Genetic Mapping ofResistance to the Cassava Mosaic Disease CMD

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Genetic Mapping of Resistance to the Cassava
Mosaic Disease (CMD)
CBN V, St.Louis, November 7, 2001
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Outline

• The Cassava Mosaic Disease(CMD)
• Genetic mapping of CMD resistance genes
• Marker-assisted selection
• Positional Cloning of CMD2
• SAGE analysis of CMD resistance

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Cassava Mosaic Disease and its white fly vector
Constraints
Diseasesand Pests
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Importance of CMD

• Number one production constraint in Africa
• A potential risk in Latin America and Asia
• Complicates germplasm exchange

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Multiple Virus Type/Strains

• African Cassava Mosaic Virus
• East African Cassava Mosaic Virus
• Ugandan Variant (Recombinant virus)
• South African Cassava Mosaic Virus
• Indian Cassava Mosaic Virus

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Genome of the Cassava Mosaic Virus
V1
A
C1
2779bp
C1
V1
C2
C2
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Inter-Center CMD Collaboration

• Genetic Mapping of Resistance to the African
  Cassava Mosaic Disease (ACMD)
• Marker-assisted breeding of CMD resistance

CIAT IITA
RF Funding
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Why Map CMD Resistance?

• To select for CMD resistant genotypes in the
  absence of the pathogen in Latin America
• To certify resistant genotypes for distribution
  to farmers Africa
• Need to pyramid different sources of resistance

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Sources of CMD Resistance

• Currently deployed CMD resistance comes from M.
  glaziovii x M. esculenta 3rd backcross derivates
• A new source of resistance that show high levels
  of resistance has been identified in Nigerian
  land race.

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CMD Mapping Populations

• TMS30572 X CM7857 (Half-sib BC1)
• 240 genotypes
• TMS30572 X TME117 (F1) - 200 genotypes
• TMS30555 X TME3 (F1 ) -178 genotypes
• TMS 30572 XTME 4 (F1 ) 200 genotypes

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CMD Mapping Populations
.

• The mapping population were established in vitro
  for easy transfer between IITA and CIAT.

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Experimental Sites

• IITA humid forest Station, Onne, Eastern Nigeria
• Transitional rain forest, Ikenne, South West
  Nigeria
• NARO mid-altitude NARO station Namulonge, Ugandan

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Evaluation of CMD Resistance in the TMS30555 X
TME3 Mapping Population
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Distribution of CMD Resistance in the TMS30555 X
TME3 Mapping Population
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Distribution of CMD Resistance in the TMS30572 x
CM7857-4 (BC1) Mapping Population
50
40
30
20
10
0.9
1.4
1.9
2.4
BC1 Pop./Ikenne
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SSR Markers (Cassava MapPairs)

• 12 SSR markers (Chavariagga et. al
• 1998
• 174 SSR markers (Mba et. al. 2000)
• 134 SSR markers (Mba et. al. 2001)
• 158 SSR markers (Fregene et.al 2001.)
• NARO mid-altitude NARO station Namulonge, Ugandan

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Bulk Segregant Analysis (BSA) of CMD resistance
in TMS30555 XTME3
NS158
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Map Location of a CMD Resistance Gene in Cassava
Location of a dominant CMD resistance gene CMD2.
Markers flanking and co-segregating with the gene
explain over 70 of phenotypic variance
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Map Location of a Second CMD Resistance Gene in
Cassava
A single genome region controls 48 of phenotypic
variance for ACMD resistance and shows a
recessive gene action
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Marker-assisted Breeding of CMD Resistance

• Introduce CMD2 resistance gene into Latin
  American cassava gene pools by MAS
• Compare MAS and conventional breeding for CMD
  resistance in African gene pools
• The use of the CMD2 marker as a diagnostic to
  certify resistant genotypes for distribution to
  farmers

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Map-Based Cloning of CMD2
Evaluation of CMD resistance
Genetic Mapping SSR markers
Fine Mapping
Genetic Linkage Map
Contig mapping using Bacterial Artificial
Chromosomes (BACs) clones (CUGI)
Fine mapping With AFLP markers
DNA Sequence
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Serial Analysis of Gene Expression (SAGE) of CMD 2

• Identify genes that are the molecular basis for
  resistance conferred by CMD2
• Use Bulk of 40 CMD resistant and 40 susceptible
  genotypes from the TMS30555 X TME3 cross
• Generate and annotate all transcripts
  differentially expressed in the CMD resistant
  bulk

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Serial Analysis of Gene Expression (SAGE)

• Velculescu et. al. 1997. Characterization of
  Yeast transcriptomes. Cell 88, 243-251
• Zhang et. al. 1997. Gene expression profiles in
  normal and cancer cells. Science 276, 1268-1272
• Polyak et. al. 1997 A model for P53-induced
  Apoptosis. Nature 389 300-305.
• Matsumura et. al. 1999 Transcript profiling in
  rice (Oryza sativa L.) seedling using serial
  analysis of gene expression. Plant Journal
  20(6)719-726

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Biotinylated cDNA synthesis from mRNA of CMD
resistant and susceptible bulks
Linker ligation, digestion and ditag ligation
Large scale ditag PCR and elution
Sequencing and analysis of gene expression
Ditag concatermerization and library construction
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Serial Analysis of Gene Expression Differential
expression of genes in resistant and susceptible
bulks
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SAGE of CMD2

• Two mRNA populations isolated from a bulk of 40
  susceptible and 40 resistant F1 progeny
• Generation of SAGE libraries from the two mRNA
  samples
• Sequencing of 5733, and 7053, tags from the
  resistant and susceptible bulks respectively
• Identification of 128 tags differentially
  expressed at a probability level of 95 in the
  resistant bulk
• Tag anotation by 3 end sequencing of ESTs to
  identify differentially expressed genes - about
  5,000 ESTs

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Conserved Toll-like-Receptors (TLR) Signal
Cascades
Cassava (CMD resistance)
Drosophila
Human
Plants
?
Leucine rich repeat
Toll Domain
?
5 4 3 2 1 Mst 18w Toll
MyD88 Tube
?
N-gene Rx
IRAK1/2 Pelle
Kinase
?
dTRAF TRAF6
?
ECSIT ECSIT
?
MEKK1/IKK ?
?
Kinase
?
NF-?B/I- ?B Dorsal/Cactus
SAR/HR
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Acknowledgement

• CIAT
• Dr M.Fregene
• Dr C.Mba
• Dr Hernan Ceballos
• Edgar Barrera
• Dr Lee Calvert
• Dr Joe Tohme
• Dr William Roca

• IITA
• Dr Alfred Dixon
• Dr Bill Khizzah

• CUGI
• Dr Jeff Tomkins

• RF
• Dr G. Toeniessen
• Dr J. Lynam

• ILTAB
• Dr C. Fauquet