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Genomics Resources 4 Crop Improvement,Ongoing Molecular Projects,Cloned and characterized virus R genes in plants,Sequence Editing and Analysis
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International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Melaku Gedil
IITA R4D Week
November 23, 2009
Ibadan, Nigeria
Comparative Genomics for Marker Development
in Cassava
Genomics Resources 4 Crop Improvement
Genome sequence
Assembly/Annotation
Gene/Marker Discovery
Assay/Validation
Application
MAB TRANSGENICS
Functional
Genomics
Proteomics
Bioinformatics
Data analysis &
mining
Melaku Gedil
1. Markers for pyramiding CMD resistance genes,
2. Molecular breeding for CBSD resistance (Application
of advanced genomics tools such as high throughput
SNP genotyping )
3. Molecular marker for drought tolerance traits
4. Marker for pVAC - comparative genomics (HP+)
5. Resistance gene analogs (RGA) – comparative
genomics
Ongoing Molecular Projects
Cloned and characterized virus R genes in plants
Gene Host species Virus AVRResistance
mechanisms
Cloning
methodReceptor structure
N N. tabacum TMV
Helicase
domain of
replicase
HRTransposon
taggingTIR-NBS-LRR
Rx1 S. tuberosum PVX CP Replication Positional cloning CC-NBS-LRR
Rx2 S. tuberosum PVX CP Replication Positional cloning CC-NBS-LRR
Sw5 S. esculentum TSWV MP HR Positional cloning CC-NBS-LRR
HRT A. thaliana TCV CP HR Positional cloning LZ-NBS-LRR
RTM1 A. thaliana TEV nd Systemic
movementPositional cloning Jacalin like seq
RTM2 A. thaliana TEV ndSystemic
movementPositional cloning Jacalin like seq
RCY1 A. thaliana CMV CP HR Positional cloning CC-NBS-LRR
Tm22 S. lycopersicum ToMV MP HRTransposon
taggingCC-NBS-LRR
Pvr21/22 C. annuum PVY VPg
Replication
cell-cell
movement,
Approximation by
homologyeIF4E
Mo11/2 L. sativa LMV ndReplication
ToleranceApproximation by
homology, eIF4E
Sbm1 P. sativumPSbM
V nd Replication
Approximation by
homologyeIF4E
Predicted domain of R genes
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Methods
Cloning/Sequencing
Sequence Analysis
Markers
PCR-Primer
(Degenerate)
NBSCC LRR
TIR NBS
NBSCC
TIR NBS
LRR
LRR
LRR
DNA/RNA extraction
Species # Acc
M. esculenta 5
M .epruinosa 1
M. glaziovii 1
M. brachyandra 1
M. tripartita 1
Other Manihots 3
Ricinus communis 1
Templates
• DNA
• …
• PCR
• Cloning
• Colony PCR
• Purify
• Sequence
* Surveys genomic DNA
• RNA
• cDNA
• PCR
• Cloning
• Colony PCR
• Purify
• Sequence
*Surveys expressed genes
PCR Amplification with degenerate primers
Amplification with degenerate primers
derived from At-NBS-LRR
Three forward and 7 reverse primers
(a total of 21 pairs) were tested
on the TME3 clone (Fig. x).
Different primer pairs yielded
different pattern of banding with
fragment sizes ranging from 500-
1500 bp.
50F-470RL was considered for
further analysis. Four DNA
templates (TME3, TME7, and
TME117-a, TME-117-b) were
amplified (Fig. xx). Amplicon
size ranged from 200 – 900
bp.
Cloning
TA-cloning, Qiagen
Purification
Sequencing
In-house on ABI 3130
Beca - Nairobi
Iowa State University
Sequence Analysis
Sequences edited
Sequences assembled
Sequence Clustered, identity matrix,
Similarity search in Genbank – BLAST
STS primers for resequencing
Marker development for MAS
Sequence Editing and Analysis - CodonCode
Sequence Editing and Analysis - BioEdit
Similarity search
BLAST
Mt_gd-tripa_582170 483 65
AY187293.1
Manihot esculenta RCa3
pseudogene, partial
sequence
Mt_gd-tripa_582172 483 67
AY187293.1
Manihot esculenta RCa3
pseudogene, partial
sequence
Mt_gd-tripa_582149 689 1
XM_002521367.1
Ricinus communis conserved
hypothetical protein,
mRNA
Mt_gd-tripa_582165 483 69
AY187293.1
Manihot esculenta RCa3
pseudogene, partial
sequence
Mt_gd-tripa_582205 482 72
AY187293.1
Manihot esculenta RCa3
pseudogene, partial
sequence
Mt_gd-tripa_594909 481 70
AY187293.1
Manihot esculenta RCa3
pseudogene, partial
sequence
Mt_gd-tripa_582206 471 45
XM_002521743.1
Ricinus communis Disease
resistance protein RFL1,
putative, mRNA
Mt_gd-tripa_582216 482 64
AY187293.1
Manihot esculenta RCa3
pseudogene, partial
sequence
Cassava Genome Database
Castor bean Genome Database
Clustering
Species Total sequences NBS-LRR
matching
Manihot esculenta -
genomic
25 10
Manihot esculenta-
cDNA
85 41
M. glaziovii 44 8
M. tripartita 96 8
M. epruinosa 140 40
M. brachyandra 16 3
Ricinus communis 51 45
Rc_gDNA_596834
Rc_gDNA_596850
Rc_gDNA_596858
Rc_gDNA_596818
Rc_gDNA_596826
Rc_gDNA_596835
Rc_gDNA_596843
Rc_gDNA_596851
Rc_gDNA_596859
Rc_gDNA_596819
Rc_gDNA_596827
Rc_gDNA_596836
Rc_gDNA_596844
Rc_gDNA_596868
Rc_gDNA_596812
Rc_gDNA_596828Rc_gDNA_596837
Rc_gDNA_596845
Rc_gDNA_596853
Rc_gDNA_596861
Rc_gDNA_596869
Rc_gDNA_596813
Rc_gDNA_596821
Rc_gDNA_596838
Rc_gDNA_596846
Rc_gDNA_596854Rc_gDNA_596862
Rc_gDNA_596822
Rc_gDNA_596830
Rc_gDNA_596839
Rc_gDNA_596847
Rc_gDNA_596855
Rc_gDNA_596815
Rc_gDNA_596823
Rc_gDNA_596831
Rc_gDNA_596848
Rc_gDNA_596864Rc_gDNA_596816
Rc_gDNA_596824
Rc_gDNA_596832
Rc_gDNA_596857
Rc_gDNA_596865
Rc_gDNA_596817
Rc_gDNA_596833
Rc_gDNA-old-rga
Mb_gDNA_580104
Mb_gDNA_580105
Mb_gDNA_580121
Me_cd-tme117-586262
Me_cd-tme117-586265
Me_cd-tme117-586266
Me_cd-tme117-586267
Me_cd-tme117-586270
Me_cd-tme117-586272
Me_cd-tme117-586274
Me_cd-tme117-586278
Me_cd-tme117-586284
Me_cd-tme117-586296
Me_cd-tme117-586297
Me_cd-tme117-594818
Me_cd-tme117-594822
Me_cd-tme117-594825
Me_cd-tme117-594828
Me_cd-tme117-594830
Me_cd-tme117-594833
Me_cd-tme117-594834
Me_cd-tme117-594835
Me_cd-tme117-594837
Me_cd-tme117-594838
Me_cd-tme117-594839
Me_cd-tme117-594843
Me_cd-tme117-594844
Me_cd-tme117-594845
Me_cd-tme117-594846
Me_cd-tme117-594847
Me_cd-tme117-594849
Me_cd-tme117-594850
Me_cd-tme117-594855
Me_cd-tme117-594859
Me_cd-tme117-594864
Me_cd-tme117-594865
Me_cd-tme117-594868
Me_cd-tme117-594872
Me_cd-tme117-594873
Me_cd-tme117-594874
Me_cd-tme117-594875
Me_cd-tme117-594876
Me_cd-tme117-594877
Me_cd-tme117-594881
Me_gD-tme6_586330
Me_gD-tme6_586323
Me_gD-tme6_586324
Me_gD-tme6_586320
Me_gD-tme6_586337
Me_gDNA_old-rga-C4Me_gDNA_old-rga-C6
Me_gDNA_old-rga-C7
Me_gDNA_old-rga-C14
Me_gDNA_old-rga-C15
Mep_gDNA_580034
Mep_gDNA_580042
Mep_gDNA_566522
Mep_gDNA_580010
Mep_gDNA_566507
Mep_gDNA_566523
Mep_gDNA_566531
Mep_gDNA_566547
Mep_gDNA_566555
Mep_gDNA_580003
Mep_gDNA_566508
Mep_gDNA_566516
Mep_gDNA_566548
Mep_gDNA_580037Mep_gDNA_566525
Mep_gDNA_566541
Mep_gDNA_580093
Mep_gDNA_580005
Mep_gDNA_580109
Mep_gDNA_580013
Mep_gDNA_580029
Mep_gDNA_580038
Mep_gDNA_566518
Mep_gDNA_580054
Mep_gDNA_580086
Mep_gDNA_580102
Mep_gDNA_580055
Mep_gDNA_566527
Mep_gDNA_566535
Mep_gDNA_566512
Mep_gDNA_580048
Mep_gDNA_566520
Mep_gDNA_580088
Mep_gDNA_580008
Mep_gDNA_580024
Mep_gDNA_566505
Mep_gDNA_566529
Mep_gDNA_580097
Mep_gDNA_580017
Mep_gDNA_580025
Mt_gd-tripa_582170Mt_gd-tripa_582172Mt_gd-tripa_582165
Mt_gd-tripa_582205
Mt_gd-tripa_594909
Mt_gd-tripa_582206
Mt_gd-tripa_582216
Mt_gd-tripa_582224
Mg_gDNA_595026
Mg_gDNA_595034
Mg_gDNA_595058Mg_gDNA_595059
Mg_gDNA_595048
Mg_gDNA_595049
Mg_gDNA_595065
Mg_gDNA_old-rga-2
Castor bean
Cassava-genomic
Cassava-cDNA
M.glaziovii
M.epruinosa
M.tripartita
M.brachyandra
Sequence divergence
Total Cluster Mean Minimum Maximum
Cassava-
genomic
10 6 181 5 325
Cassava-cDNA 41 5 177 1 357
Glaziovii 8 5 146 61 231
Brachy 3 2 197 4 294
Epruinosa 40 8 222 1 326
Tripartita 8 4 236 1 328
Castor 45 16 184 1 336
Between species distance based on nucleotide differences
Rc Mb Me-
cDNA
Me-
geno
Mep Mt
castor
brachyandr 215
cas-cDNA 214 170
cas-geno 205 221 194
epruinosa 222 193 201 220
tripartita 223 192 199 219 224
glaziovii 174 192 184 179 193 195
SNP Identification
Identification of nucleotide polymorphism
Position 153
C vs T
Position 115
G vs C/G het
Position 297
A vs G
C6
C4
Clone C4 and C6 primer position
Work in Progress
(WIP)
and
Applications
Work in progress
• Sequence analysis and characterization
• Search on cassava and castor genome
• Re-sequencing primers from candidate
sequences (STS) for marker discovery
– in a panel of R and S cultivars
– BSA analysis of potential primers
– Develop PCR-based markers
* Allele Specific-PCR
* CAPS marker
Applications
1.Molecular markers (SNP, STS)
2.Gene discovery
3.Physical mapping (Qiu 2007 leaf rust R in wheat)
4.NBS profiling of genetic diversity – a modification
of AFLP (Mantovani 2006)
5.Host-pathogen interaction/pathways e.g. ATP-
binding or hydrolysis
6.Genome wide survey of Resistance genes
(Arabidopsis, Rice, poplar, Grape, papaya)
Advantages
1.Protocol is adaptable to other crops….e.g.
yam, cowpea
2.All resistance genes – insights to the
structure & organization
3.Starting material for comparative
genomics based data mining of R genes
4.Easy and cost-effective to generate data
5.Markers are gene-based (not random
association)
Melaku Gedil
Application of Markers in Breeding
1.Gene mining in genetic resources (germplasm
evaluation)
• Selection of parents (diversity analysis/heterotic
group)
• Cultivar identity (‘branding’), hybrid validation
2. Introgression: minimizes linkage drag, saves time
(e.g. AB-QTL)
3.Pyramiding – traits from multiple parents
4.New approaches such as
• Marker-assisted Recurrent selection (MARS)
• Genome-wide selection (GWS)
Melaku Gedil
Key issues in implementation of MAB1. Availability of genomic resources
2. Cost-effective genotyping systems
• Declining cost of genotyping and a choice of genotyping
technologies and markers
• Capital costs not necessary, (subcontract to service providers -
GCP)
3. Multienvironment phenotyping (GxE, epistasis)
• Genotyping no longer an issue
4. Accurate Marker-trait association methods (LD, QTL)
• Begin with less complex traits
• Advances in genomics (structural, functional) and other -omics,
and other disciplines, will elucidate the genetic mechanism of
complex traits of economic importance.
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Thank you
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