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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DNA fragments amplified by Sc OPO18 marker

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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