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