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I.Moreno, J. Tomkins, P. Hurtado, E. Okogbenin, A. Dixon & M.Fregene

Progress in Positional Cloning of CMD2 the Gene that Confers High level of Resistance to the Cassava Mosaic Disease (CMD). I.Moreno, J. Tomkins, P. Hurtado, E. Okogbenin, A. Dixon & M.Fregene Global Cassava Partnership GCP-I July 22, 2008. Outline. Introduction Fine Mapping

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I.Moreno, J. Tomkins, P. Hurtado, E. Okogbenin, A. Dixon & M.Fregene

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  1. Progress in Positional Cloning of CMD2 the Gene that Confers High level of Resistance to the Cassava Mosaic Disease (CMD). I.Moreno, J. Tomkins, P. Hurtado, E. Okogbenin, A. Dixon & M.Fregene Global Cassava Partnership GCP-I July 22, 2008

  2. Outline • Introduction • Fine Mapping • Screening of the TME-3 BAC library • Chromosome walking • Conclusions

  3. Cassava: A Tropical Crop of World-Wide Importance • Fifth staple after rice, wheat, maize, and potato • World-wide production exceeds 180million tons/year, over half is produced in sub-Saharan Africa (SSA) • Demand is expected to increase 2% per year for the next 3 years

  4. Constraints:PestsandDiseases Green spider mite Cassava Brown Streak Disease Cassava Mosaic Disease Whiteflies Cassava mealybug Cassava bacterial blight

  5. P. manihoti P. herreni Cassava Green Mite Hornworm E. ello Mealybugs 18 – 64% 21-53% 34 – 88% Potential Yield Losses due to Cassava Pests Brown Streak Disease Whiteflies 33 – 79% Cassava Mosaic Disease 40-100% 30-100%

  6. The Cassava Mosaic Disease (CMD) • A viral disease endemic in SS-Africa and India • Not recorded in the Americas • Vectored by the white fly (Bemisia tabaci)

  7. The Cassava Mosaic Disease (CMD) • A viral disease and number one production constraint in SS Africa, yield loss estimated at >US$1billion/year • A potential risk in South America and South East Asia, and complicates germplasm transfer from the center of diversity to to SS-Africa • Viruses can recombine to more virulent forms and some have associated satellite molecules known to breakdown resistance • The most efficient form of control is resistant varieties which take 6-10 years to develop

  8. Background IITA (1930) - Sources of resistance I. 3rd Backcrossing : Cassava X Manihot glaziovii (1947) II. Local Nigerian varieties (1990) CIAT (1997) • Fregene 2000 (gen CMD1)Recessive gene • Akano et al 2002 (gen CMD2) Dominant gene • Linkage SSRY28 y NS158 (Akano 2002; Zárate 2002)

  9. SSRY NS158 TMS30555 XTME3 Dist Marker cM Name Resistant Susceptible rGY115 7.9 RP/SP/RB/SB rGY9 D 200bp 15.6 175bp R 150bp rGY1 Y66 CMD2 18.5 A single genome region explains 48% of phenotypic variance for CMD resistance and recessive gene action. 16.1 rI18b 20.5 rSSRY28 rJ1a 11.3 20.0 Resistant Susceptible Ai19 rGY57 21.0 rGY25 21.2 SSRY9 23.9 SSRY3 CMD1 16.2 SSRY 40 SSRY23 TMS30572 X CM7857-4 A single genome region explains >70% of phenotypic variance for CMD resistance and dominance gene action.

  10. Fine mapping of the CMD2 region 1.Segregant population ( > 1000 individuals) • Molecular markers of high resolution. 3. Bulk Segregant Analysis (BSA). • Evaluation of molecular markers candidates in recombinant individuals

  11. SUSCEPTIBLE PARENTAL RESISTANT PARENTAL TMS 30572 TME-3 X (1690 Individuals) 1. Fine Mapping Population

  12. 2. Phenotypical Evaluation Seedling trial- low pressure - Mokwa (IITA) Clonal trial- High pressure of disease (IITA)

  13. R R R S S S R R R S S S Polymorphic profile Monomorphic profile PCR 3. Bulk Segregant Analysis (BSA) Susceptible Bulk Recombinant Resistant Bulk Recombinant Susceptible Bulk Resistant Bulk 1st Level of selection: Parentals and Bulks 2nd Level of Selection: Open Bulks and recombinants individuals

  14. F1 POPULATION (TME-3 X TMS 30572) PHENOTYPICAL EVALUATION DNA EXTRACTION ( Dellaporta.1993) Scheme of Fine Mapping IDENTIFICATION OF RESISTANCE, SUSCEPTIBLES AND RECOMBINANTS INDIVIDUALS MAPPING OF SSRY28-NS158 MARKERS. BULK SEGREGANT ANALYSIS (“BSA”) MAPPING OF MOLECULAR MARKERS SELECTION OF MOLECULAR MARKERS ASSOCIATED TO CMD2 DATA ANALYSIS

  15. Linkage map of molecular markers associated to CMD2 MAPMAKER 2.0 ( θ= 0.3; LOD=5.0) New linkage map ( SSRY28, NS158, RME-1 y RME-2) 27.9 cM

  16. Screening of the TME-3 BAC Library • Library construction • Characterization • PCR screening of BAC-Pools

  17. Purification and cloning HindIII partial digestion 388b 194kb 48.5kb Size selection Ligation and Sizing of clones Clone picking by Robotics 97kb 48.5kb 6.9kb Sizing of clones 1.Cassava BAC Library Construction Mega base pair-sized DNA in Agarose Plugs Cloning vector BAC LIBRARY (73,728 clones In Micro-titre plates and high density filters)

  18. 2. Characterization Coverage 10X N= Ln ( 1-P)/ Ln ( 1- L/G) P= 99% • 73,728 clones • 4 high density filters • Inserts average = 93 kb (25-250)

  19. MARKER LOCATION # 90 N18 NS158 189 M19 MARKER LOCATION # 12 M14 17 N21 34 L16 47 O3 51 I1 52 A23 RME - 1 54 I1 85 J23 130 D18 136 J8 139 G22 155 K24 173 F1 3. PCR Screening of the TME-3 library 2 NS158 positive clones 13 RME-1 positivos clones

  20. Chromosome Walking • Restriction profile • Contig construction • BAC-ends Mapping

  21. 1. Restriction profile Positive clones were digested with Hind III

  22. 2. Contig Construction “Fingerprinting Contig” (“ FPC”) ( Marra et al 1997)

  23. 3. BAC-ends Mapping Identification of BAC ends and primer design (RME=6,NS158=4) Cloning and sequencing of amplification products Identification of SSCP markers Sequence analysis Design of alleloespecific primers

  24. BS PR PS BR BS BR BRecS Results 1. SSCP-SNP marker =Bac33b 2. SSCP-SNP marker =SBac33c

  25. Fine mapping SBAC 33c BAC 33b RME--1 SSRY-28 RME--2 NS158 CMD2 BAC 33 BAC 18 BAC 35 BAC 36 BAC 23 BAC 9

  26. Contig Construction SBac33c “Fingerprinting Contig” (“ FPC”) ( Marra et al 1997)

  27. Ongoing work Sequencing Minimum tilling path Annotation Candidate genes Genetic Complementation

  28. Conclusions • Application of Positional cloning in Cassava for a dominant gene (Fine mapping, Screening of BAC library and Chromosome walking). • Identification of new molecular markers associated to CMD2: RME-1, RME-2, BAC33b and SBAC33c. • Successive screening of the BAC library led to the construction of a contig of BAC clones that stretches for about 500Kb around CMD2. • Five BAC clones that traverse a 500 Kb region around the gene are currently being sequenced to search for candidate R genes that can be tested by genetic complementation.

  29. Acknowledgements CIAT Cassava Genetics (Jaime Marin, Edgar Barrera, Paola Alfonso) Myriam Cristina Duque-Statistical support Fernando Rojas- Bioinformatics support CUGI (B. Blackmon, Michael Atkins, Mónica Muñoz, Maria Delgado, Michael Saski) University of Maryland (Pablo Rabinowicz- Comparative Genomics) Cornell University (Sharon Mitchel and Bunmi Olasanmi) University of California-Davis (Ming Cheng Luo and Yanquin Ma).

  30. Thank You

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