Comparative Genomics for Marker Development in Cassava


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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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Comparative Genomics for Marker Development in Cassava

  1. 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 –
  2. 2. Genomics Resources 4 Crop Improvement Genome sequenceBioinformatics Functional Assembly/Annotation GenomicsData analysis & mining Proteomics Gene/Marker Discovery Assay/Validation Application MAB TRANSGENICS
  3. 3. 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 genomicsMelaku Gedil
  4. 4. Cloned and characterized virus R genes in plants Resistance CloningGene Host species Virus AVR Receptor structure mechanisms method Helicase TransposonN N. tabacum TMV domain of HR tagging TIR-NBS-LRR replicaseRx1 S. tuberosum PVX CP Replication Positional cloning CC-NBS-LRRRx2 S. tuberosum PVX CP Replication Positional cloning CC-NBS-LRRSw5 S. esculentum TSWV MP HR Positional cloning CC-NBS-LRRHRT A. thaliana TCV CP HR Positional cloning LZ-NBS-LRR SystemicRTM1 A. thaliana TEV nd Positional cloning Jacalin like seq movement SystemicRTM2 A. thaliana TEV nd Positional cloning Jacalin like seq movementRCY1 A. thaliana CMV CP HR Positional cloning CC-NBS-LRR TransposonTm22 S. lycopersicum ToMV MP HR tagging CC-NBS-LRR Replication Approximation byPvr21/22 C. annuum PVY VPg cell-cell homology eIF4E movement, Replication Approximation byMo11/2 L. sativa LMV nd eIF4E Tolerance homology, PSbM Approximation by
  5. 5. Predicted domain of R genes
  6. 6. MethodsInternational Institute of Tropical Agriculture – Institut international d’agriculture tropicale –
  7. 7. TIR NBS LRR CC NBS LRR TIR NBS LRR CC NBS LRR PCR-Primer (Degenerate) Cloning/SequencingSequence Analysis Markers
  8. 8. DNA/RNA extraction Species # AccM. esculenta 5M .epruinosa 1M. glaziovii 1M. brachyandra 1M. tripartita 1Other Manihots 3Ricinus communis 1
  9. 9. Templates• DNA • RNA• … • cDNA• PCR • PCR• Cloning • Cloning• Colony PCR • Colony PCR• Purify • Purify• Sequence • Sequence* Surveys genomic *Surveys expressed DNA genes
  10. 10. PCR Amplification with degenerate primersAmplification with degenerate primers 50F-470RL was considered for derived from At-NBS-LRR further analysis. Four DNAThree 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.
  11. 11. Cloning TA-cloning, QiagenPurification
  12. 12. SequencingIn-house on ABI 3130Beca - NairobiIowa State University
  13. 13. Sequence AnalysisSequences editedSequences assembledSequence Clustered, identity matrix,Similarity search in Genbank – BLASTSTS primers for resequencingMarker development for MAS
  14. 14. Sequence Editing and Analysis - CodonCode
  15. 15. Sequence Editing and Analysis - BioEdit
  16. 16. Similarity search
  17. 17. BLAST
  18. 18. Mt_gd-tripa_582170 483 65 Manihot esculenta RCa3 AY187293.1 pseudogene, partial sequenceMt_gd-tripa_582172 483 67 Manihot esculenta RCa3 AY187293.1 pseudogene, partial sequenceMt_gd-tripa_582149 689 1 Ricinus communis conserved XM_002521367.1 hypothetical protein, mRNAMt_gd-tripa_582165 483 69 Manihot esculenta RCa3 AY187293.1 pseudogene, partial sequenceMt_gd-tripa_582205 482 72 Manihot esculenta RCa3 AY187293.1 pseudogene, partial sequenceMt_gd-tripa_594909 481 70 Manihot esculenta RCa3 AY187293.1 pseudogene, partial sequenceMt_gd-tripa_582206 471 45 Ricinus communis Disease XM_002521743.1 resistance protein RFL1, putative, mRNAMt_gd-tripa_582216 482 64 Manihot esculenta RCa3 AY187293.1 pseudogene, partial sequence
  19. 19. Cassava Genome Database
  20. 20. Castor bean Genome Database
  21. 21. Clustering
  22. 22. Species Total sequences NBS-LRR matchingManihot esculenta - 25 10genomicManihot esculenta- 85 41cDNAM. glaziovii 44 8M. tripartita 96 8M. epruinosa 140 40M. brachyandra 16 3Ricinus communis 51 45
  23. 23. Mt_gd-tripa_582216Castor 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_594909Cassava-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_580105Cassava-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_566518M.glaziovii Mep_gDNA_566522 Mep_gDNA_566523 Mt_gd-tripa_582205 Mep_gDNA_580048 Mep_gDNA_580055 Mep_gDNA_580042 Me_cd-tme117-586278M.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_580093M.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_596823M.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
  24. 24. Sequence divergence Total Cluster Mean Minimum MaximumCassava- 10 6 181 5 325 genomicCassava-cDNA 41 5 177 1 357Glaziovii 8 5 146 61 231Brachy 3 2 197 4 294Epruinosa 40 8 222 1 326Tripartita 8 4 236 1 328Castor 45 16 184 1 336
  25. 25. 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
  26. 26. SNP Identification
  27. 27. 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
  28. 28. Work in Progress (WIP) and Applications
  29. 29. 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
  30. 30. Applications1. Molecular markers (SNP, STS)2. Gene discovery3. 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 hydrolysis6. Genome wide survey of Resistance genes (Arabidopsis, Rice, poplar, Grape, papaya)
  31. 31. Advantages1.Protocol is adaptable to other crops….e.g. yam, cowpea2.All resistance genes – insights to the structure & organization3.Starting material for comparative genomics based data mining of R genes4.Easy and cost-effective to generate data5.Markers are gene-based (not random association)
  32. 32. 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
  33. 33. 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
  34. 34. Thank youInternational Institute of Tropical Agriculture – Institut international d’agriculture tropicale –