Genomics Resources 4 Crop Improvement,Ongoing Molecular Projects,Cloned and characterized virus R genes in plants,Sequence Editing and Analysis

1. Comparative Genomics for Marker Development
in Cassava
Melaku Gedil
IITA R4D Week
November 23, 2009
Ibadan, Nigeria
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org

5. Genomics Resources 4 Crop Improvement
Genome sequence
Bioinformatics Functional
Assembly/Annotation Genomics
Data analysis &
mining Proteomics
Gene/Marker Discovery
Assay/Validation
Application
MAB TRANSGENICS

6. Ongoing Molecular Projects
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
Melaku Gedil

7. Cloned and characterized virus R genes in plants
Resistance Cloning
Gene Host species Virus AVR Receptor structure
mechanisms method
Helicase
Transposon
N N. tabacum TMV domain of HR tagging
TIR-NBS-LRR
replicase
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
Systemic
RTM1 A. thaliana TEV nd Positional cloning Jacalin like seq
movement
Systemic
RTM2 A. thaliana TEV nd Positional cloning Jacalin like seq
movement
RCY1 A. thaliana CMV CP HR Positional cloning CC-NBS-LRR
Transposon
Tm22 S. lycopersicum ToMV MP HR tagging
CC-NBS-LRR
Replication
Approximation by
Pvr21/22 C. annuum PVY VPg cell-cell homology
eIF4E
movement,
Replication Approximation by
Mo11/2 L. sativa LMV nd eIF4E
Tolerance homology,
PSbM Approximation by

8. Predicted domain of R genes

9. Methods
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org

10. TIR NBS LRR
CC NBS LRR
TIR NBS LRR
CC NBS LRR
PCR-Primer
(Degenerate)
Cloning/Sequencing
Sequence Analysis
Markers

11. 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

12. Templates
• DNA • RNA
• … • cDNA
• PCR • PCR
• Cloning • Cloning
• Colony PCR • Colony PCR
• Purify • Purify
• Sequence • Sequence
* Surveys genomic *Surveys expressed
DNA genes

13. PCR Amplification with degenerate primers
Amplification with degenerate primers 50F-470RL was considered for
derived from At-NBS-LRR further analysis. Four DNA
Three forward and 7 reverse primers templates (TME3, TME7, and
(a total of 21 pairs) were tested TME117-a, TME-117-b) were
on the TME3 clone (Fig. x). amplified (Fig. xx). Amplicon
Different primer pairs yielded
size ranged from 200 – 900
different pattern of banding with
fragment sizes ranging from 500- bp.
1500 bp.

14. Cloning
TA-cloning, Qiagen
Purification

15. Sequencing
In-house on ABI 3130
Beca - Nairobi
Iowa State University

16. Sequence Analysis
Sequences edited
Sequences assembled
Sequence Clustered, identity matrix,
Similarity search in Genbank – BLAST
STS primers for resequencing
Marker development for MAS

17. Sequence Editing and Analysis - CodonCode

18. Sequence Editing and Analysis - BioEdit

19. Similarity search

21. BLAST

23. Mt_gd-tripa_582170 483 65 Manihot esculenta RCa3
AY187293.1 pseudogene, partial
sequence
Mt_gd-tripa_582172 483 67 Manihot esculenta RCa3
AY187293.1 pseudogene, partial
sequence
Mt_gd-tripa_582149 689 1 Ricinus communis conserved
XM_002521367.1 hypothetical protein,
mRNA
Mt_gd-tripa_582165 483 69 Manihot esculenta RCa3
AY187293.1 pseudogene, partial
sequence
Mt_gd-tripa_582205 482 72 Manihot esculenta RCa3
AY187293.1 pseudogene, partial
sequence
Mt_gd-tripa_594909 481 70 Manihot esculenta RCa3
AY187293.1 pseudogene, partial
sequence
Mt_gd-tripa_582206 471 45 Ricinus communis Disease
XM_002521743.1 resistance protein RFL1,
putative, mRNA
Mt_gd-tripa_582216 482 64 Manihot esculenta RCa3
AY187293.1 pseudogene, partial
sequence

24. Cassava Genome Database

25. Castor bean Genome Database

28. Clustering

29. Species Total sequences NBS-LRR
matching
Manihot esculenta - 25 10
genomic
Manihot esculenta- 85 41
cDNA
M. glaziovii 44 8
M. tripartita 96 8
M. epruinosa 140 40
M. brachyandra 16 3
Ricinus communis 51 45

30. Mt_gd-tripa_582216
Castor bean Mep_gDNA_580003
Mep_gDNA_580008
Mb_gDNA_580104
Me_cd-tme117-594825
Me_cd-tme117-594881
Me_cd-tme117-594850
Me_cd-tme117-594843
Mt_gd-tripa_594909
Cassava-genomic Me_cd-tme117-594872
Me_cd-tme117-594830
Me_cd-tme117-594837
Me_cd-tme117-594874
Me_cd-tme117-594855
Me_cd-tme117-594834
Me_cd-tme117-594828
Mb_gDNA_580105
Cassava-cDNA Me_cd-tme117-594864
Me_cd-tme117-586284
Mep_gDNA_566535
Mep_gDNA_566531
Mep_gDNA_566508
Mep_gDNA_566547
Mep_gDNA_566512
Mep_gDNA_566518
M.glaziovii Mep_gDNA_566522
Mep_gDNA_566523
Mt_gd-tripa_582205
Mep_gDNA_580048
Mep_gDNA_580055
Mep_gDNA_580042
Me_cd-tme117-586278
M.epruinosa Mep_gDNA_566505
Rc_gDNA_596848
Rc_gDNA_596835
Rc_gDNA_596858
Rc_gDNA_596819
Mep_gDNA_580097
Me_cd-tme117-594859
Mep_gDNA_580093
M.tripartita Me_cd-tme117-594865
Me_cd-tme117-594833
Mep_gDNA_580005
Mep_gDNA_566555
Mep_gDNA_580025
Mep_gDNA_580024
Me_cd-tme117-586272
Rc_gDNA_596823
M.brachyandra Mg_gDNA_595065
Rc_gDNA_596861
Rc_gDNA_596834
Rc_gDNA_596854
Rc_gDNA_596862
Rc_gDNA_596864
Rc_gDNA_596816
Rc_gDNA_596833
Rc_gDNA_596859
Rc_gDNA_596836
Mg_gDNA_595049
Rc_gDNA_596812
Rc_gDNA_596851
Rc_gDNA_596830
Rc_gDNA_596832
Rc_gDNA_596817
Rc_gDNA_596837
Rc_gDNA_596828
Rc_gDNA_596831
Rc_gDNA_596827
Rc_gDNA_596850
Rc_gDNA_596824
Rc_gDNA_596853
Rc_gDNA_596843
Rc_gDNA_596845
Rc_gDNA_596839
Rc_gDNA_596826
Rc_gDNA_596857
Me_gDNA_old-rga-C6
Me_gDNA_old-rga-C4
Me_gDNA_old-rga-C14
Mep_gDNA_566525
Mep_gDNA_580037
Mg_gDNA_old-rga-2
Rc_gDNA-old-rga
Rc_gDNA_596815
Me_gDNA_old-rga-C7
Rc_gDNA_596847
Mep_gDNA_580010
Me_gD-tme6_586320
Me_cd-tme117-594844
Me_cd-tme117-594835
Me_cd-tme117-594818
Mb_gDNA_580121
Me_cd-tme117-586270
Me_cd-tme117-586265
Me_cd-tme117-594868
Mep_gDNA_580088
Mep_gDNA_580017
Mep_gDNA_580034
Me_cd-tme117-586267
Me_cd-tme117-594838
Mep_gDNA_580054
Me_cd-tme117-594846
Rc_gDNA_596868
Me_cd-tme117-586297
Me_cd-tme117-594849
Me_cd-tme117-594845
Me_cd-tme117-586296
Rc_gDNA_596821
Me_gD-tme6_586337
Me_gD-tme6_586330
Me_cd-tme117-586266
Me_cd-tme117-594877
Me_cd-tme117-594847
Me_cd-tme117-594822
Mep_gDNA_580102
Me_cd-tme117-594873
Me_cd-tme117-594839
Mep_gDNA_566541
Mep_gDNA_566507
Mep_gDNA_566529
Mep_gDNA_566516
Mt_gd-tripa_582165
Mt_gd-tripa_582172
Mt_gd-tripa_582170
Mep_gDNA_580086
Rc_gDNA_596844
Rc_gDNA_596818
Rc_gDNA_596846
Rc_gDNA_596813
Rc_gDNA_596865
Mg_gDNA_595034
Mg_gDNA_595048
Mg_gDNA_595026
Mep_gDNA_566548
Me_gD-tme6_586324
Mep_gDNA_566527
Mep_gDNA_566520
Mep_gDNA_580013
Me_cd-tme117-594875
Me_cd-tme117-586274
Mep_gDNA_580029
Me_cd-tme117-594876
Me_gD-tme6_586323
Me_cd-tme117-586262
Mt_gd-tripa_582224
Mep_gDNA_580109
Mt_gd-tripa_582206
Mep_gDNA_580038
Mg_gDNA_595059
Mg_gDNA_595058
Me_gDNA_old-rga-C15
Rc_gDNA_596838
Rc_gDNA_596822
Rc_gDNA_596869
Rc_gDNA_596855

31. Sequence divergence
Total Cluster Mean Minimum Maximum
Cassava- 10 6 181 5 325
genomic
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

32. Between species distance based on nucleotide differences
Rc Mb Me- Me- Mep Mt
cDNA geno
castor
215
brachyandr
214 170
cas-cDNA
205 221 194
cas-geno
222 193 201 220
epruinosa
223 192 199 219 224
tripartita
174 192 184 179 193 195
glaziovii

33. SNP Identification

34. Identification of nucleotide polymorphism
C6
C4
Clone C4 and C6 primer position
Position 115 Position 153 Position 297
G vs C/G het C vs T A vs G

36. Work in Progress
(WIP)
and
Applications

37. 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

38. 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)

39. 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)

40. 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

41. Key issues in implementation of MAB
1. 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.
Melaku Gedil

42. Thank you
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org