Utilization of ESTs from Cassava: Progress on SSR marker development and a Oligo DNA Microarray I Ingelbrecht Central Biotechnology Laboratory IITA, Ibadan, Nigeria Global Cassava Partnership, First Scientific Meeting Ghent, Belgium 22 July 2008

USDA, Fargo, USA J Anderson Centre for Biotechnology, Turku, Finland S Rudd KU, Copenhagen, Denmark S Bak, K Jorgensen, J Gorodkin, B Moller DSMZ, Braunschweig, Germany S Winter, M Koerbler IITA, Ibadan, Nigeria I Ingelbrecht, A Raji, Y Lokko, M Gedil, A Dixon Partners

Cassava Resilient to adverse growth conditions (soil, drought) Flexibility in harvesting time Adaptable to range of agroecologies Low maintenance High yield potential (80 ton/ha) Highly heterozygous Vegetatively propagated Low fertility Pest/disease susceptibility, PPD, other + -

Cassava DNA Microarray a tool for gene discovery and transcriptome analysis Applications: 1.Understanding function of genes/alleles/gene networks 2.Understanding allelic differences between gene families/varieties 3.Diagnostics Essential tool for cassava genome sequencing effort trait improvement through genetic transformation reverse genetics (genotype phenotype)

ESTs Inexpensive to produce Input for (oligo) DNA microarray Source of molecular markers (COS, SNP, SSR)

Target traits drought response plant-virus interactions cyanogenesis other

Microarray design Gene Expression Analysis Screen for markers (SSR, SNP, COS) Mol genetic applications Approach Develop and 5’sequencing of cDNA libraries Combine in house ESTs with public cassava sequences Clustering and assembly of a unigene set Functional annotation of the unigene set (GO)

- cDNA libraries from control and water stressed tissues - leaf, root and stem tissues sampled - 18,166 ESTs sequenced (5’end) - Clustering and assembly of 8,577 unigene set EST development

- Annotation:

Some outcomes: - Study suggested involvement of class of dehydrins and thus also for a prominent role of osmotic adjustment in drought response in cassava - Gene families with multiple alleles is frequent occurrence in cassava: allele discovery in addition to gene discovery!

Number of (EST)-SSR for cassava are limited; typically many 1000ds available for non orphan crops 646 putative EST-SSR identified in silico from EST unigene dataset Validation: PCR amplification optimized Screened for polymorphism using diversity panels analyzed on SFR agarose, PAGE and ABI3100 SSR marker development

Total number of EST sequences investigated: 18,166 Total number of unique SSR loci appropriate for primer modeling: 646 (3.3%) Number of candidate SSR investigated : 96 PCR successful: 91 (95%) Failed PCR: 5 (5%) Fragments > 500 bp: 4 Size monomorphic within cassava panel: 46 (53%) Size polymorphic within cassava panel: 41 (47%) PCR products with expected sizes: 74 (81%) Amplification of introns: 17 (19%) Number of unigenes used for in silico identification of SSRs: 8,577 Workflow

192 SSRs out of 646 validated; so far ~80 new SSR markers Diversity study using EST-SSR confirms known genetic relationships Outcomes See poster C16. Raji et al.

- oligo DNA microarray (Agilent platform) - Workflow: ? Microarray selection Probe Selection Microarray order Sample preparation Hybrid- ization Feature extraction Informatics Microarray scanning Biological question Design Protocol Data Cassava DNA microarray

~ 40,000 cassava sequences assembled, clustered: 18,177 in house ESTs 5,000 ESTs from root specific library remainder from public databases (EST, genomic, etc) plus: ACMV and CBSV ORFs Km ORF 1. Design and probe selection Unigene set established, orientation determined Total probes; 13,865; corresponds to % of cassava transcriptome

- Array architecture uploaded (eArray) - 8-array format 2. Microarray selection

3. Hybridization and scanning

GENOTYPECHARACTERISTICS TME 3 TME 117 Landrace, CMD resistant, parent of mapping population Landrace, source of majority of ESTs TMS 96/0160IITA breeding line, adopted in DR Congo, CBSD suscep. TMS 30572IITA breeding line, widely adopted in SSA, CMD tol. TMS 96/1089AIITA breeding line, resistant to CMD & CBSD* KibahaTanzanian landrace, susceptible to CBSD Albert Tanzanian cultivar, susceptible to CBSD A.Diversity study: gene expression analysis of 7 different cassava genotypes Cassava Transcriptome Analysis

B. Greenhouse versus in vitro grown plant (TMS 96/0160) C. Diploid versus tetraploid cassava (TMS 30572) D. Healthy versus virus infected plant: ACMV and CBSV using TME 117 and TMS 96/0160

Genotype vs TC: dot plot: fold change vs adjusted P value

TC GH Clustering plot: genotypes in GH versus TC

Tetraploid cassava vs diploid

Virus infected vs healthy

Description: TMS 96/160_ohneS - TMS96/0160 FDR=0.05; |Contrast|>=2 Clone IDGene Name Fold change gb_CL1576Contig1.1.KVL45FFC7CB gb_CBSV_6K BM gb_CL1046Contig1.1.KVL45FFC7CB gb_CL198Contig2.1.KVL45FFC7CB B CK gb_CL1734Contig1.1.KVL45FFC7CB DV gb_CL1351Contig1.1.KVL45FFC7CB CK BI gb_CL1647Contig1.1.KVL45FFC7CB gb_CBSV_CP4.9

Cassava Transcriptome Analysis - Summary 1.Cassava specific oligo DNA microarray with 13,865 probes developed 2.Microarray passed all QC, hybridization and detection limit is as expected 3.Some preliminary findings: - Varietal differences fewer than difference between growth conditions -Genome duplication (tetraploid) from same genotype generates more diversity than differences between diverse African genotypes -Sensitivity comparable or exceeds that of PCR: diagnostics tool

Thank you!