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“Wheat Science to Textbooks” Workshop CIMMYT El Batan December 5-10, 2010

“Wheat Science to Textbooks” Workshop CIMMYT El Batan December 5-10, 2010. Practical Issues in Marker-Assisted Selection in Durum Wheat. Karim Ammar & Susanne Dreisigacker Global Wheat Program CIMMYT-Mexico. Outline. Why are we using molecular markers in the durum program at CIMMYT?

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“Wheat Science to Textbooks” Workshop CIMMYT El Batan December 5-10, 2010

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  1. “Wheat Science to Textbooks” WorkshopCIMMYTEl Batan December 5-10, 2010 Practical Issues in Marker-Assisted Selection in Durum Wheat Karim Ammar & Susanne Dreisigacker Global Wheat Program CIMMYT-Mexico

  2. Outline • Why are we using molecular markers in the durum program at CIMMYT? • What markers are we using? • Practical issues faced in implementing MAS: • Marker optimization needs • Special population/data management needs • When to use MAS? • Issues of segregation distortion • Need for phenotypic validation • Example of a practical MAS program • Conclusions & practical recommendations

  3. Reasons for using MASGenetic vulnerability for leaf rust resistance • CIMMYT germplasm was becoming susceptible in the world but not in Mexico, until… • March 2001: BBG/BN overcomes a major gene protecting most CIMMYT germplasm • 85%CIMMYT elite material susceptible • Genetic Vulnerability Demonstrated

  4. Reasons for using MASGenetic vulnerability for stem rust resistance, Ug99 + Ethiopian Durum races • Less than 3% of CIMMYT elite germplasm resistant in Ethiopia • Direct threat to durum production areas in central India • Possible threat for North Africa, Turkey, Middle-East

  5. Reasons for using MASRelying on major genes still necessary… so Pyramiding required! Sus 1 RES 2 RES 3 RES • Ideally, a minor-genes based resistance should provide durable resistance, but still a long-term solution • Available/effective major genes still the short to medium term option: • Gene pyramiding required for durability • Phenotypic selection fails to detect accumulation of major genes

  6. MAS for Pyramiding Diseases Resistance GenesMolecular Markers linked to know genes of interest rr Rr RR Co-dominant Susceptible Resistant rr Rr RR Dominant

  7. MAS for Pyramiding Diseases Resistance GenesMolecular Markers independently identifying different genes r1r1 R1R1 Marker 1, linked to R-gene 1 R1R1R2R2R3R3 Marker 2, linked to R-gene 2 r2r2 R2R2 Marker 3, linked to R-gene 3 r3r3 R3R3

  8. Markers Used in CIMMYT’s Durum BreedingList and general use BW/DW: Originally introduced from bread wheat but CIMMYT used a durum wheat source BW:from bread wheat, introduced to durum at CIMMYT DW: from durum wheat

  9. Markers Used Routinely in CIMMYT’s Durum BreedingLr19/Sr25 • Effectiveness: Against all known durum wheat LR races and all SR races (including Ug99+). LR Virulence on BW has been reported • Marker:wms63 (Dubcovsky et al.) • Chromosome:7AL, for durum • Source: Originally from Thinopyrum sp., now available from various bread + durum wheat • Type: CO-DOMINANT • Reliability:Good • Added benefit: yellow color • Use at CIMMYT: used widely for gene pyramiding for leaf (+Lr14a+Lr47) and stem rust resistance (+Sr22). Effectiveness validated against Ethiopian durum SR races

  10. Markers Used Routinely in CIMMYT’s Durum BreedingLr47 • Effectiveness: Against all known durum wheat LR races. Virulence on BW has been reported • Marker:PS10 (Dubcovsky et al. 1998) • Chromosome:7AS • Source: Originally from T. speltoides, now available from various bread + durum wheat • Type:DOMINANT (Co-dominant?) • Reliability:Good • Use at CIMMYT: used widely for gene pyramiding for leaf rust resistance (+Lr14a+Lr19)

  11. Markers Used Routinely in CIMMYT’s Durum BreedingLr14a • Effectiveness: Against durum wheat LR races, except in France. Virulence on BW very common. • Marker:wms344+wms146 (Herrera et al., 2008) • Chromosome:7BL • Source: Originally from Yaroslav Emmer, present in many bread and durum wheat • Type:DOMINANT • Reliability:Fair • Use at CIMMYT: used widely for gene pyramiding for leaf resistance (+Lr19+Lr47) but mostly for parent characterization & avoiding presence at monogenic state • Lr14a is the basis of resistance of most currently LRres durum germplasm (CIMMYT, ICARDA, France, Italy…), often in monogenic state, resulting in global vulnerability

  12. Markers Used Routinely in CIMMYT’s Durum BreedingSr22 • Effectiveness: Against all known SR races, including Ug99+ • Marker:CFA2123 (Khan et al., 2005) • Chromosome:7AL • Source: Originally from T. monoccocum sp., now available from various bread + durum wheat • Type: CO-DOMINANT • Reliability:Good • Use at CIMMYT: used widely for gene pyramiding for stem rust resistance (+Sr25), effectiveness validated against Ethiopian durum races.

  13. Markers Used Routinely in CIMMYT’s Durum BreedingCre5 (Lr37)+ Cre1 + Rln1 • Gene: Cre1, Cereal Cyst Nematodes, Marker:Cre1 M19, (Ogbonnaya et al., 2001) • Chromosome:2AS • Source: Bread wheat cv.Silverstar • Type: DOMINANT • Reliability:Acceptable, needs phenotypic validation • Gene:Cre 5 (also Lr37/Yr17/Sr38), Cereal Cyst Nematodes, Marker:Ln2+Ventriup (Helguera et al., 2003) • Chromosome: 2AS • Source: Bread wheat cv. Trident • Type: DOMINANT • Reliability:Acceptable, needs phenotypic validation for CCN • Gene:Rln 1, Marker:FC3 • Chromosome: 7BL • Source: Bread wheat cv. Krichauff • Type: DOMINANT • Reliability:Marginal, needs phenotypic validation

  14. Markers Used in CIMMYT’s Durum BreedingStatistics of marker use

  15. Markers Used in CIMMYT’s Durum BreedingStatistics of marker use by objective – 2009-10 • Leaf samples: 11,275 • Data-points: 21,821

  16. Practical Issues Faced in MAS in Durum WheatMarker optimization… Need for constant work • All markers need significant optimization work and constant checking/verification: • Experimental conditions • Chemicals/water • Primers • DNA quality • Even once optimized the best of markers have failed us at one point or another during the last 3 years! • The availability of a dedicated person for troubleshooting/problem solving is CRITICAL! • Permanent dialogue between lab & breeder is required

  17. Practical Issues Faced in MAS in Durum WheatSpecial population & data handling • Regular breeding material are handled on a population/family basis, MAS populations need to be handled on a per-plant basis • Individual plant tagging and record-keeping • Selection, harvest, threshing and handling more complex and cautious • Accurate record-keeping at the lab level • Tubes, plates, gel-lanes • Reading and transcription of results • More proficiency in computer processing of data

  18. Practical Issues Faced in MAS in Durum WheatSpecial population & data handling: MAS before crossing

  19. Practical Issues Faced in MAS in Durum WheatSpecial population & data handling: MAS after phenotypic selection

  20. Practical Issues Faced in MAS in Durum WheatWhich generation should we use MAS? And for how many generations? Parents (Donors + Recipients) Ensuring correct crosses and marker combinations F1s (if donor is not fixed for sure) Simple Cross (AxB) F2: Phenotype + MAS F3: Phenotype + MAS F4: Phenotype F5: Phenotype F6 (line derivation): Phenotype + MAS 3- or 4-way Crosses (AxBxC), [(AxB)x(CxD)] F1TOP/F1DOB:Phenotype + MAS F2: Phenotype + MAS F3: Phenotype + MAS F4: Phenotype F5: Phenotype F6 (line derivation): Phenotype + MAS • As early as segregation starts • Cycles of MAS depend on: • Marker number • Marker type (dominant/co-dominant) • Reliability of each MAS cycle • Resources

  21. Practical Issues Faced in MAS in Durum WheatPractical example of pyramiding 3 LR resistance genes Elite 1 Lr14a x Source Lr19 Elite 2 Lr14a x Source Lr47 F1-1 Lr14a + Lr19 x F1-2 Lr14a + Lr47 F1-double cross population App. 200-250 plants - MAS on app. 80-100 plants preselected in field for Ag. type and LR resistance - Bulk plant positive for 3 markers Lr14a +Lr19 +Lr47 F2 Population Enriched for Lr14a +Lr19 +Lr47

  22. Practical Issues Faced in MAS in Durum WheatPractical example of pyramiding 3 LR resistance genes F2 population Enriched for Lr14a +Lr19 +Lr47 App. 1000-1200 plants - MAS on app. 80-100 plants preselected in field for Ag. type and LR resistance - Bulk plant positive for 3 markers Lr14a +Lr19 +Lr47 F3population Enriched for Lr14a +Lr19 +Lr47 App. 200-300 plants - MAS on app. 50-70 plants preselected in field for Ag. type and LR resistance - Bulk plant positive for 3 markers Lr14a +Lr19 +Lr47

  23. Practical Issues Faced in MAS in Durum WheatPractical example of pyramiding 3 LR resistance genes F4 Bulk Population Enriched for Lr14a +Lr19 +Lr47 NO-MAS F5Individual Plants – NO MAS F6 (PYT) MAS confirmation ofLr14a +Lr19 +Lr47 on individual head-rows

  24. Practical Issues Faced in MAS in Durum WheatNeed for phenotypic validation • If you know you have the marker … make sure to also have the gene! • Marker-gene recombination can occur! • Need for confirmatory phenotypic evaluation • Positive phenotypic confirmation for • Lr19/Sr25 • Sr22 • Lr14a • Lr47 • Lr53 (so far) • GPC-B1 - marker + marker

  25. Practical Issues Faced in MAS in Durum WheatNeed for phenotypic validation • If you know you have both the marker and the gene, make sure the gene is expressed in its new background • Genes introduced from other species can be suppressed in new backgrounds (FHB-3BS, not effective in durum wheat, Sensibility gene?) • Genes can have their expression altered in new backgrounds (Lr46?, Cre1?, Cre5?) • Need for phenotypic confirmation of gene expression at desired level

  26. Practical Issues Faced in MAS in Durum WheatSegregation distortion • Not all markers always behave as typical Mendelian factors, segregation distortion is common: • Cross-combinations • Phenotypic pre-selection (sampled not random samples) • Often from alien translocations

  27. Conclusions & Recommendations • To breeders: • Relax! … markers won’t replace us, let’s keep an open mind to the possibility of being more efficient • To markers people: • Relax! … the world is convinced, let’s stop overselling it • To all of us: “… We have not made our contribution until it is in farmers’ fields!” • Any technology that would accelerate the process should be used • Any person who can help should be enlisted

  28. Conclusions & Recommendations • Have a clear purpose & justification for their use: • Using them because they are available is a waste of time & resources… • Know your marker: • Type, inheritance, reliability, cost… • Have a clear plan of work: • How many crosses, at which generation to screen, how many plants/population… • Handle MAS population separately from the normal breeding populations • Always keep contact lab/breeder, processes always need to be optimized

  29. Conclusions & Recommendations • Check your sources carefully: • Donors of markers and resulting F1need to be confirmed by plants (before investing considerable resources on the F2 or F1 top screening) • Ensure accurate, plant-by-plant, record keeping during MAS screening • MAS is for one/few gene/s only! Ensure enough genetic variability remains in the cross after selection for the presence of the marker

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