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IRRI MAS CASE STUDY 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 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’ • Many popular and widelygrown 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 • Adopt backcrossing strategy for incorporating genes/QTLs into ‘mega varieties’ • Utilize DNA markers for backcrossing for greater efficiency – marker assisted backcrossing (MAB) Conventional backcrossing • High yielding • Susceptible for 1 trait P1 Elite cultivar x P2 Donor Desirable trait e.g. disease resistance P1 x F1 • Called recurrent parent (RP) P1 x BC1 P1 x BC2 Discard ~50% BC1 Visually select BC1 progeny that resemble RP Repeat process until BC6 P1 x BC3 P1 x BC4 P1 x BC5 P1 x BC6 BC6F2 Recurrent parent genome recovered Additional backcrosses may be required due to linkage drag MAB: 1ST 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 Concept of ‘linkage drag’ TARGET LOCUS c Donor/F1 BC1 BC3 RECURRENT PARENT CHROMOSOME DONOR CHROMOSOME TARGET LOCUS BC10 LINKED DONOR GENES • Large amounts of donor chromosome remain even after many backcrosses • Undesirable due to other donor genes that negatively affect agronomic performance • Markers can be used to greatly minimize the amount of donor chromosome….but how? Conventional backcrossing TARGET GENE F1 BC1 c c BC2 BC3 BC10 BC20 Marker-assisted backcrossing TARGET GENE c Ribaut, J.-M. & Hoisington, D. 1998 Marker-assisted selection: new tools and strategies. Trends Plant Sci. 3, 236-239. F1 BC1 BC2 MAB: 2ND 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 1 2 3 4 RECOMBINANT SELECTION Step 1 – select target locus BC1 Step 2 – select recombinant on either side of target locus OR Step 3 – select target locus again BC2 Step 4 – select for other recombinant on either side of target locus * * OR * 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 • 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 Theoretical proportion of the recurrent parent genome is given by the formula: 2n+1 - 1 2n+1 Where n = number of backcrosses, assuming large population sizes Percentage of RP genome after backcrossing Important concept: although the average percentage of the recurrent parent is 75% for BC1, some individual plants possess more or less RP than others CONVENTIONAL BACKCROSSING P1 x P2 MARKER-ASSISTED BACKCROSSING P1 x P1 x F1 P1 x F1 BC1 BC1 VISUAL SELECTION OF BC1 PLANTS THAT MOST CLOSELY RESEMBLE RECURRENT PARENT BC2 P2 USE ‘BACKGROUND’ MARKERS TO SELECT PLANTS THAT HAVE MOST RP MARKERS AND SMALLEST % OF DONOR GENOME BC2 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 A major QTL on chrom. 9 for submergence tolerance – Sub1 QTL LOD score 0 IR40931-26 PI543851 OPQ1 600 OPN41200 OPAB16 850 20 Sub-1(t) C1232 RZ698 15 OPS14 900 RG553 R1016 RZ206 50cM OPH7 950 10 RZ422 5 100cM C985 0 1 2 3 4 5 6 7 Submergence tolerance score 8 9 RG570 150cM Segregation in an F3 population Xu and Mackill (1996) Mol Breed 2: 219 RG451 RZ404 10 20 30 40 Make the backcrosses X Swarna Popular variety IR49830 Sub1 donor F1 X Swarna BC1F1 Seeding BC1F1s Pre-germinate the F1 seeds and seed them in the seedboxes Collect the leaf samples - 10 days after transplanting for marker analysis Genotyping to select the BC1F1 plants with a desired character for crosses Seed increase of tolerant BC2F2 plant Selection for Swarna+Sub1 Swarna/ IR49830 F1 376 had Sub1 21 recombinant Select plant with fewest donor alleles BC2F2 937 plants Swarna Plant #242 BC1F1 697 plants Plants #246 and #81 Swarna BC2F1 320 plants Plant #227 158 had Sub1 5 recombinant Swarna Plant 237 BC2F2 1 plant Sub1 with 2 donor segments BC3F1 18 plants Time frame for “enhancing” megavarieties • Name of process: “variety enhancement” (by D. Mackill) • Process also called “line conversion” (Ribaut et al. 2002) May need to continue until BC3F2 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 Graphical genotype of Swarna-Sub1 BC3F2 line Approximately 2.9 MB of donor DNA 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 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