New tools for the characterization and improvement of cassava
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  • 1. New tools for the characterization and improvement of cassava Contract Review 15 April 2009 I Ingelbrecht IITA, Ibadan, Nigeria
  • 2. Outline 1. Work plan achievement 2. Quality of science 3. Communication 4. Capacity building 5. Resource mobilization 6. Project management 7. Other professional activities 8. Personal effectiveness
  • 3. Cassava Root crop; ranks 6th as source of carbohydrates globally (163 M ton/yr) Allopolyploid with disomic inheritance; 2n=36 C=700-800 Mbp Vegetatively propagated Grown in (sub)tropics of South Am., Asia, and Sub Saharan Africa; introduced in SSA in 16th century
  • 4. Cassava +• Resilient to adverse growth conditions (soil, drought)• Adaptable to range of agroecologies• Low maintenance• High yield potential (80 ton/ha) -• Pest & disease: virus (CMD & CBSD), whitefly, other• Highly heterozygous, vegetatively propagated• Root: main use, has low nutritional value• Long breeding cycle, shy flowering
  • 5. Work plan achievement 1. Workplan achievement 2. Quality of science 3. Communication 4. Capacity building 5. Resource mobilization 6. Managing resources 7. Other professional activities 8. Personal effectiveness
  • 6. Developing new tools for cassavaWhy?- Contribute to IITA mission & community effort to enhance cassava R4D- Develop tools that will be useful to IITA for various applications and also by other groups; reduce dependenceHow?- Strengthen local skills & capacities; balance outsourcing and in house researchWhat?- Regeneration and genetic transformation protocols for African landraces- A new vector for Agro-mediated transformation of dicots with derivatives- A cassava-specific DNA microarray: a tool for reverse genetics/gene discovery- EST-derived SSR markers for cassava
  • 7. 1.Transformation of farmer-preferred cassava for CBSD resistance Equatorial Guinea Uganda Kenya ROC DRC Tanzania Malawi Zambia Mozambique CBSD devastating CBSD damaging CBSD reported A transgenics approach for resistance to Potyviruses previously used and grown commercially: eg papaya resistant to Papaya ringspot virus
  • 8. Why target cassava landraces?All current transformation protocols are for ‘model’ genotypes, notused by farmers or breeders in Africa: excellent research tool butlimited application in the fieldBottleneck since current protocols are highly genotype-dependent Develop protocol for (African) farmer-preferred linesOngoing efforts on cassava landrace transformation(unpublished): - DDPSC, USA using FEC (based on Schopke et al., 1996) - KU, Denmark using cotyledons (based on Li et al., 1996) - CIAT, OSU, others?
  • 9. Experimental Approach- Produce SEs for cassava landraces (IITA)- Develop new protocol for genetic transformation of cassava landrace (using GUS reporter gene) (IITA)- Determine CBSV sequence from viral isolates from different countries (public domain; DSMZ)- Make R-gene constructs; multiple constructs based on RNAi to aim for resistance to different viral isolates (IITA; DSMZ)- Test R-genes in N benthamiana (DSMZ)- Transform farmer-preferred cassava using CBSD resistance gene(s) (IITA)
  • 10. Three basic steps in genetic transformation protocol:1. In vitro shoot regeneration method2. Gene transfer method: Agrobacterium-mediated3. Selection and/or screening for transgenic shootsIdeally, all steps are efficient (high % of success) and applicable to a rangeof genotypes
  • 11. Cassava regeneration/transformation Explants; e.g. immature leaf lobes Embryogenesis Shoot organogenesis Secondary SE * Primary SE Multiple shoots Embryogenesis Cotyledonary Friable embryogenic stage SE * Callus * Adventitious shoots Embryos Protoplasts Embryogenic suspensions PlantletsFig. modified from Zhang et al. 2006
  • 12. Cassava genotypes In use by cultivar CBSD Origin farmers Albert S Tanzania ? Kibaha S Tanzania Yes TME 12 ND WCA Yes TMS 96/0160 S IITA Yes TME 117 T Nigeria Yes TME 1 S Nigeria Yes Kibandameno S ECA Yes
  • 13. Somatic embryogenesisSE produced for 7 genotypes: 3 landraces from ESA 3 landraces from WCA 1 IITA elite line cv Albert TME12
  • 14. Summary cassava regeneration responses Explants; e.g. immature leaf lobes Embryogenesis +/- Shoot organogenesis Secondary SE * Primary SE ++++ ++++ Multiple shoots ++++ Embryogenesis ++ Cotyledonary Friable embryogenic stage SE * Callus * Adventitious shoots Embryos Protoplasts +/- Embryogenic +/- 0 - 40% suspensions Plantlets ++ 20 - 40%Fig. modified from Zhang et al. 2006 ++++ >90%
  • 15. Genetic transformation- Optimized using GUS reporter gene from pOYE153- Using organogenesis pathway with selection on Geneticin, recovered transgenic TME12 aka ‘Tokunbo’ (TE<0.1%); transgenics with uniform expression levels obtained: LEAF ROOT STEM STRONG WEAK
  • 16. Transgenic ‘Tokunbo’ in greenhouse
  • 17. GUS expressed in leaf, stem and petiole
  • 18. GUS expression in tuber and fibrous roots
  • 19. GUS expression in propagated ‘Tokunbo’ transgenic~70 clones tested: 1 14 28 13 27 41 expression remains stable and high in all plants after ratooning
  • 20. Also chimeric transgenics?
  • 21. 2. Transformation vector with CsVMV promoter cassetteObjectiveDevelop new Agro transformation vector with two different,constitutive promoters; CaMV 35S and CsVMVCharacteristics• Generic vector, can be used for various traits in various dicot species: - CBSD resistance - cassava - starch modification - tobacco (N benthamiana) - herbicide tolerance, etc• Promoters are oriented towards the border sequences to reduce unwanted gene silencing effects• No repeats within the T-DNA to reduce gene silencing effects
  • 22. - pCAMBIA2300 backbone- Km gene for selection- pCsVMV promoter cassette with polylinker for cloning gene of interest p35S pCsVMV nptll pING71 polylinker for cloning: 3’nos - GUS 9.5 kb LB - virus resistance RB - starch - etc
  • 23. p35S pCsVMV GUSnptll With GUS ORF for testing functionality pOYE153 of the construct 3’nos 11.5 LB RB
  • 24. pCAMBIA2301 pOYE153TobaccoCassava
  • 25. F1 transgenic tobacco plants pOYE153 pCAMBIA2301 pScVMV drives higher expression levels compared to p35S of pCAMBIA2301
  • 26. p35S pCsVMV Intron 3’nosnptll pING71-IV RB 9.7 With intron sequence for RNAi constructsLB (DSMZ) p35S pCsVMV CBSV-IR pRAJ42 With CBSV Inverted Repeatnptll 3’nos 11.1 for CBSD resistanceLB (IITA) RB + 2 other constructs targeting different viral isolates
  • 27. Status cassava landrace transformation for CBSD- Produce SEs for cassava landraces (IITA) OK- Develop new protocol for genetic transformation of cassava OK landrace (using GUS reporter gene) (IITA; KU) (TME12)- Determine CBSV sequence from viral isolates from different OK countries (public domain; DSMZ)- Make R-gene constructs; multiple constructs based on RNAi OK to aim for resistance to different viral isolates (IITA; DSMZ)- Test R-genes in N benthamiana (DSMZ) ongoing- Transform farmer-preferred cassava using CBSD resistance ongoing gene(s) (IITA)
  • 28. CBSD resistance is strain specific: eg miRNA CBSV-Kenya in N benthamianaTransgenic miRNA-Ke Empty vector miRNA-Ke No NoInfection CBSV-Ke CBSV-Ke CBSV-Moz CBSV-Ke No R S!
  • 29. 3. a 14K custom cassava long oligo array a tool for gene discovery and transcriptome analysisGeneric tool reverse genetics (genotype phenotype) complements QTL & association mapping approaches cassava genome sequencing effort trait improvement through genetic transformationApplications1. Understanding function of genes/alleles/gene networks2. Understanding allelic differences between gene families/varieties3. Diagnostics
  • 30. Target traits drought response plant-virus interactions cyanogenesis other
  • 31. Previously- Normalized cDNA libraries produced from control and water stressed tissues (leaf, root and stem tissue)- 18,166 ESTs sequenced (5’end) and assembled in 8,577 unigene set with functional annotation Metabolism 10.2% Metabolism 10.2% Energy Energy 25.7% 4.0% 25.7% 4.0% Cell growth, division DNA synthesis Cell growth, division DNA synthesis 2.1% 2.1% TranscriptionTranscription 6.1% 6.1% Protein synthesis Protein synthesis Protein destination 3.5% 3.5% destination Protein Transport Facilitation 4.7% Facilitation Transport 4.7% Cellular transport11.2% Cellular transport 11.2% 3.2% Cellular Biogenesis 3.2% Cellular Biogenesis 3.1% 0.2% Cellular communication/signal 3.1% transduction 0.2% 3.1% Cellular communication/signal 1.6% transductionCell rescue, defense, death and 8.7% 3.1% 6.1% ageing 1.6% 0.1% 6.4% Cell rescue, defense, death and 8.7% 6.1% ageing Ionic homeostasis 0.1% 6.4% Ionic homeostasis Cellular Organization
  • 32. - long oligo array: Agilent platform- Why Agilent? * flexibility * accessibility- Workflow: Design Microarray selection ? Informatics Data Probe Feature Selection Biological extraction question Microarray Microarray order scanning Sample Hybrid- preparation ization Protocol
  • 33. - Design and probe selection Input: ~ 40,000 cassava sequences: * 18,177 in house ESTs * ~ 5,000 ESTs from root specific library (unpublished) * remainder from public databases (EST, genomic, etc) * ACMV and CBSV ORFs; Km ORF Unigene set established, orientation determined
  • 34. Design summary Input Targets 14113 Targets with Probe 13865 Probe Length Shortest Probe 60.0 Length Mean 60.0 Length SD 0.0 BC Scores (1 = good; 4= bad) BC_1 13473 BC_2 360 BC_3 13 BC_4 19 Output: 13,865 unique probes ~ 14K BC_poor 0 or ~ 25-50 % of cassava transcriptome Total Probes 13865
  • 35. - Microarray selection- Array architecture uploaded (eArray)- 8x15K array format
  • 36. - Hybridization and scanning- Analysis: R Bioconductor
  • 37. Transcriptome AnalysisA. ‘Diversity’: expression profiling of different cassava genotypes GENOTYPE CHARACTERISTICS TME 3 Landrace, CMD resistant, parent of mapping population TME 117 Landrace, source of majority of ESTs TMS 96/0160 IITA breeding line, adopted in DR Congo, CBSD suscep. TMS 30572 IITA breeding line, widely adopted in SSA, CMD tol. TMS 96/1089A IITA breeding line, resistant to CMD & CBSD* Kibaha Tanzanian landrace, susceptible to CBSD Albert Tanzanian cultivar, susceptible to CBSD
  • 38. B. Different growth conditions: greenhouse versus in vitro (TMS 96/0160)C. Healthy versus virus infected plant: ACMV and CBSV Eg TME 4 CMD resist CBSD suscept
  • 39. Genotype vs TC: dot plot: fold change vs adjusted P value
  • 40. Virus infected vs healthy
  • 41. Candidate gene lists Setname Contrast Cut Off1 Short List TMS_96/0160_mitS-Control 0.05/2.0/1104 TMS_96/0160_ohneS-Control 0.05/2.0/168 TMS_96/0160_mitS-TMS_96/0160_ohneS 0.05/2.0/635 TME117_ohneS-Control 0.05/2.0/406 TME117_mitS-Control 0.05/2.0/937 1p-Value threshold/Contrast threshold/Number of candidates
  • 42. Description: TMS 96/0160_ohneS - control FDR=0.05; |Contrast|>=2 FoldClone ID Gene Name change79002281 gb_CL1576Contig1.1.KVL45FFC7CB0000... 2.679008123 gb_CBSV_6K2 2.679001735 BM260324.1 2.679011094 gb_CL1046Contig1.1.KVL45FFC7CB0000... 2.679013282 gb_CL198Contig2.1.KVL45FFC7CB0000019B 2.879014250 CK640993.1 2.979004173 gb_CL1734Contig1.1.KVL45FFC7CB0000... 3.079006640 DV447666.1 3.079004438 gb_CL1351Contig1.1.KVL45FFC7CB0000... 3.179011755 CK652281.1 3.179015613 BI325199.1 3.179009153 gb_CL1647Contig1.1.KVL45FFC7CB0000... 3.879006299 gb_CBSV_CP 4.9
  • 43. Cassava Transcriptome Analysis - Summary1. 14K Cassava-specific long oligo microarray developed2. Microarray passed all QC, hybridization and detection limit is as expected3. Results:- Differential gene expression between varieties limited (~0.1% DEG)- Profound effect of growth conditions on differential gene expression- Sensitivity comparable or exceeds that of PCR: diagnostics tool
  • 44. 4. Marker developmentObjectiveNumber of markers for cassava limited; eg current map has~ 400 markers; typically many 1000ds for non orphan cropsContribute to the community effort to develop additional molecularmarkers for cassava• In silico identification of COS, SNPs and SSRs from EST unigene dataset• 646 candidate EST-SSRs; duplicates with existing SSRs (CIAT collection) eliminated; primers designed for 346 ESTs• Candidate SNP markers + trace files provided to CBL colleagues
  • 45. Workflow EST-SSR validation Total number of EST sequences investigated: 18,166 Number of unigenes used for in silico identification of SSRs: 8,577 Total number of unique SSR loci appropriate for primer modeling: 646 (3.3%) Number of candidate SSR investigated : 346 PCR successful: ~ 90% Failed PCR: ~ 10% PCR products with expected sizes Amplification of introns > 500 bp Eliminate Screen on diversity panel
  • 46. Two panels‘Africa’ panel: cassava elite lines and landraces from Africa‘global’ panel: cassava from Africa, LA, Asia plus wild species, and castor bean plus leafy spurgeMarkers screened for polymporhism Different levels of resolution: SFR < PAGE < ABI3100 < DNA sequence
  • 47. SFR M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 M Used for PCR optimization + screen for P using diverse panel: All 346 primer pairs PCR optimized and screened on SFR gels
  • 48. Polymorhism & cross species transferabilitySET 1 – panel 2 SET 3 – panel 2 M 20 (23%) M 26 (33%) P 66 (77%) P 53 (67%) NA 3 NA 11 NS 2 NS 2 LONG 6 LONG 3For set 1 and 3; a total of 119 markers are PFor set 3: 80 of 85 markers amplify wild Manihot species ~ 94% 13 of 85 and 9 of 85 amplify castor and leafy spurge resp. or 15 and 10% TME117 TME419 M. M. M. Castor Leafy epruinosa brachyandra glaziovii bean spurge 97% 94% 85% 87% 91% 15% 11%
  • 49. Fluorescent genotyping (ABI3100)On a subset of P markers To estimate # allele and their sizes To develop fingerprinting kit
  • 50. Primer Primer # alleles No name per locus Allele sizes Predicted size 4 AT27 2 168, 170 166 5 AT45 2 210, 213 212 6 AT47 7 128, 132, 136, 153, 155, 157, 175 154 8 AT101 8 147, 149, 152, 157, 158, 163, 160 157 12 AT158 7 200, 202, 205, 209, 210, 211, 225 208 13 AAG54 8 157, 158, 159, 161, 167, 170, 173 165 15 AGA49 6 183, 190, 193,196, 199,204 198 16 AGA87 3 184, 198, 200 199 18 AGA157 4 249, 252, 260 250 20 CT19 4 169, 174, 184 183 21 CT22 5 207, 209, 214, 222 212 24 CT65 8 216, 243, 247, 249, 251, 253, 247 25 CT75 4 188,191,192,197 190 26 CT83 7 139, 145, 146, 142, 147, 148, 149, 152 150 27 CT109 3 164,168, 170 169 28 CT118 4 188, 192, 200, 202, 201 29 CT129 9 192, 198, 200, 204, 208, 210, 212, 216, 218 201 30 CAT46 4 220, 223, 226, 229 229 32 CTT15 3 170, 173, 177 173 34 GCA94 2 177, 182 179
  • 51. DNA sequencing of allelesEg CT109 Genotype # clones sequenced # alleles Allele sizes Nachinaya 4 3 169, 171 CM6740-7 6 5 169, 171 TMS30572 3 3 167, 169, 171 M epruinosa 5 5 166, 171 Overall size range: 166-171 Cassava only size range: 167-171 Overall unique alleles: 12 Cassava only unique alleles 7 Overall allele sizes: 166, 167, 169, 171 Cassava only allele sizes: 167, 169, 171
  • 52. Eg TC31 Genotype # clones sequenced # alleles TME7 8 7 MTai7 4 4 TMS30572 3 3 M glaziovii 5 4 or 5 M brachyandra 9 8 M epruinosa 5 5 Overall size range: 168-194 Cassava only size range: 168-182 Cassava only unique alleles 14
  • 53. TC31 allele phylogenetic tree MTai3 MTai4 TME117 TMS30572_ 1 TMS30572_ 2 TMS30572_ 3 MTai2 MTai1 TME7_4 cassava TME7_6 TME7_3 TME7_1 TME7_2 TME7_7 TME7_8 TME7_5 Mglaz4 Mglaz5 Mglaz3 Mglaz1 Mglaz2 Mepru 1 Mepru 2 Mepru 3 Mepru 4 wild Mbrac h5 Mbrac h3 manihot Mbrac h1 Mepru 5 Mbrac h8 Mbrac h2 Mbrac h4 Mbrac h6 Mbrac h74.9 4 2 0 Nucleotide Sub stitutions (x100)
  • 54. Conclusions- In total ~180 new polymorphic SSR markers (SFR)- EST-SSRs transferable to other Manihot species but less to other genera in Euphorbs TME117 TME419 M. M. M. Castor Leafy epruinosa brachyandra glaziovii bean spurge 97% 94% 85% 87% 91% 15% 11%- More than 2 alleles/genotype in the marker/genotype combinations examined so far! all multigene families or ploidy in cassava higher than generally accepted
  • 55. Quality of science 1. Workplan achievement 2. Quality of science 3. Communication 4. Capacity building 5. Resource mobilization 6. Managing resources 7. Other professional activities 8. Personal effectiveness
  • 56. Bibliography- Six articles published in refereed journals- One article in R4D Review, 2nd Ed.- Cowpea transposon sequences submitted to NCBI, USA with Acc No.- Eleven abstracts (9 with poster) at various meetings- Five manuscripts in preparation for refereed journalsInvited presentations- Six invited presentations at (inter)national meetings in Uganda, Mozambique, Tanzania, Belgium and USA (2).Paper review (external)- Eight manuscripts for international Scientific Journals- Two proposals for granting agencies (NSF, USA; AARI, Canada)
  • 57. Communications 1. Workplan achievement 2. Quality of science 3. Communication 4. Capacity building 5. Resource mobilization 6. Managing resources 7. Other professional activities 8. Personal effectiveness
  • 58. • Attended to CBL visitors (donors, collaborators, etc) with on average one visit every 1 to 2 weeks• Gave two interviews on agricultural biotechnology, to NTA and BBC• Wrote one article for ‘R4D review, 2nd Ed (2009); provided inputs for a second• Contributed to DVD on IITAs R4D program: ‘Award winning Research for Development’
  • 59. Capacity building 1. Workplan achievement 2. Quality of science 3. Communication 4. Capacity building 5. Resource mobilization 6. Managing resources 7. Other professional activities 8. Personal effectiveness
  • 60. Trained 4 undergraduate students and 6 graduate students (4 MSc and 2 Phd)Trained 3 technical staff in various biotechnologiesHosted 4 external professionals for hands-on capacity buildingResource person at workshop on ‘Recent Advances in the Applications ofMolecular Markers in Tropical Agriculture’ and invited the WABWS to jointly organizethis workshop with IITALab Safety Training: 59 lab users attended the CBL Lab Safety Trainingbetween June 2006 and March 2009; also used at NRCRI, NigeriaOrganized training course on ‘Working with radioactive chemicals’ at IITA withexternal resource people from the NNRA
  • 61. Resource mobilization 1. Workplan achievement 2. Quality of science 3. Communication 4. Capacity building 5. Resource mobilization 6. Managing resources 7. Other professional activities 8. Personal effectiveness
  • 62. Projects funded• BioCassava Plus. Supplemental Grant. Bill and Melinda Gates Foundation. With Dr Maziya-Dixon. 2008-2010• Cassava genetic transformation for the longevity of cassava brown streak resistance in Tanzania. Partners: IITA-Tanzania; Mikocheni Agricultural Research Institute, Tanzania. RF. 2007-2009. With Drs Herron, Ndunguru Linking phenotypes with genotypes: development and validation of a genome-wide DNA microarray as a reverse genetics tool in cassava (Manihot esculenta L Crantz). 2009 IITA Opportunity Grant. With Drs Gedil, Raji, Hearne and FrancoProposal submitted Enhancement of iron and zinc contents of cassava (Manihot esculenta Crantz) by soil bacteria and bacterial secondary metabolites. With ETH, Switzerland(Additional 5 proposals or CN submitted; not funded/considered)
  • 63. Managing resources 1. Workplan achievement 2. Quality of science 3. Communication 4. Capacity building 5. Resource mobilization 6. Managing resources 7. Other professional activities 8. Personal effectiveness
  • 64. • Established charge back system in CBL through bench fee and service charges for cost recovery as recommended by the IITA admin ($ 67,861 recovered for 2007-2008)• Streamlining of procurement and inventories in CBL; worked with Supply Chain for inventory of the CBL chemical and supply stores• Balanced special projects budgets
  • 65. Other professional activities 1. Workplan achievement 2. Quality of science 3. Communication 4. Capacity building 5. Resource mobilization 6. Managing resources 7. Other professional activities 8. Personal effectiveness
  • 66. • Assistant Editor: In Vitro Cellular and Developmental Biology – Plant• Ad hoc reviewer of papers for international scientific journals and granting agencies such as NSF, USA.• Member RDC• Collaboration with IITA Genebank, MARI-Tanzania and WARDA, Benin
  • 67. Personnel effectiveness 1. Workplan achievement 2. Quality of science 3. Communication 4. Capacity building 5. Resource mobilization 6. Managing resources 7. Other professional activities 8. Personal effectiveness
  • 68. • Manage facilities, oversee procurement and inventory of common items and the CBL support staff: • hold regular lab meetings with CBL scientists • regular updating of booklet ‘Operational Guidelines of CBL’ • re-established a hot lab facility and renewed license for use of radiochemical at IITA with support from DDG-Support and IITA Safety committee • jointly with colleagues, developed draft plan for CBL refurbishment • CBL has been accident-free with enabling environment for biotech research• Act for IITA admin when requested• Timely response to requests for inputs by CGO, PPS, Supply Chain and IITA admin Member of IITA’s procurement committee
  • 69. Future planning- Use tools for product development; move from ‘output’ to ‘outcome’ eg - cassava landrace with useful traits via genetic transformation - microsatellite-based fingerprinting kit for the characterization of cassava genetic resources- Expand role of the Biotech Lab in Ibadan to serve as a research center for national programs, other institutions (beyond traditional IITA mandate crops)
  • 70. AcknowledgementsIITA, Nigeria A Raji O Oyelakin B Odeseye F Kolade J Opabode U Okechukwu DSMZ, Germany S Winter KU, Denmark S Bak K Jorgensen J Gorodkin B Moller