Slide 1: MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT Bert Collard & David Mackill Plant Breeding, Genetics and Biotechnology (PBGB) Division, IRRI firstname.lastname@example.org & email@example.com LECTURE OUTLINE: LECTURE OUTLINE MARKER ASSISTED SELECTION: THEORY AND PRACTICE MAS BREEDING SCHEMES IRRI CASE STUDY CURRENT STATUS OF MAS SECTION 1 MARKER ASSISTED SELECTION (MAS): THEORY AND PRACTICE: SECTION 1 MARKER ASSISTED SELECTION (MAS): THEORY AND PRACTICE Definition:: Definition: Marker assisted selection (MAS) refers to the use of DNA markers that are tightly-linked to target loci as a substitute for or to assist phenotypic screening Assumption: DNA markers can reliably predict phenotype Slide 5: F 2 P 2 F 1 P 1 x large populations consisting of thousands of plants PHENOTYPIC SELECTION Field trials Glasshouse trials Donor Recipient CONVENTIONAL PLANT BREEDING Salinity screening in phytotron Bacterial blight screening Phosphorus deficiency plot Slide 6: F 2 P 2 F 1 P 1 x large populations consisting of thousands of plants Resistant Susceptible MARKER-ASSISTED SELECTION (MAS) MARKER-ASSISTED BREEDING Method whereby phenotypic selection is based on DNA markers Advantages of MAS: Advantages of MAS Simpler method compared to phenotypic screening Especially for traits with laborious screening May save time and resources Selection at seedling stage Important for traits such as grain quality Can select before transplanting in rice Increased reliability No environmental effects Can discriminate between homozygotes and heterozygotes and select single plants Potential benefits from MAS: Potential benefits from MAS more accurate and efficient selection of specific genotypes May lead to accelerated variety development more efficient use of resources Especially field trials Crossing house Backcross nursery Slide 9: (1) LEAF TISSUE SAMPLING (2) DNA EXTRACTION (3) PCR (4) GEL ELECTROPHORESIS (5) MARKER ANALYSIS Overview of ‘marker genotyping’ Considerations for using DNA markers in plant breeding: Considerations for using DNA markers in plant breeding Technical methodology simple or complicated? Reliability Degree of polymorphism DNA quality and quantity required Cost** Available resources Equipment, technical expertise Markers must be tightly-linked to target loci!: Markers must be tightly-linked to target loci! Ideally markers should be <5 cM from a gene or QTL Using a pair of flanking markers can greatly improve reliability but increases time and cost Marker A QTL 5 cM RELIABILITY FOR SELECTION Using marker A only: 1 – r A = ~ 95% Marker A QTL Marker B 5 cM 5 cM Using markers A and B: 1 - 2 r A r B = ~ 99.5% Markers must be polymorphic: Markers must be polymorphic 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 RM84 RM296 P 1 P 2 P 1 P 2 Not polymorphic Polymorphic! DNA extractions: DNA extractions DNA EXTRACTIONS LEAF SAMPLING Porcelain grinding plates High throughput DNA extractions “Geno-Grinder” Mortar and pestles Wheat seedling tissue sampling in Southern Queensland, Australia. PCR-based DNA markers: PCR-based DNA markers Generated by using P olymerase C hain R eaction Preferred markers due to technical simplicity and cost GEL ELECTROPHORESIS Agarose or Acrylamide gels PCR PCR Buffer + MgCl 2 + dNTPS + Taq + Primers + DNA template THERMAL CYCLING Agarose gel electrophoresis: Agarose gel electrophoresis http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/agardna.html UV light UV transilluminator Acrylamide gel electrophoresis 1: UV light UV transilluminator Acrylamide gel electrophoresis 1 Acrylamide gel electrophoresis 2: Acrylamide gel electrophoresis 2 SECTION 2 MAS BREEDING SCHEMES: SECTION 2 MAS BREEDING SCHEMES Marker-assisted backcrossing Pyramiding Early generation selection ‘Combined’ approaches 2.1 Marker-assisted backcrossing (MAB): 2.1 Marker-assisted backcrossing (MAB) MAB has several advantages over conventional backcrossing: Effective selection of target loci Minimize linkage drag Accelerated recovery of recurrent parent 1 2 3 4 Target locus 1 2 3 4 RECOMBINANT SELECTION 1 2 3 4 BACKGROUND SELECTION TARGET LOCUS SELECTION FOREGROUND SELECTION BACKGROUND SELECTION 2.2 Pyramiding: 2.2 Pyramiding Widely used for combining multiple disease resistance genes for specific races of a pathogen Pyramiding is extremely difficult to achieve using conventional methods Consider: phenotyping a single plant for multiple forms of seedling resistance – almost impossible Important to develop ‘durable’ disease resistance against different races Slide 21: F 2 F 1 Gene A + B P 1 Gene A x P 1 Gene B MAS Select F2 plants that have Gene A and Gene B Genotypes P 1 : AA bb P 2 : aa BB F 1 : A a B b F 2 AB Ab aB ab AB AABB AABb AaBB AaBb Ab AABb AAbb AaBb Aabb aB AaBB AaBb aaBB aaBb ab AaBb Aabb aaBb aabb Process of combining several genes, usually from 2 different parents, together into a single genotype x Breeding plan Hittalmani et al. (2000). Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in riceTheor. Appl. Genet. 100: 1121-1128 Liu et al. (2000). Molecular marker-facilitated pyramiding of different genes for powdery mildew resistance in wheat. Plant Breeding 119: 21-24. 2.3 Early generation MAS: 2.3 Early generation MAS MAS conducted at F2 or F3 stage Plants with desirable genes/QTLs are selected and alleles can be ‘fixed’ in the homozygous state plants with undesirable gene combinations can be discarded Advantage for later stages of breeding program because resources can be used to focus on fewer lines References: Ribaut & Betran (1999). Single large-scale marker assisted selection (SLS-MAS). Mol Breeding 5: 21-24. Slide 23: F 2 P 2 F 1 P 1 x large populations (e.g. 2000 plants) Resistant Susceptible MAS for 1 QTL – 75% elimination of (3/4) unwanted genotypes MAS for 2 QTLs – 94% elimination of (15/16) unwanted genotypes Slide 24: P1 x P2 F1 PEDIGREE METHOD F2 F3 F4 F5 F6 F7 F8 – F12 Phenotypic screening Plants space-planted in rows for individual plant selection Families grown in progeny rows for selection. Preliminary yield trials. Select single plants. Further yield trials Multi-location testing, licensing, seed increase and cultivar release P1 x P2 F1 F2 F3 MAS SINGLE-LARGE SCALE MARKER-ASSISTED SELECTION (SLS-MAS) F4 Families grown in progeny rows for selection. Pedigree selection based on local needs F6 F7 F5 F8 – F12 Multi-location testing, licensing, seed increase and cultivar release Only desirable F3 lines planted in field Benefits: breeding program can be efficiently scaled down to focus on fewer lines 2.4 Combined approaches: 2.4 Combined approaches In some cases, a combination of phenotypic screening and MAS approach may be useful To maximize genetic gain (when some QTLs have been unidentified from QTL mapping) Level of recombination between marker and QTL (in other words marker is not 100% accurate) To reduce population sizes for traits where marker genotyping is cheaper or easier than phenotypic screening ‘Marker-directed’ phenotyping: ‘Marker-directed’ phenotyping BC 1 F 1 phenotypes: R and S P 1 (S) x P 2 (R) F 1 (R) x P 1 (S) Recurrent Parent Donor Parent 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 … SAVE TIME & REDUCE COSTS *Especially for quality traits* MARKER-ASSISTED SELECTION (MAS) PHENOTYPIC SELECTION (Also called ‘tandem selection’) Use when markers are not 100% accurate or when phenotypic screening is more expensive compared to marker genotyping References: Han et al (1997). Molecular marker-assisted selection for malting quality traits in barley. Mol Breeding 6: 427-437. Slide 27: Any questions SECTION 3 IRRI MAS CASE STUDY: SECTION 3 IRRI MAS CASE STUDY 3. Marker-assisted backcrossing for submergence tolerance: 3. Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar, Darlene Sanchez, Endang Septiningsih & Abdel Ismail Photo by Abdel Ismail Abiotic stresses are major constraints to rice production in SE Asia: Abiotic stresses are major constraints to rice production in SE Asia Rice is often grown in unfavourable environments in Asia Major abiotic constraints include: Drought Submergence Salinity Phosphorus deficiency High priority at IRRI Sources of tolerance for all traits in germplasm and major QTLs and tightly-linked DNA markers have been identified for several traits ‘Mega varieties’: ‘Mega varieties’ Many popular and widely-grown rice varieties - “Mega varieties” Extremely popular with farmers Traditional varieties with levels of abiotic stress tolerance exist however, farmers are reluctant to use other varieties poor agronomic and quality characteristics BR11 Bangladesh CR1009 India IR64 All Asia KDML105 Thailand Mahsuri India MTU1010 India RD6 Thailand Samba Mahsuri India Swarna India, Bangladesh 1-10 Million hectares Backcrossing strategy: Backcrossing strategy Adopt backcrossing strategy for incorporating genes/QTLs into ‘mega varieties’ Utilize DNA markers for backcrossing for greater efficiency – marker assisted backcrossing (MAB) Conventional backcrossing: Conventional backcrossing x P 2 P 1 Donor Elite cultivar Desirable trait e.g. disease resistance High yielding Susceptible for 1 trait Called recurrent parent (RP) P 1 x F 1 P 1 x BC1 P 1 x BC2 P 1 x BC3 P 1 x BC4 P 1 x BC5 P 1 x BC6 BC6F2 Visually select BC1 progeny that resemble RP Discard ~50% BC1 Repeat process until BC6 Recurrent parent genome recovered Additional backcrosses may be required due to linkage drag MAB: 1ST LEVEL OF SELECTION – FOREGROUND SELECTION: MAB: 1 ST LEVEL OF SELECTION – FOREGROUND SELECTION Selection for target gene or QTL Useful for traits that are difficult to evaluate Also useful for recessive genes 1 2 3 4 Target locus TARGET LOCUS SELECTION FOREGROUND SELECTION Slide 35: Donor/F1 BC1 c BC3 BC10 TARGET LOCUS RECURRENT PARENT CHROMOSOME DONOR CHROMOSOME TARGET LOCUS LINKED DONOR GENES Concept of ‘linkage drag’ Large amounts of donor chromosome remain even after many backcrosses Undesirable due to other donor genes that negatively affect agronomic performance Slide 36: Conventional backcrossing Marker-assisted backcrossing F1 BC1 c BC2 c BC3 BC10 BC20 F1 c BC1 BC2 Markers can be used to greatly minimize the amount of donor chromosome….but how? TARGET GENE TARGET GENE Ribaut, J.-M. & Hoisington, D. 1998 Marker-assisted selection: new tools and strategies. Trends Plant Sci. 3 , 236-239. MAB: 2ND LEVEL OF SELECTION - RECOMBINANT SELECTION: MAB: 2 ND LEVEL OF SELECTION - RECOMBINANT SELECTION Use flanking markers to select recombinants between the target locus and flanking marker Linkage drag is minimized Require large population sizes depends on distance of flanking markers from target locus) Important when donor is a traditional variety RECOMBINANT SELECTION 1 2 3 4 Slide 38: OR Step 1 – select target locus Step 2 – select recombinant on either side of target locus BC1 OR BC2 Step 4 – select for other recombinant on either side of target locus Step 3 – select target locus again * * * Marker locus is fixed for recurrent parent (i.e. homozygous) so does not need to be selected for in BC2 MAB: 3RD LEVEL OF SELECTION - BACKGROUND SELECTION: MAB: 3 RD LEVEL OF SELECTION - BACKGROUND SELECTION Use unlinked markers to select against donor Accelerates the recovery of the recurrent parent genome Savings of 2, 3 or even 4 backcross generations may be possible 1 2 3 4 BACKGROUND SELECTION Background selection: Background selection Percentage of RP genome after backcrossing Theoretical proportion of the recurrent parent genome is given by the formula: Where n = number of backcrosses, assuming large population sizes 2 n+1 - 1 2 n+1 Important concept: although the average percentage of the recurrent parent is 75% for BC1, some individual plants possess more or less RP than others Slide 41: P 1 x F 1 P 1 x P 2 CONVENTIONAL BACKCROSSING BC1 VISUAL SELECTION OF BC1 PLANTS THAT MOST CLOSELY RESEMBLE RECURRENT PARENT BC2 MARKER-ASSISTED BACKCROSSING P 1 x F 1 P 1 x P 2 BC1 USE ‘BACKGROUND’ MARKERS TO SELECT PLANTS THAT HAVE MOST RP MARKERS AND SMALLEST % OF DONOR GENOME BC2 Breeding for submergence tolerance: Breeding for submergence tolerance Large areas of rainfed lowland rice have short-term submergence (eastern India to SE Asia); > 10 m ha Even favorable areas have short-term flooding problems in some years Distinguished from other types of flooding tolerance elongation ability anaerobic germination tolerance Screening for submergence tolerance: Screening for submergence tolerance A major QTL on chrom. 9 for submergence tolerance – Sub1 QTL: A major QTL on chrom. 9 for submergence tolerance – Sub1 QTL Segregation in an F3 population Xu and Mackill (1996) Mol Breed 2: 219 Make the backcrosses: Make the backcrosses Swarna Popular variety X IR49830 Sub1 donor F1 X Swarna BC1F1 Seeding BC1F1s: Pre-germinate the F1 seeds and seed them in the seedboxes Seeding BC1F1s Collect the leaf samples - 10 days after transplanting for marker analysis: Collect the leaf samples - 10 days after transplanting for marker analysis Genotyping to select the BC1F1 plants with a desired character for crosses: Genotyping to select the BC1F1 plants with a desired character for crosses Seed increase of tolerant BC2F2 plant: Seed increase of tolerant BC2F2 plant Selection for Swarna+Sub1: Selection for Swarna+ Sub1 Swarna/ IR49830 F1 Swarna BC1F1 697 plants Plant #242 Swarna X X 376 had Sub1 21 recombinant Select plant with fewest donor alleles 158 had Sub1 5 recombinant Swarna X Plant #227 BC3F1 18 plants 1 plant Sub1 with 2 donor segments BC2F1 320 plants Plants #246 and #81 Plant 237 BC2F2 BC2F2 937 plants Slide 51: Time frame for “enhancing” mega-varieties May need to continue until BC3F2 Name of process: “variety enhancement” (by D. Mackill) Process also called “line conversion” (Ribaut et al. 2002) Mackill et al 2006. QTLs in rice breeding: examples for abiotic stresses. Paper presented at the Fifth International Rice Genetics Symposium. Ribaut et al. 2002. Ribaut, J.-M., C. Jiang & D. Hoisington, 2002. Simulation experiments on efficiencies of gene introgression by backcrossing. Crop Sci 42: 557–565. Swarna with Sub1: Swarna with Sub1 Graphical genotype of Swarna-Sub1: Graphical genotype of Swarna- Sub1 BC3F2 line Approximately 2.9 MB of donor DNA Slide 54: Swarna 246-237 Percent chalky grains Chalk(0-10%)=84.9 Chalk(10-25%)=9.1 Chalk(25-50%)=3.5 Chalk(>75%)=2.1 Chalk(0-10%)=93.3 Chalk(10-25%)=2.3 Chalk(25-50%)=3.7 Chalk(>75%)=0.8 Average length=0.2mm Average length=0.2mm Average width=2.3mm Average width=2.2mm Amylose content (%)=25 Gel temperature=HI/I Gel consistency=98 Amylose content (%)=25 Gel temperature=I Gel consistency=92 IBf locus on tip of chrom 9: inhibitor of brown furrows: IBf locus on tip of chrom 9: inhibitor of brown furrows Some considerations for MAB: Some considerations for MAB IRRI’s goal: several “enhanced Mega varieties” Main considerations: Cost Labour Resources Efficiency Timeframe Strategies for optimization of MAB process important Number of BC generations Reducing marker data points (MDP) Strategies for 2 or more genes/QTLs SECTION 4 CURRENT STATUS OF MAS: OBSTACLES AND CHALLENGES: SECTION 4 CURRENT STATUS OF MAS: OBSTACLES AND CHALLENGES Current status of molecular breeding: Current status of molecular breeding A literature review indicates thousands of QTL mapping studies but not many actual reports of the application of MAS in breeding Why is this the case? Some possible reasons to explain the low impact of MAS in crop improvement: Some possible reasons to explain the low impact of MAS in crop improvement Resources (equipment) not available Markers may not be cost-effective Accuracy of QTL mapping studies QTL effects may depend on genetic background or be influenced by environmental conditions Lack of marker polymorphism in breeding material Poor integration of molecular genetics and conventional breeding Cost - a major obstacle: Cost - a major obstacle Cost-efficiency has rarely been calculated but MAS is more expensive for most traits Exceptions include quality traits Determined by: Trait and method for phenotypic screening Cost of glasshouse/field trials Labour costs Type of markers used How much does MAS cost?: How much does MAS cost? Institute Country Crop Cost estimate per sample* (US$) Reference Uni. Guelph Canada Bean 2.74 Yu et al. (2000) CIMMYT Mexico Maize 1.24–2.26 Dreher et al. (2003) Uni. Adelaide Australia Wheat 1.46 Kuchel et al. (2005) Uni. Kentucky, Uni. Minnesota, Uni. Oregon, Michigan State Uni., USDA-ARS United States Wheat and barley 0.50–5.00 Van Sanford et al. (2001) *cost includes labour Yu et al. 2000 Plant Breed. 119 , 411-415; Dreher et al. 2003 Mol. Breed. 11 , 221-234; Kuchel et al. 2005 Mol. Breed. 16 , 67-78; and Van Sanford et al. 2001 Crop Sci. 41 , 638-644. How much does MAS cost at IRRI?: How much does MAS cost at IRRI? Consumables: Genome mapping lab (GML) ESTIMATE USD $0.26 per sample ( minimum costs) Breakdown of costs: DNA extraction: 19.1%; PCR: 61.6%; Gel electrophoresis: 19.2% Estimate excludes delivery fees, gloves, paper tissue, electricity, water, waste disposal and no re-runs GAMMA Lab estimate = USD $ 0.86 per sample Labour: USD $0.06 per sample (Research Technician) USD $0.65 per sample (Postdoctoral Research Fellow) TOTAL: USD $0.32/sample (RT); USD $0.91/sample (PDF) Cost of MAS in context: Example 1: Early generation MAS: F 2 P 2 F 1 P 1 x 2000 plants USD $640 to screen 2000 plants with a single marker for one population Cost of MAS in context: Example 1: Early generation MAS Cost of MAS in context: Example 2 - Swarna+Sub1: Cost of MAS in context: Example 2 - Swarna+ Sub1 Swarna/ IR49830 F1 Swarna BC1F1 697 plants Plant #242 Swarna X X 376 had Sub1 21 recombinant Background selection – 57 markers 158 had Sub1 5 recombinant 23 background markers BC2F1 320 plants Estimated minimum costs for CONSUMABLES ONLY. Foreground, recombinant and background BC1- BC3F2 selection = USD $2201 Plant #246 Swarna X BC3F1 18 plants 11 plant with Sub1 10 background markers Swarna+ Sub1 Cost of MAS in context: Cost of MAS in context Example 1: Pedigree selection (2000 F2 plants) = USD $640 Philippines (Peso) = 35,200 India (Rupee) = 28,800 Bangladesh (Taka) = 44,800 Iran (Tuman) = 576,000 Example 2: Swarna+Sub1 development = USD $2201 (*consumables only) Philippines (Peso) = 121,055 India (Rupee) = 99,045 Bangladesh (Taka) = 154,070 Iran (Tuman) = 1,980,900 Costs quickly add up! A closer look at the examples of MAS indicates one common factor: : A closer look at the examples of MAS indicates one common factor: Most DNA markers have been developed for…. In other words, not QTLs!! QTLs are much harder to characterize! An exception is Sub1 MAJOR GENES! Reliability of QTL mapping is critical to the success of MAS : Reliability of QTL mapping is critical to the success of MAS Reliable phenotypic data critical! Multiple replications and environments Confirmation of QTL results in independent populations “Marker validation” must be performed Testing reliability for markers to predict phenotype Testing level of polymorphism of markers Effects of genetic background need to be determined Recommended references: Young (1999). A cautiously optimistic vision for marker-assisted breeding. Mol Breeding 5: 505-510. **Holland, J. B. 2004 Implementation of molecular markers for quantitative traits in breeding programs - challenges and opportunities. Proceedings of the 4th International Crop Sci. Congress., Brisbane, Australia. Breeders’ QTL mapping ‘checklist’: Breeders’ QTL mapping ‘checklist’ What is the population size used for QTL mapping? How reliable is the phenotypic data? Heritability estimates will be useful Level of replication Any confirmation of QTL results? Have effects of genetic background been tested? Are markers polymorphic in breeders’ material? How useful are the markers for predicting phenotype? Has this been evaluated? LOD & R 2 values will give us a good initial idea but probably more important factors include: Integration of molecular biology and plant breeding is often lacking: Integration of molecular biology and plant breeding is often lacking Large ‘gaps’ remain between marker development and plant breeding QTL mapping/marker development have been separated from breeding Effective transfer of data or information between research institute and breeding station may not occur Essential concepts in may not be understood by molecular biologists and breeders (and other disciplines) Advanced backcross QTL analysis: Advanced backcross QTL analysis Combine QTL mapping and breeding together ‘Advanced backcross QTL analysis’ by Tanksley & Nelson (1996). Use backcross mapping populations QTL analysis in BC2 or BC3 stage Further develop promising lines based on QTL analysis for breeding References: Tanksley & Nelson (1996). Advanced backcross QTL analysis: a method for the simultaneous discovery and transfer of valuable QTLs from unadapted germplasm into elite breeding lines. Theor. Appl. Genet. 92: 191-203. Toojinda et al. (1998) Introgression of quantitative trait loci (QTLs) determining stripe rust resistance in barley: an example of marker-assisted line development. Theor. Appl. Genet. 96: 123-131. x P 2 P 1 P 1 x F 1 P 1 x BC1 BC2 QTL MAPPING Breeding program Future challenges: Future challenges Improved cost-efficiency Optimization, simplification of methods and future innovation Design of efficient and effective MAS strategies Greater integration between molecular genetics and plant breeding Data management Future of MAS in rice?: Future of MAS in rice? Most important staple for many developing countries Model crop species Enormous amount of research in molecular genetics and genomics which has provided enormous potential for marker development and MAS Costs of MAS are prohibitive so available funding will largely determine the extent to which markers are used in breeding Food for thought: Food for thought Do we need to use DNA markers for plant breeding? Which traits are the highest priority for marker development? When does molecular breeding give an important advantage over conventional breeding, and how can we exploit this? How can we further minimize costs and increase efficiency? Slide 74: Thank you!