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Development of powdery mildew resistant and lodging tolerant Pisum sativum through marker assisted selection

Abdul GHAFOORInstitute of Agr-Biotechnology and genetic ResourcesNARC, Islamabad

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Crop of interest!Diseasehost-plant interactionpathogen variationdamage, economics Genetics of resistanceverticalhorizontal Screening techniquesGermplasmextent of diversitysource of resistance Breeding decisionconventional biotechnologyMarkers, sources, development! Germplasm improvement for disease resistance

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Pea Pisum sativum vegetable, fresh pods, fresh/dry seedsGenetic diversity linear relationship with crop improvement Powdery mildew up to 35% losses, low quality seed productionLodging a problem associated with peas

Conventional breedingMarker Assisted Selection

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Background knowledgeDiversityIdentificationUtilization

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Germplasm preserved in Genebank 800 accessionsIndigenous genetic resources56 accessionsAcquired from exotic sources744 accessions (from more than 40 countries)Characterized 412 accessionsVariation observedFlower color, Fresh pod surface, Fresh seed color, Dry seed testa color, Hilum color, Seed surface, Seed shape, Cotyledon color, Spots on testaEvaluation (two years)398 accessionsVariation observed Flower initiation, flower completion, mature pod picking, pod completion, dry pods appearance, harvesting, branches, plant height, fresh pod weight, fresh pod width, fresh pod length, dry pods width, dry pod weight, dry pod length, grain yield, biomass, harvest index

Background knowledge

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Thirty four bands were observed, 75% were polymorphic. The germplasm collected from Punjab, and KPK exhibited 70%, and 60% variation, respectively.

Low to moderate level of association between genetic diversity and geographic pattern of the genotypes [Nisar et al., 2009. Russian Journal of Genetics 45, 805810].

No association of protein markers with powdery mildewDiversity in indigenous Pisum sativum for seed protein profiles

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Relationship among Pakistani germplasm with germplasm obtained from 37 countries for seed storage protein profile among 222 genotypes.GD ranged from 0 71% across the countriesbroad base of genetic resources>60% genetic similarity of Pakistani genotypes with ICARDA, UK, India, Ethiopia and Australia. The germplasm of Ecuador, Guinea, Iraq, Uzbekistan, Lebanon, Norway, and Morocco showed less >19% genetic similarity. >80% genetic similarity was calculated between the germplasm of Pakistan with ICARDA and India.No association with powdery mildew

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Source GenotypesPowdery mildew resistant10603, 10628, Fallon, PS9910238, PS0010128, DMR 4, DMR 7, DMR 20Vegetable use10303, 10567, 10478, 10628, DMR-4 Grain use88 P 001-4-9, 10609, P 75/87, 88 P 007-2-1, 10604 Dual useDMR-20, 88 P 090-5-21, 10634 Powdery mildew resistant and lodging tolerant lines PSO735 M91, PSO735 M61, PSO735 M39, PSO 735 M116Suitable for split dalPSO735 M86

Identification of elite lines

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Evaluation of elite lines of Pisum sativum

Germplasm catalog published by IABGR

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Source Causal organism Gene/alleleMarker ReferenceVigna radiataErysiphe polygoni DCTwo genesPMR1, PMR2 RFLP, AFLPHumphry et al. 2003; Chaitieng et al. 2002; Miyagi et al. 2004Glycine maxErysipheSingle gene (Rmd)RFLP, RAPDLohnes & Bernard 1992; Polzin et al. 1994Phaseolus vulgarisErysipheTwo major genesRAPD, AFLPRezende et al. 1999; Johnson et al. 1995Triticum aestivum Erysiphe DCMultiple loci (more than 30 loci)RAPD, SSR, SCAR, AFLP, RFPLHuang et al. 2000a; Chantret et al. 2001; Hordeum vulgare Blumeria graminis (DC) EO Speer f.sp. hordeiMultiple lociRFLPKurth et al. 2001Rosa spp.Podosphaera pannosa Single gene (Rpp1)AFLPs, RGAsLinde and Debener 2003; Linde et al. 2006Malus pumila Mill.Podosphaera leucotrichaSingle gene (Pl-w)Isozymes, SCAR, SSR, AFLP, RAPDEvans and James 2003; Liebhard et al. 2002; Hemmat et al. 1994; Batlle and Alston 1996Lycopersicon parviflorum Oidium lycopersiciTwo AFLP, RFLP, CAPBai et al. 2003; Huang et al. 2000b

Molecular markers linked to powdery mildew resistance in various crops including legumes

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Screening techniques

Under natural field conditionsSpores collected from the susceptible checks and inoculated the test entries mechanically in greenhouseDetached leaf disk assayAny other?

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Screening against powdery mildew and regeneration of pea genetic resources from ICARDA, Syria under greenhouse

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Screening for powdery mildew under greenhouse

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Sources of powdery mildew in PeaDMR 4, DMR 7, DMR 20, 10603, 10599, 3279, 3196, 10628, Fallon, PS9910238, PS0010128, PS0735 M116, PS0735 M108, PS0735 M92, PS0735 M83, PS0735 M79, PS0735 M86, PS0735 M102

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Worldwide sources resistance to powdery mildew in PeaVarieties OriginReferenceGlenroy (er1), Kiley (er1) (er1), Mukta (er1), M257-3-6 (er1), M257-5-1 (er1), PSI 11, ATC 1181 (er1)AustraliaLiu et al. 2003LE 25 (er1), ATC 823 (er1), KPMR- 10 (er1), T- 10, P- 185, 6533 (er1), 6587 (er1), 6588 (er1), JI 210 (er1), DMR 4 (Unknown), DMR 7 (Unknown), DMR 20 (Unknown)IndiaLiu et al. 2003; Mishra and Shukla 1984; Tiwari et al. 1997a; Iqbal et al. 2001ATC 649 (er1), ATC 1036 (er1), SVP 950 (er1), JI 1210 (er1), JI 2302 Stratagem (er1), Fallon (er1), PS99102238 (er1), PS0010128 (er1)USALiu et al. 2003; Heringa et al. 1969; Tiwari et al. 1997a; Nisar et al. 2006JI 1559 Mexique 4 (er1) MexicoTiwari et al. 1997aATC 767, Highlight (er1), SwedenLiu et al. 2003; Tiwari et al. 1997a; Tiwari et al. 1998ATC 1121(er1)NetherlandsLiu et al. 2003955180 (er1), AC Tamor (er1), Tara (er1)CanadaEk et al. 2005; Tiwari et al. 1997aJI 2480 (er2)UKTiwari et al. 1997aJI 1951 (er1)ChinaTiwari et al. 1997aErygelFranceDirlewanger et al. 1994JI 82 (er1)Afghanistan Tiwari et al. 1997a10603 (Unknown), 10628 (Unknown)Pakistan Ahmad et al. 2001Quantum (Unknown)New ZealandViljanen-Rollinson et al. 1998

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CrossExpected segregantsDMR4/13240Powdery mildew resistanceFallon/11760-3Powdery mildew resistancePS610152/11760-3Powdery mildew resistant and lodging tolerantPS99102238/11760-3Powdery mildew resistant and lodging tolerant19634/18340Pod size

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Linkage of a RAPD marker with powdery mildew resistance er1 gene in Pisum sativum L.Among 43 RADP primers, 21 were polymorphic and one was linked with er1.Falloner and 11760-3ER selected under heavy infestation with Erysiphe pisi through artificial inoculation under greenhouse. F1 plants (Fallon/11760-3) indicated dominance of the susceptible allele, F2 plants segregated in 3:1 ratio (susceptible: resistant). A RAPD maker OPB18 (5-CCACAGCAGT-3) was linked to the er1 gene with 83% probability, and was located at a distance of 11.2 cM from the er1 gene.

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ScOPO18 reported linkage at 0cM by Tiwari et al. 1998, but not linked across the genotypes.Check prior to use Have universal population for basic study STMS associated to er1

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RAPD still valid when converted to SCAR (Sequence characterized amplified region) Markers are crop, population and locus specific, not universal!Should be critical for marker to be implied for!MappingMASFingerprintingGenotyping Cost-effective?

Molecular markers for MAS

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Powdery mildew resistant and lodging tolerantadvanced line derived from the hybrid PS99102238/11760-3 using OPB18.

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DNA markers reported for MAS for powdery mildew in peaMarker Distance (cM) from erMarker typeReferenceer geneSc-OPO-1812000.0RAPD/SCARTiwari et al. 1998er1OPL-62.0RAPDTiwari et al. 1998er1OPD 106502.1RAPD/SCARTimmerman et al. 1994er1Sc-OPD106503.4SCARJanila and Sharma 2004erSc-OPE-1616004.0RAPD/SCARTiwari et al. 1998er1P2369.8RFLPDirlewanger et al. 1994erPSMPSAD6010.4SSREk et al. 2005er1PSMPSAA374e11.6SSREk et al. 2005er1PSMPA514.9SSREk et al. 2005er1ScX1714002.6SCARKatoch et al. 2010er2SCAB18742.8SCARFonddevilla et al. 2008Er3BC2108.2RADP/SCARTonguc and Weeden (2010)er1OPB1843011.2RAPDNisar and Ghafoor 2011er1

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Powdery mildew resistant (right) and susceptible (left) lines of Pisum sativum. Powdery mildew symptoms on leaves (lower left) and on pods (lower right)

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Powdery mildew resistant and lodging tolerant promising lines of Pisum sativum developed at IABGR

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Progress/salient achievementsEstablished diverse germplasm collection of more than 800 accessions from various sourcesCharacterized and evaluated half of the materialPublished pea catalog with evaluation and passport informationIdentified powdery mildew resistant linesDeveloped powdery mildew resistant and lodging resistant genotypes using molecular techniques coupled with conventional breedingDistributed >500 accessions at multiple time to breeders, students and researchers Mapped powdery mildew resistant gene linked to OPB18430 in the cross Falloner/11760-3ER, Fallon being exotic and resistant to powdery mildewUtilized powdery mildew resistant sources and enhanced breeding populations through Single Seed Descent (SSD) method

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ConclusionThe er1 and er2 characterized, er1 widely used, could be risky [three genes and new pathogens [Ondrej et al., 2005; Attanayake et al. 2010]. Molecular markers linked to er1 and er2 are available, the gene er2 is not commonly used, expression is influenced by temperature and leaf age. Newly identified Er3 gene is needed to characterize for utilization, the RAPD markers linked to Er3 have been converted into SCARs. Molecular markers are useful for a variety of purposes relevant to crop improvement including disease resistance through MAS. Due to complexity and the high initial cost, Collaborative efforts be strengthened. Once markers are established, be tested for wider application. Comm