
PVE=5%
PVE=10%
PVE=20%
PVE=30%
MD= 40 cM
Huihui Li, Sarah Hearne, Yunbi Xu, Marianne Bänziger, Zhonglai Li, & Jiankang Wang, Heredity in press
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
MAS versus
GWSMAS
Only track significant markers
GWS
All markers have value
Which is optimal?
How many cycles of selection?
Best estimate?
BLUE (GxE), BLUP (no GxE but
marker by marker)
Selection index?
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
MAS versus
GWSSimulations
Cerón-Rojas, J. J., Crossa, J., Alvarado, G., Burgueño, J., Wang, J., Atlin, G., Bänziger, M., Hearne, S. J. & Davenport, G., F., Sub Crop Sci
Fig.1
-5
0
5
10
15
20
25
30
325
330
335
340
345
350
355
360
0 1 2 3 4 5 6Cycle of selection
YB 400MM BLUE YB 40MM BLUE
YA 400MM BLUE YB 400MM BLUP
YB 40MM BLUP YA 400MM BLUP
EPP
GY
ASI
GY
(gra
ms
pe
rp
lot)
EP
P (
nu
mb
er)
AS
I (d
ays)
Fig. 1a
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
MAS versus
GWSGWS better than MAS in all situations
400 markers better than 40
BLUP better than BLUE when looking at
well watered environments
BLUE better than BLUP when looking at
drought stressed environments
Genetic gain for grain yield up to cycle six
Fig.1
-5
0
5
10
15
20
25
30
325
330
335
340
345
350
355
360
0 1 2 3 4 5 6Cycle of selection
YB 400MM BLUE YB 40MM BLUE
YA 400MM BLUE YB 400MM BLUP
YB 40MM BLUP YA 400MM BLUP
EPP
GY
ASI
GY
(gra
ms
pe
rp
lot)
EP
P (
nu
mb
er)
AS
I (d
ays)
Fig. 1a
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Three years-
outputs and
outcomes
Maize molecular breeding
projects, methodologies
applications
Plans for the future
Maize
Cowpea
Cassava
Musa
Striga
Some highlights
Maize genetic diversity
global diversity
Striga
distribution, diversity
pathogenicity
Resource development and use
cowpea, cassava and musa
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Maize diversity Maize from Americas, Africa, Asia, Europe
and teosintes
Group of collaborators from ten different
institutes
Population geneticists, breeders, genebank
Curators, molecular geneticists, GIS
specialists
How has maize migrated across the
globe?
What is the pattern of global diversity?
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Maize diversity Maize from Americas, Africa, Asia, Europe
and teosintes
Group of collaborators from ten different
institutes
Population geneticists, breeders, genebank
Curators, molecular geneticists, GIS
specialists
How has maize migrated across the
globe?
What is the pattern of global diversity?
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Maize diversity
Africa
Globally landraces are in general tropical
in origin
Twelve main landrace clusters exist in the
world
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Maize diversity
Africa
Western Sub-Saharan Africa -tropical
maize
Diversity data strongly supports hypothesis
of introduction via Portuguese slave
West Africa very distinct - Sao Tome and
Cape Verde – groups originating from both
can be defined:- rapid differentiation of
original gene pools- environment and
human uses
East Africa – data suggests direct diffusion
of US maize after World War II – potential
replacement of tropical landraces
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Mir, C., Warburton, M.L., Taba, S., Bedoya, C., Franco, J.,Zhang, S., Xie, C., Prasanna, B.M., Hearne, S., Muthamia, Z., Yunus, M., Cuong,
B.M., and Charcosse. In Prep
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Three years-
outputs and
outcomes
Maize molecular breeding
projects, methodologies
applications
Plans for the future
Maize
Cowpea
Cassava
Musa
Striga
Some highlights
Maize genetic diversity
global diversity
Striga
distribution, diversity
pathogenicity
Resource development and use
cowpea, cassava and musa
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Striga
-from the parasite
perspective
Striga is one of the most significant biotic
constraints to maize production in SSA
Hearne, Pest Mgmt Sci, 65, 2009
Striga sp., while often lumped together have
distinct and different breeding systems. S.
asiatica is inbreeding species while S.
hermonthica is self incompatible and is highly
outbreeding in nature.
These differences have implications on diversity
within and between parasite populations per se
and also has implications control technology
efficacy from region to region – be that host
resistance and tolerance to parasite or herbicide
tolerant germplasm.
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Striga
-from the parasite
perspective
Surveying Striga endemic areas in Nigeria, DRC
and Kenya. In 2011 survey Tanzania.
Taking basic farmer perception data and
collecting Striga leaf and seed samples from
individuals and populations to assess parasite
diversity.
Using location data to prepare and atlas of Striga
distribution working with GIS
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Striga
-from the parasite
perspective
Pilot project completed assessing diversity of S.
hermonthica in Kenya using SSR markers
Within population variation contributed to some
95.89% of the total variance with only 4.11%
being due to among populations. High even for
an allogamous species.
The total heterozygosity observed across all
markers and populations was very high at
0.72054, within population heterozygosities
ranged from 0.4829 to 0.73988.
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Striga
-from the parasite
perspective
The level of heterozygosity may reflect the
obligate out-breeding nature of the species and
the level of population diversity needed to ensure
fitness
Marker availability for Striga is limited – 8
informative SSR markers
Link with evolutionary biologist, Claude de
Pamphilis at Penn State on NSF initiative to
sequence parasitic plant ESTs
Striga tissue collected from diverse sources in
Nigeria and sent to US for RNA isolation and
sequencing using 454 Titanium sequencing
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Striga
-from the parasite
perspective
Assemblies being constructed – mine for SNP
and polymorphic SSR to conduct further studies
Claude using data to complete the Striga
chloroplast genome and construct a mitochondrial
genome – understand evolution of parasitism
across parasitic plants – Striga, Alectra,
Orobanche
Use new markers to evaluate within and between
population S. hermonthica diversity. SSR in
Ibadan and SNP at Kbiosciences.
Data analysis, Sarah, Jorge and GIS
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Striga Pathogenicity work – implication of
diversity?
NSF proposal to sequence Striga genome – great
resource to start to understand parasitism and
pathogenicity at the genomic level
First need a system for maintaining the discrete
Striga populations collected in the field
Working with Mike Timko, U . Virginia to look at
methodologies for inter population mating to
maintain population diversity. Working on
Nigerian isolates of Striga
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Striga-host
interactions
Field screening $, uses only one or two populations of
Striga and does not provide an understanding of the
mechanisms of tolerance / resistance seen
Implemented medium and high throughput lab and
screenhouse based phenotyping protocols to assess
the Striga-host interaction at biochemical, physiological
and morphological levels.
Assess resistance at germination, haustorial initiation,
attachment and post attachment stages
Enable improved selection of favorable recombinants
and facilitate studies of gene action
Integrate these assays with current molecular breeding
work to identify molecular markers for key traits of
interest
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Three years-
outputs and
outcomes
Maize molecular breeding
projects, methodologies
applications
Plans for the future
Maize
Cowpea
Cassava
Musa
Striga
Some highlights
Maize genetic diversity
global diversity
Striga
distribution, diversity
pathogenicity
Resource development and use
cowpea, cassava and musa
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Marker
development
JCVI, ILRI, UC
Riverside
Program of EST sequencing projects – cowpea,
cassava and musa
Generated:-
Cowpea-
41949 sequences
3367 putative SNP
1805 putative SSR, 916 di- and trinucleotide repeats
Cassava
5046 sequences (41173)
2699 putative SSR
2486 putative SNP
Musa
5494 sequences (52907)
1937 putative SSR
28815 putative SNP
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Marker development
Consolidation and
utilization of EST
HarvEST cowpea database - 17 libraries -
Two IITA libraries, 12 UCR libraries.
http://harvest.ucr.edu/
HarvEST cassava database - 17 libraries -
Two IITA libraries, 12 UCR libraries.
HarvEST musa database - 17 libraries - Two
IITA libraries, 12 UCR libraries.
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Marker
development and
use
Cowpea
Developed Illumina Goldengate SNP assay
IITA Reference collection, breeders lines, bi-
parental populations
IITA reference collection encompassed genetic
diversity assessed using SNP
Ref set and additional germplasm extensively
phenotyped – tool for allele mining
Development of consensus genetic map of
cowpea, and synteny with soybean and Medicago
Provides genomic framework for identification of
marker trait association, map-based cloning,
selection of markers for assessment of genetic
diversity, association mapping
Muchero, W., Diop, N., Bhat, P., Fenton, R., Pottorff, M., Hearne, S., Ndiaga, C., Fatokun, C., Ehlers, J.,
Roberts, P., Close,T. 2009. PNAS
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Marker
development
Consolidation and
utilization of EST
410 EST derived SSR - Xu et al 2009, Molecular
breeding
Mike Timko’s group compared EST with GSS
data
Determine the gene discovery rate Timko et al
2008 BMC Genomics
Predict open reading frames for the genes (oligos)
used to create a cowpea microarray – 385k.
Microarray used to study cowpea-Striga
interaction- Li et al 2009 Pest Mgmt Sci
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Three years-
outputs and
outcomes
Maize molecular breeding
projects, methodologies
applications
Plans for the future
Maize
Cowpea
Cassava
Musa
Striga
Some highlights
Maize genetic diversity
global diversity
Striga
distribution, diversity
pathogenicity
Resource development and use
cowpea, cassava and musa
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Building on good
foundations
Breeding
DTMA DTMA DTMA
TLI TLII
DTMA
Cassava
Musa Yam
Adapt and apply know how to achieve
rapid advances in other IITA crops
Genome sequence
ESTs
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Be brave and
look at
complexity
In farmers fields plant are subject to
multiple stresses
Start looking at the basis of stress
tolerance resistance. Then look at
complexes of commonly occurring
stresses
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
We have a bright future
Thank you