powdery mildew resisatnt peas
TRANSCRIPT
Development of powdery mildew resistant and lodging tolerant Pisum sativum through marker assisted selection
Abdul GHAFOORInstitute of Agr-Biotechnology and genetic ResourcesNARC, Islamabad
Crop of interest!Disease
host-plant interactionpathogen variationdamage, economics
Genetics of resistanceverticalhorizontal
Screening techniquesGermplasm
extent of diversitysource of resistance
Breeding decisionconventional biotechnology
Markers, sources, development!
Germplasm improvement for disease resistance
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
Background knowledge
DiversityIdentificationUtilization
Germplasm preserved in Genebank
800 accessions
Indigenous genetic resources
56 accessions
Acquired from exotic sources
744 accessions (from more than 40 countries)
Characterized 412 accessionsVariation observed Flower color, Fresh pod surface, Fresh seed color,
Dry seed testa color, Hilum color, Seed surface, Seed shape, Cotyledon color, Spots on testa
Evaluation (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
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, 805–810].
No association of protein markers with powdery mildew
Diversity 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
Source GenotypesPowdery mildew resistant 10603, 10628, Fallon, PS9910238,
PS0010128, DMR 4, DMR 7, DMR 20Vegetable use 10303, 10567, 10478, 10628, DMR-4 Grain use 88 P 001-4-9, 10609, P 75/87, 88 P
007-2-1, 10604 Dual use DMR-20, 88 P 090-5-21, 10634
Powdery mildew resistant and lodging tolerant lines
PSO735 M91, PSO735 M61, PSO735 M39, PSO 735 M116
Suitable for split “dal” PSO735 M86
Identification of elite lines
Evaluation of elite lines of Pisum sativum
Germplasm catalog published by IABGR
Source Causal organism
Gene/allele Marker Reference
Vigna radiata Erysiphe polygoni DC
Two genesPMR1, PMR2
RFLP, AFLP Humphry et al. 2003 ; Chaitieng et al. 2002 ; Miyagi et al. 2004
Glycine max Erysiphe Single gene (Rmd)
RFLP, RAPD Lohnes & Bernard 1992; Polzin et al. 1994
Phaseolus vulgaris Erysiphe Two major genes
RAPD, AFLP Rezende et al. 1999; Johnson et al. 1995
Triticum aestivum Erysiphe DC Multiple loci (more than 30 loci)
RAPD, SSR, SCAR, AFLP, RFPL
Huang et al. 2000a; Chantret et al. 2001;
Hordeum vulgare Blumeria graminis (DC) EO Speer f.sp. hordei
Multiple loci RFLP Kurth et al. 2001
Rosa spp. Podosphaera pannosa
Single gene (Rpp1)
AFLPs, RGAs
Linde and Debener 2003; Linde et al. 2006
Malus pumila Mill. Podosphaera leucotricha
Single gene (Pl-w)
Isozymes, SCAR, SSR, AFLP, RAPD
Evans and James 2003; Liebhard et al. 2002; Hemmat et al. 1994; Batlle and Alston 1996
Lycopersicon parviflorum
Oidium lycopersici
Two AFLP, RFLP, CAP
Bai et al. 2003; Huang et al. 2000b
Molecular markers linked to powdery mildew resistance in various crops including legumes
Screening techniques
Under natural field conditions
Spores collected from the susceptible checks and inoculated the test entries mechanically in greenhouse
Detached leaf disk assay
Any other?
Screening against powdery mildew and regeneration of pea genetic resources from ICARDA, Syria under greenhouse
Screening for powdery mildew under greenhouse
Sources of powdery mildew in Pea
DMR 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
Worldwide sources resistance to powdery mildew in PeaVarieties Origin ReferenceGlenroy (er1), Kiley (er1) (er1), Mukta (er1), M257-3-6 (er1), M257-5-1 (er1), PSI 11, ATC 1181 (er1)
Australia Liu et al. 2003
LE 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)
India Liu et al. 2003; Mishra and Shukla 1984; Tiwari et al. 1997a; Iqbal et al. 2001
ATC 649 (er1), ATC 1036 (er1), SVP 950 (er1), JI 1210 (er1), JI 2302 Stratagem (er1), Fallon (er1), PS99102238 (er1), PS0010128 (er1)
USA Liu et al. 2003; Heringa et al. 1969; Tiwari et al. 1997a; Nisar et al. 2006
JI 1559 Mexique 4 (er1) Mexico Tiwari et al. 1997aATC 767, Highlight (er1), Sweden Liu et al. 2003; Tiwari et al. 1997a;
Tiwari et al. 1998ATC 1121(er1) Netherlands Liu et al. 2003955180 (er1), AC Tamor (er1), Tara (er1) Canada Ek et al. 2005; Tiwari et al. 1997a
JI 2480 (er2) UK Tiwari et al. 1997aJI 1951 (er1) China Tiwari et al. 1997aErygel France Dirlewanger et al. 1994JI 82 (er1) Afghanistan Tiwari et al. 1997a10603 (Unknown), 10628 (Unknown) Pakistan Ahmad et al. 2001
Quantum (Unknown) New Zealand Viljanen-Rollinson et al. 1998
Cross Expected segregantsDMR4/13240 Powdery mildew resistanceFallon/11760-3 Powdery mildew resistancePS610152/11760-3 Powdery mildew resistant and
lodging tolerantPS99102238/11760-3 Powdery mildew resistant and
lodging tolerant19634/18340 Pod size
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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Resistant to powdery mildew Susceptible to powdery mildew
1200 (bp)
DNA fragments amplified by Sc OPO18 marker
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PSMPSAD-51 PSMPSAD-141
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DNA fragments amplified by STMS primers
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
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
Powdery mildew resistant and lodging tolerantadvanced line derived from the hybrid PS99102238/11760-3 using OPB18.
DNA markers reported for MAS for powdery mildew in pea
Marker Distance (cM) from er
Marker type Reference er gene
Sc-OPO-181200 0.0 RAPD/SCAR Tiwari et al. 1998 er1OPL-6 2.0 RAPD Tiwari et al. 1998 er1OPD 10650 2.1 RAPD/SCAR Timmerman et al. 1994 er1Sc-OPD10650 3.4 SCAR Janila and Sharma 2004 erSc-OPE-161600 4.0 RAPD/SCAR Tiwari et al. 1998 er1P236 9.8 RFLP Dirlewanger et al. 1994 erPSMPSAD60 10.4 SSR Ek et al. 2005 er1PSMPSAA374e 11.6 SSR Ek et al. 2005 er1PSMPA5 14.9 SSR Ek et al. 2005 er1ScX171400 2.6 SCAR Katoch et al. 2010 er2SCAB1874 2.8 SCAR Fonddevilla et al. 2008 Er3BC210 8.2 RADP/SCAR Tonguc and Weeden (2010) er1OPB18430 11.2 RAPD Nisar and Ghafoor 2011 er1
Powdery mildew resistant (right) and susceptible (left) lines of Pisum sativum. Powdery mildew symptoms on leaves (lower left) and on pods (lower right)
Powdery mildew resistant and lodging tolerant promising lines of Pisum sativum developed at IABGR
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
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. Common mapping populations.
Screening for powdery mildew could be with a leaf disk assay, detached leaf assay, tapping spores and mechanical inoculation, or other in-vitro method. There is a need to reconfirm DNA markers for use in breeding pea for powdery mildew resistance.
Molecular markers have potential scope for target plant breeding – the induced evolution.Molecular markers are not equal and none is ideal, select the best one/s. Some are better for some purposes than others. All are generally preferable to morphological markers for mapping, frequency and polymorphism high.DNA markers are not universal, need conformation or development!Integrated approach involving breeders and molecular geneticists.
Take home message
Powdery mildew resistant, lodging tolerant high yielding cultivar of pea with low water requirement
Marker assisted selection (MAS) for developing powdery mildew resistant pea cultivars. Euphytica (DOI: 10.1007/s10681-011-0596-6).Linkage of a RAPD marker with powdery mildew resistance er-1 gene in Pisum sativum L. RJG, 47: 300-304. Phenotypic variation in the agronomic and morphological traits of Pisum sativum L. germplasm obtained from different parts of the world. RJG, 47: 19-25. First proteomic assay of Pakistani Pisum sativum L. germplam relation to geographic pattern. RJG, 45: 807-812.Inheritance Studies of Pisum sativum F1, F2 and F3 Generation Based Morphological Traits and Selection of High Yielding Powdery Mildew Resistant Lines. Molecular Plant Breeding, 7: 1-6.Screening of Pisum sativum (L.) Germplasm against Erysiphe pisi Syd. in relation with vegetative traits and SDS-PAGE profile. Acta Biologica Cracoviensia, Series Botanica, 48: 33-37.Novel protocol for albumin and globulin detection in Pisum sativum genotypes using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). PJB, 43: 1733-1734.Genetic diversity for determining yield potential and selection criteria in Pisum sativum (L.) genetic resources. PJB, 41: 2987-2993.Genetic similarity of Pakistani pea (Pisum sativum L.) germplasm with world collection using cluster analysis and Jaccard’s similarity index. J Chem Soc Pak, 31: 138-144.Seed protein profiling of Pisum sativum (L) germplasm using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) for investigation of biodiversity. PJB, 40: 2315-2321.Evaluation of genetic diversity of pea germplasm through phenotypic trait analysis. PJB, 40: 2081-2086.Evaluation of genetic diversity present in pea (Pisum sativum L.) germplasm base on morphological traits, resistance to powdery mildew and molecular characteristics. PJB, 39: 2739-2747.Genetic diversity in Pisum sativum and a strategy for indigenous biodiversity conservation. PJB, 37: 71-77. Evaluation of local and exotic pea (Pisum sativum) germplasm for vegetable and dry grain traits. PJB, 34):419-427.Yield potential of local and exotic germplasm with special reference to powdery mildew disease in peas [Pisum sativum (L.)]. PJB, 33(3): 251-255.
Knowledge generated
AcknowledgementsFarmers and international genebanks for collection and acquisition of genetic resources
Dr. Muhammad Nisar, Zahir Ali, Muhammad Sajid (R & D)
Dr. Sheikh Muhammad Iqbal, Dr. Asif Javaid (R & D)
IABGR for executing experimentations