Application of biotechnology in breeding lentil for resistance to biotic and abiotic stress

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  • Euphytica (2006) 147: 149165DOI: 10.1007/s10681-006-7108-0 C Springer 2006

    Application of biotechnology in breeding lentil for resistance to biotic andabiotic stress

    Frederick J. Muehlbauer1,, Seungho Cho3, Ashutosh Sarker4, Kevin E. McPhee1,Clarice J. Coyne2, P.N. Rajesh1 & Rebecca Ford51U.S. Department of Agriculture, Agricultural Research Service, 303 Johnson Hall, Washington State University,Pullman, WA 99164-6434, U.S.A.; 2U.S. Department of Agriculture, Agricultural Research Service, 59 JohnsonHall, Washington State University, Pullman, WA 99164-6402, U.S.A.; 3Department of Agronomy and Plant Genetics,University of Minnesota, St. Paul, MN, U.S.A.; 4Germplasm Program, International Center for Agricultural Researchin the Dry Areas, Aleppo, Syria; 5School of Agricultural and Food Systems, the University of Melbourne, Melbourne,Victoria, Australia (author for correspondence: e-mail: 31 December 2004; accepted 31 January 2006

    Key words: genetic mapping, genomics, Lens culinaris, marker-assisted-selection, synteny


    Lentil is a self-pollinating diploid (2n = 14 chromosomes) annual cool season legume crop that is produced through-out the world and is highly valued as a high protein food. Several abiotic stresses are important to lentil yields worldwide and include drought, heat, salt susceptibility and iron deficiency. The biotic stresses are numerous and include:susceptibility to Ascochyta blight, caused by Ascochyta lentis; Anthracnose, caused by Colletotrichum truncatum;Fusarium wilt, caused by Fusarium oxysporum; Sclerotinia white mold, caused by Sclerotinia sclerotiorum; rust,caused by Uromyces fabae; and numerous aphid transmitted viruses. Lentil is also highly susceptible to severalspecies of Orabanche prevalent in the Mediterranean region, for which there does not appear to be much resistancein the germplasm. Plant breeders and geneticists have addressed these stresses by identifying resistant/tolerantgermplasm, determining the genetics involved and the genetic map positions of the resistant genes. To this endprogress has been made in mapping the lentil genome and several genetic maps are available that eventually willlead to the development of a consensus map for lentil. Marker density has been limited in the published genetic mapsand there is a distinct lack of co-dominant markers that would facilitate comparisons of the available genetic mapsand efficient identification of markers closely linked to genes of interest. Molecular breeding of lentil for diseaseresistance genes using marker assisted selection, particularly for resistance to Ascochyta blight and Anthracnose, isunderway in Australia and Canada and promising results have been obtained. Comparative genomics and syntenyanalyses with closely related legumes promises to further advance the knowledge of the lentil genome and providelentil breeders with additional genes and selectable markers for use in marker assisted selection. Genomic toolssuch as macro and micro arrays, reverse genetics and genetic transformation are emerging technologies that mayeventually be available for use in lentil crop improvement.


    Lentil (Lens culinaris Medik.) is a highly valued annualfood legume crop that coevolved with wheat, barley and

    other cool season pulses in the Near East arc about 8000years ago (Cubero, 1981; Ladizinsky, 1979). WorldProduction of lentil is estimated at 3.3 million met-ric tons from an estimated 3.8 million hectares with

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    an average yield of 850 kg/ha FAOSTAT, 2005). Theseyields seem small, but generally lentil is produced onmarginal lands that are relatively dry and without thebenefit of fertilizer inputs or irrigation. Attempts to im-prove yields through breeding are underway through-out the world and especially at the International Centerfor Agricultural Research in the Dry Areas (ICARDA),USA, Canada, Australia, Turkey, South Asia and manycountries of the West Asia-North Africa (WANA) re-gion. Major producing countries are India, Turkey,Canada, Australia, Ethiopia, Morocco, Spain, Chileand Argentina. Of these, the major exporting countriesare Canada, Australia and the U.S. The countries of theMiddle East and North Africa are major consumers.

    The genus Lens comprises seven taxa in fourspecies (Ferguson & Erskine, 2001; Ferguson et al.,2000). Lens orientalis is the presumed progenitor ofcultivated L. culinaris and the two species are fullycrossable and produce fully fertile progenies. How-ever, except for L. odemensis, there is difficulty incrossing the other wild species to the cultigen. Fromthe stand point of crossability for use in breeding, theLens species can be divided into two groups (Ladizin-sky, 1999): L. culinarisL. odemensis and L. ervoidesL. nigricans that for convenience can be assigned toprimary and secondary gene pools. Crosses betweenmembers of different groups fail because of hybrid em-bryo abortion; however, embryo rescue has been usedsuccessfully to obtain viable hybrids between groups(Ladizinsky et al., 1985).

    Several abiotic stresses such as cold, drought,heat, salinity, nutrient deficiency and nutrient tox-icity adversely affect lentil yields world wide(Monti et al., 1994; Saxena, 1993; Slindard et al.,1994). Numerous biotic stresses that adversely af-fect lentil include: susceptibility to Ascochyta blight,caused by Ascochyta lentis; Anthracnose, caused byColletotrichum truncatum; Fusarium wilt, caused byFusarium oxysporum; root rots caused by Fusariumsolani, Aphanomyces eutieches, Pythium ultimum,Rhizoctonia solani and possibly other pathogens; Scle-rotinia white mold, caused by Sclerotinia sclerotiorum;rust, caused by Uromyces fabae; and numerous aphidtransmitted viruses. Lentil is also highly susceptible toseveral species of Orabanche prevalent in the Mediter-ranean region, for which there does not appear to bemuch resistance in the germplasm. Resistance to someof these stresses has been found in germplasm collec-tions. See Erskine et al. (1994b) for a review of strate-gies for breeding lentil for resistance to biotic and abi-otic stresses.

    Molecular markers and gene tagging has pro-vided plant breeders with a means to accelerate breed-ing programs and an efficient tool for indirect selectionfor traits that would otherwise be difficult to se-lect for using standard procedures. Biotechnologicaltechniques such as Microarrays, TILLING, Genetictransformation and others hold promise for improv-ing our knowledge of the lentil genome and acceler-ating progress through breeding. Our objectives wereto review the status of available and emerging biotech-nologies and their current and potential application forovercoming major biotic and abiotic stresses of lentilcrops.

    Constraints to production of lentil

    Abiotic stresses

    The major abiotic stresses affecting cool season foodlegumes were the subject of a comprehensive review(Singh & Saxena, 1993). Stresses that affect lentil werelisted as cold, drought, heat, salinity, nutrient deficiencyand nutrient toxicity. Of these stresses, drought and heatare considered the most important world wide (Turneret al., 2001). Cold stress was considered important inthe West Asia-North Africa (WANA) region. Salinityis an important stress factor in the Indian sub-continentand to some extent in WANA. Nutrient deficiency andnutrient toxicity is of lesser importance world widebut important in localized regions. For breeding ap-proaches for crop improvement in stress environmentssee the review by Buddenhagen and Richards (1998).

    Susceptibility to cold temperatures has limited pro-duction of lentil in cold highland areas of the world.However, germplasm is available that has useful degreeof tolerance to cold temperatures which makes it pos-sible to breed winter hardy cultivars that can be plantedin the fall with a reasonable expectation of survivingthe winter (Erskine et al., 1981; Spaeth & Muehlbauer,1991). Kahraman et al. (2004a) reported that winterhardiness in lentil is conferred by several genes. In agenetic analysis of winter hardiness, Kahraman et al.(2004b) showed that the combined effects of severalquantitative trait loci accounted for 42% of the varia-tion in winter survival. Molecular markers associatedwith those QTL have potential use in a marker assistedselection program for winter hardiness, but first mustundergo validation.

    Lentil is able to produce something of value in manyof the semi-arid regions primarily through drought

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    avoidance (Erskine & Saxena, 1993; Turner et al.,2001). Early senescence and crop maturity forced bydrought conditions that are often more severe due tothe usually associated high temperatures. Rainfall ac-counted for 41% and 55% of the variation in yieldsof two lentil cultivars, respectively, in Syria. Variousprocedures have been attempted for screening lentilgermplasm and breeding material for drought toler-ance. Field screening using line source irrigation todevelop a moisture gradient has been attempted atICARDA. Also, covering field plot areas to restrictmoisture to form moisture differential areas, the useof late planting to increase exposure of the plants tohigher temperatures and greater moisture stress haveall been attempted with inconclusive results. Increasedrooting depth as a means of drought avoidance has beensuggested; however, that approach may be at consider-able expense to succeeding crops, most likely wheat.Molecular approaches such as marker assisted selectionmay have merit; however, considerable work is neededto identify the important regions of the genome, mostlikely through a QTL analysis, and validation of asso-ciated molecular markers.

    Heat stress often accompanies drought causing dif-ficulties in separating the two stresses and their effectson lentil growth and yield. There is general agreementthat heat affects the distribution of dry matter to re-productive growth and that high temperatures have anadverse effect on lentil yields. Research is needed tocritically define the effects of heat on the reproduc-tive stages of lentil and the effects on yield. Evalua-tion of the world collection of lentil germplasm hasindicated useful genetic variation is available for im-proving adaptation to environmental extremes (Erskineet al., 1990, 1994b).

    Salinity problems with lentil are not wide spreadbut can be acute in certain regions of South Asia,the Nile delta of Egypt and in some areas of Turkey.Canada also has some difficulty in high salinity areas ofSaskatchewan. Of the legumes, lentil is more salt sensi-tive when compared to faba bean and soybean (Katerjiet al., 2001, 2003). Salt stress can adversely affect nodu-lation and N2 fixation (Rai & Singh, 1999; Rai et al.,1985) presumably by restricting growth of the root hairsand the potential sites of infection by Rhizobium. Somegermplasm with tolerance to salt stress has been identi-fied (Ashraf & Waheed, 1993; Ashraf et al., 1990; Jana& Slinkard, 1979).

    Nutrient deficiencies and toxicities are important inspecific situations. Iron deficiency symptoms appear ingermplasm introduced to other areas from the Indian

    subcontinent and Ethiopia and has been shown to have agenetic basis ( Erskine, 1997; Erskine et al., 1993). Theacute yellowing symptomatic of iron deficiency canbe alleviated by germplasm efficient in the utilizationof iron (Erskine, 1997; Erskine et al., 1993). Borontoxicity has also been problematic and there appears tobe some tolerant germplasm (Yau & Erskine, 2000).

    Biotic stresses

    Foliar diseases are the most serious biotic stresses af-fecting lentil crops. Ascochyta blight caused by A.lentis is problematic to various degrees in all lentilgrowing regions of the world, but especially dam-aging in Canada (Ahmed & Morrall, 1996; Ahmedet al., 1996), Australia and Middle Eastern countries(Johansen et al., 1994). Symptoms of Ascochyta blightinclude lesions on all above ground parts of the plant,stem girdling, pod and seed lesions and resistance hasbeen found in the germplasm (Andrahennadi et al.,1996). Other major biotic stresses of lentil include An-thracnose caused by C. truncatum, Botrytis grey moldcaused by Botrytis fabae and B. cinerea, Stemphyliumblight caused by Stemphylium botryosum, lentil rustcaused by U. fabae and Sclerotinia white mold causedby S. sclerotiorum. See Tivoli et al. this volume fora description of Ascochyta blight, Anthracnose andBotrytis grey mold.

    Stemphylium blight is a major threat to lentilin South Asia and North America (ICARDA, 2004;Vandenberg: Personal communication). The pathogencauses a leaf blight, plant defoliation and death. Thedisease is poorly understood but apparently there issome resistance available in the germplasm; however,the genetics of resistance is still to be determined.

    Lentil rust caused by U. fabae is widespread inSouth Asia, Morocco and Ethiopia and is character-ized by lesions on the stems and leaves, leaf drop andpremature plant death (Ahmed & Morral, 1996; Ahmedet al., 1996). Losses from the disease, estimated at upto 70%, have been reported (Erskine and Sarker, 1997;Negussie et al., 1998). Resistance to the disease hasbeen identified in germplasm line ILL5588 and is cur-rently being used as a source of resistance in breedingprograms and in studies to determine the inheritance ofresistance and to map the important genes (Ahmed &Morral, 1996; Ahmed et al., 1996).

    Sclerotinia white mold caused by S. sclerotiorumhas been responsible for extensive damage to lentilcrops in areas that are relatively moist and humid.Dense crop canopies also contribute to the severity

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    of the disease. The pathogen affects 148 known plantgenera and there is little resistance to the disease. Re-search is underway by the USDA-ARS Grain LegumeBreeding program at Pullman to determine the genet-ics of resistance/tolerance found in lentil cultivars andgermplasm. Research is currently underway towardsconducting a Quantitative Trait Loci analysis using re-combinant inbred lines from crosses of presumed re-sistant cultivars with highly susceptible cultivars.

    Root rots and wilts usually attack lentil plants inthe seedling stage and cause seed rot, damping off,wilt, destruction of the root system and rotting of lowerstems. These disease problems have been extensivelyreviewed (Khare, 1981; Kraft et al., 1988, 1994). Fusar-ium wilt caused by F. oxysporum is considered to bethe most damaging soil borne disease problem facinglentil world wide (Khare, 1981) and several races of thepathogen can cause the disease. Hamwieh et al. (2005)developed a genetic map of lentil that comprises over300 molecular markers and used the map to determinethe location of the gene for resistance to Fusarium wilt(Figure 1).

    Molecular markers and marker assisted selection

    The current state of molecular breeding in lentil

    Lentil is a relatively small crop compared to wheat,rice, maize and soybean and ranks third among the coolseason food legumes in area harvested (4.08 millionhectares) annually (, and be-hind pea and chickpea. Muc...