1. Sigrid HeuerGCP meeting, Hyderabad, Sept 2011+P -PDeveloping rice with dual tolerance of P deficiency and Al toxicity

2. The major QTL Pup1 was mapped in Kasalath in aP-deficient field in Japan under rainfed conditionsNipponbareKasalath02468101214Puptake(mgplant-1)Screening of 30 rice accessionsDularWissuwa et al (1998), Theor Appl GenetPup1QTL mapping in Kasalath x Nipponbare NILsMatthias Wissuwa: Several years of efforts to understand the function in relation to P uptake No physiological mechanisms or (consistent ) root traits detected No candidate genes in the Pup1 Nipponbare genomic region 3. Kasalath Pup1 sequence: complex hot spot of TEPup1 molecular markers (dominant INDEL markers only):+ + + + + + K + + + ++++ ++ + + +++ +++ ++ + + + +++ +++ ++ + ++382 bp918 bp912 bp523 bp847 bp505 bp550 bpNK42K46K41K43K48K52K595 10 15 20 25 30 35 40 45 50 551 58Pup1markerK= Kasalath (Pup1 donor); N= Nipponbare (intolerant)genotypeT5-4 Ba76H14_7154K41-K43 K46 K48 K52 K59K20 K30INDELdominantco-dominantK01 K05co-dominantK67co-dominantNipponbare(intolerant; 154 kb)Kasalath(tolerant; 272 kb)Pup1 is present indrought-tolerantbreeding linesbreedersunknowinglyselected for Pup1Chin et al 2010, Theor Appl Genet 4. co-dominantdominantK N IR64 IR74 B D SB H2Oamplicon (bp)K / NOsPupK20-2OsPupK46-2OsPupK29-1K1K5K20-1K20-1MseK20-2K20-2BspK29-1K29-2K29-3K41K42K43K45K46-1K46-2K48K52K59125 / 122272 / 280240 / 243201 / 243982 / 995231+349+402 / 413+582212 / 206219 / 212236 / 248382 / none918 / none912 / none276 / none523 / none227 / none ~550 unspecific847 / none505 / none ~700 unspecific550 / noneCore set of best Pup1 markers(dominant and co-dominant)Chin et al 2011, Plant Physiology 5. 0.1AUS 196IRAT 77AzucenaPratao PrecoceIR 69502-6-SRN-3-UB-1BIR 78966-B-10-B-B-B-1IR 78933-B-24-B-B-4B6144F-MR-6-0-0IR 65192-48-10-3-BApoIR 78997-B-16-B-B-B-2KasalathVary Lava 701AUS 257DularIAC 25IAC 47IR 78937-B-3-B-B-2IR 78937-B-3-B-B-3IR 84144-11-12UPL RI 7VandanaCT 6510-24-1-2IR 55419-04IR 55423-01IR 78875-131-B-1-1IR 78937-B-20-B-B-2IR 78937-B-3-B-B-1IR 74371-46-1-1IR 78942-B-2-B-B-2IR 78933-B-24-B-B-1IR 78940-B-22-B-B-B-1IR 78966-B-10-B-B-B-2IR 79008-B-11-B-B-1IR 79008-B-11-B-B-2UPL RI 5IR 78940-B-22-B-B-B-2Way RaremIR 78933-B-24-B-B-2IR 80340-23-B-12-6-BPM-36PS 36IR 9764-45LemontYS 27Arkansas FortunaIR 71525-19-1-1IR 73678-6-9-BIR 73685-8-11-16-1-2-3-3-BSri KuningIR 36IR 72Gaisen Ibaraki 2IR 49830-7-1-2-3IR 75870-5-8-5-B-2-BIR 70358-84-1-1IR 73382-80-9-3-13-2-2-1-3-BAshoka 228IR 63295-AC209-7-BIR 78942-B-2-B-B-1IR 74IR 78933-B-24-B-B-3K36-5-1-1BBNerica 4PS 12BalaCT 99993-5-10-MIR 62266-42-6-2IR 64IR 66424-1-2-1-5IR 72418-3B-B-14-9-2K36-3-1-1MoroberekanIR 24637-38-2-2-1IR 3646-8-1-2IR 42IR 73678-8-1-3-3-2-BAkihikariNipponbareIR 20IR 66K1K5K20-1MseK20-2BspK29-1K29-2K29-3K41K42K43K45K46-1K46-2K48K52K59Group IGroup IIGroup IIIEcotypeNon-Kasalath alleleHeterozygousKasalath alleleInconsistent or missing dataGenotypeEcotypeUpland (UL)Upland and lowlandLowland (LL)Core marker set Chin et al 2011,Plant PhysiologyGermplasm survey with Pup1 core markersrainfedirrigated 6. Lowland UplandP+ P- P+ P- Well-watered DroughtP T SM G P T SM G P T SM G P T SM G P T SM G P T SM GP: plant heightT: tiller numberS: straw weightM: maturityG: grain yieldPup1Markers (#380)by chromosomeGreen: higher regression coefficient (r2) of the corresponding markers to thephenotypes.SNP data confirm that Pup1 is associated with good performanceunder field conditions in diverse genotypes (JH Chin; ongoing)(core markers) 7. Marker assisted backcrossing (MABC): Principle approach1 2 1 12 2242 bp123 bp94 bpSub1A allele-specific markerMarkers:backgroundflankingSub1 foregroundflankingbackgroundSSR or SNPbackgroundChr. 9further BC andselfingSub1 donor recipient (local variety)F1 and backcross to local varietyPup1 8. csister 4 (IR64-KK-NN) IR64-Pup1 BC2F3= PH218-5-3 ?BC2F4IR64-Pup1 IR64BC2F4IR74-Pup1 IR74K20-2BspK29-1K46-1GAPDHalleleIR74-Pup1+Pup1IR64-Pup1+Pup1IR74-Pup1 IR64-Pup1N N K N N KSelection with Pup1 foregroundand SNP background markers(i) MABC of IR64-Pup1 and IR74-Pup1 ricePhenotyping (+P/-P, flooded field)IR74 IR64+25% 9. Chin et al 2011, Plant Physiol(ii) MABC of Indonesian Pup1-varietiesNurul Hidarayam(PhD student at IRRI from ICABIOGRAD): Final selection and genotyping completed GH phenotypic data collected Seed increase next seasonJoko Prasetiono:Parallel screening ongoing at ICABIOGRAD 10. Pup1 region93-11 (742 bp)Kasalath (278 kb)Nipponbare (154 kb)Connecting linesPercent identityLEGEND 68 predicted genes with RiceGAAS (incl. TEs) many unclear gene models due to transposable elements no P-transporter or P-uptake related geneHeuer et al (2009), Plant Biotechnology JournalPup1 is a complex genomic region and a hot spot of transposon integration54 % TEs54.6 % TEs45.2 % TEsII. Identification of the Pup1 major tolerance gene(s) 11. 2029Kasalath4, 5Nipponbare6753491protein kinasehypothetical proteindirigent gene(PupK-21n)fatty acid DOX/unknown proteinasparticproteinaseZn knuckle andZn finger proteinDUF568Short listed Pup1 candidate genes 12. Chromosome 12 region has been mapped in other studies: 13. Summary Final set of Pup1 gene/allele specific markers available Development of Pup1-breeding lines by MABC completed (2 irrigated, 2(3) rainfed) First field data confirm beneficial effect of Pup1 Seed increase ongoing at IRRI and ICABIOGRAD Pup1 major tolerance gene is a constitutive enhancer of root growth Acts upstream of genes with key function in root growth and stress responseUpdate on mapping of Aluminum-toxicity tolerance at IRRI(PI: A. Ismail)Nurul Hidayatun from ICABIOGRAD is now PhD student at UP(i) Seeds of mapping populations are on the way from Indonesia(ii) Optimization of screening assay is ongoing***