Indian Journal of BiotechnologyVol 2, January 2003, pp 99-109

Molecular Diagnosis and Application of DNA Markers in the Management ofFungal and Bacterial Plant Diseases

T R Sharma*National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 1[00[2, India

Successful management of plant diseases is mainly dependent on the accurate and efficient detection of plantpathogens, amount of genetic and pathogenic variability present in a pathogen population, development of diseaseresistant cultivars and deployment of effective resistance genes in different epidemiological regions. Besideconventional methods of pathogen detection and breeding resistant cultivars, recent developments in molecularbiology techniques particularly the advent of various DNA markers have greatly influenced the plant protectionmethods. Pathogen detection has relied on isolation of microorganisms and observations of symptoms they induce onsusceptible hosts. In many situations cultural, morphological and chemical markers have been used to studyvariation in pathogen populations. Such markers are limited, often unstable and are influenced by environmentalconditions. Molecular detection of plant pathogens and characterization of genetic variability by using different DNAmarkers have offered additional tools in the hands of plant pathologists and plant breeders. Various PCR based andhybridization based DNA marker techniques can be used for the characterization of genetic variability in pathogensand molecular tagging of disease resistance genes. DNA markers linked to specific resistance gene can be used inmarker-assisted-selection for resistance breeding, gene pyramiding and map-based cloning of the resistance genes. Inthis communication various uses of DNA markers in pathogen diagnostics and mapping, pyramiding and map-basedcloning of disease resistance genes have been discussed.

Keywords: chromosome walking, DNA fingerprinting, molecular tagging, gene pyramiding, resistance gene cloning

IntroductionManagement of plant diseases has been a high

priority area of research worldwide. In India, duringthe post 'Green Revolution' era, productivity of highyielding cultivars of various crops has been seriouslyaffected by pest and diseases, which can only besustained by the use of better plant protectionstrategies. Chemical management of plant diseases,though, quite easy and effective option, has variousinherent problems. The most important problemassociated with this method is environmentalpollution and health hazards caused by toxicchemicals used for the disease management. With theincreased awareness and health consciousness among

*Tcl: 91-11-25824787 Ext-302; Fax: 91-11-25823984E-mail: trsharma @nrcpb.orgAbbreviations:RFLPs: Restriction fragment length polymorph isms; RAPD:Random amplified polymorphic DNA, AFLPs: Amplifiedfragment length polymorph isms; STS: Sequence tagged sites;SJ Ps: Single nucleotide polymorph isms; PCR: Polymerase chainreaction; PFGE: Pulsed field gel electrophoresis; CHEF: Clampedhomogenous electric field; YAC: Yeast artificial chromosome,BAC: Bacterial artificial chromosome; MAS: Marker assistedselection; NILs: Near isogenic lines; RILs: Recombinant inbredlines.

the people, produce of organic farming is beingpreferred for consumption in the developed nations.Precise identification of disease-causing-organisms isthe basic requirement of deciding various diseasemanagement options. Disease management throughhost resistance involves characterization of pathogendiversity for the effective and prolonged life ofR-genes under natural conditions. Development anddeployment of cultivars with durable resistance toplant diseases is a very economic and easy method ofplant disease management. However, to develophomozygous resistant and agronomically superiorcultivars, it takes about 6-7 years of rigorous multilocations disease testing at the 'Hot Spots'. Molecularbiology tools are now being used to facilitate theconventional disease resistance-breeding programmeand to shorten the duration required to develop aresistant cultivar in different crops (Bent, 1999).Biotechnological approaches are now being used tofacilitate the existing disease management strategies.

DNA markers like RFLPs (Botstein et al, 1980),RAPD (Williams et al, 1991), AFLPs (Vos et aI,1995) and STS (Powell et al, 1996) are commonlybeing used for the molecular characterization of plant

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pathogens and mapping of disease resistance genes.Besides, molecular markers are also used in markerassisted selection, gene pyramiding and map-basedcloning of R-genes. Various R-genes have alreadybeen mapped and cloned by using molecular markers.The predicted products of R-genes have also beendeduced (Hulbert et al, 2001). These cloned R-genesare now being transferred and mobilized in the highyielding susceptible cultivars. The presentcommunication reports uses of molecular markers indiagnostics and characterization of genetic variabilityin plant pathogens, mapping, pyramiding and mapbased cloning of disease resistance genes.

Molecular Detection of Plant PathogensAccurate diagnosis and identification of plant

pathogens is a pre-requisite of disease management tosustain high yield potential of crops. Therefore,continuous efforts are being made to develop asimple, reliable, rapid and safe method for the diseasediagnosis. Visual identification of plant diseasesthough a very rapid method but hard to perform byinexperienced personnel and is limited particularly todiseases affecting aerial parts of the plants. It cannotbe performed effectively in case of soil and seedborne diseases, where several species of pathogensmay cause similar symptoms. Microscopicexamination of diseased tissues and identification ofpathogen on the basis of their morphologicalcharacteristics though a preferred method of diseasediagnosis, requires highly specialized taxonomists.

Some of the major advances occurred in plantpathogen detection during the past decades with thedevelopment of monoclonal antibodies (Kohler &Milstein, 1975) and enzyme linked immunosorbantassay (Clark & Adams, 1977). These are rapidmethods of viral and bacterial pathogen detection.However, the technique still has certain limitationsand sometimes-false epitopic detection leads toerroneous results.

Molecular detection and identification of pathogensusing nucleic acids based methods have been in usefor the past few years. These methods overcomevarious problems associated with microscopical andimmunological detection of plant pathogens. Mostimportantly, DNA based methods can be used at anydevelopmental stage of the pathogen, since everypathogen propagule/cell contains the entire set ofnucleic acids of the organism. Precise detection andidentification of plant pathogens can be performed bythe use of specific DNA probes in infected tissues and

identification at genus/species or even at race level.There are various methods used for making DNAprobes (Sharma et al, 2002a). These probes have beenused by various workers for pathogen detection(Sharma et al, 1999). Various non-radioactive DNAprobes used in the detection of plant pathogens by dotblot hybridization can also be developed by PCR(Sharma et al, 2002a). Such probes have already beendeveloped and used for the detection of manypathogens including Pythium ultimum (Levesque etal, 1994), Rhynchosporium secalis (Sharma et at,1996) and Alternaria brassicae (Sharma & Tewari,1996a, 1998).

PCR is a very rapid and sensitive method for themolecular diagnostics of plant pathogens, which areotherwise difficult to identify morphologically.Pathogen detection by using PCR is mainly dependenton the design of primers. Specific, semi- specific andarbitrary primers can be used for the PCR. Arbitraryprimers are used in RAPD to produce characteristicprofiles of amplified products. However, DNAsequences of plants and other organisms are alsoamplified with random primers, which lead toambiguous results. Thus, plant pathogens must beisolated from their hosts or reservoirs and purifiedbefore the DNA extraction. Hence, RAPD analysismay not be useful for the direct detection of plantpathogens in infected tissues or for the detection ofobligate parasites. However, longer and species-specific PCR primers allow detection of targetsequences in crude specimens of infected tissues.

Any DNA or RNA sequence that is specific for aparticular organism can be used to design specificprimers for the detection of that organism using PCR.Primers have also been designed on the basis ofpathogen -specific plasmid sequences. Such primershave been used to develop PCR based diagnosticassays for Xanthomonas campestris pv. phaseoli andX. c. pv. phaseoli var fuscans (Audy et al. 1994),Erwinia amylovora and X. campestris pv. cirri(McManus & Jones, 1995; Hartung et al, 1996).Genes controlling the specific properties (e.g. toxinproduction & pathogenecity) of pathogens have alsobeen used as target sequences for. pecific detection ofplant pathogen. PCR amplification of Tox gene region(phaseolotoxin producing gene) for the detection ofPseudomonas syringae pv. phaseolicola (Schaad etal, 1995) and efe (ethylene forming enzyme) gene forthe detection of P. syringae pvs. cannabina andsesami (Sato et al, 1997) have been successfully used.

SHARMA: MOLECULAR MARKERS AND PLANT DISEASE MANAGEMENT

Subtractive hybridization is another method ofpathogen detection, which can be used to selectpathogen and even strain specific sequences that arepotential targets for designing primers. The techniqueenriches nucleic acid sequences specific to aparticular organism or strain by hybridization andsubsequent removal of sequences that are commonwith other organisms. Specific detection ofClavibacter michigan ens is ssp. sepedonicum wasdone by amplification of these unique DNAsequences isolated by subtractive hybridization(Millis et al, 1997). Serological techniques can alsobe combined with PCR e.g. irnrnuno-capture PCR andimmuno-PCR. In immuno-capture PCR antigen isconcentrated by use of specific antibodies, which isthen subjected to PCR, while, immuno-PCR enhancesthe antigen-antibody reaction. Hartung et al (1996)used the immuno-capture technique for the detectionof X. campestris pv. citri.

Recent advances in genomics and molecularbiology have uncovered the complete genomesequence of two important plant pathogens, Xyllelafastidiosa (Simpson et al, 2000) and Ralstoniasolanacearum (Salanoubat et al, 2002). Using theunique sequence data of the pathogens DNA, probesand primers could be designed for the differentialdetection of pathogen and their characterization atmolecular level. Research in agricultural genomics isgenerating massive genome sequence data, which canbe used for the simultaneous detection of thousands ofplant pathogens and beneficial micro-organisms.Micro-arrays made from unique sequences from awide range of pathogens and beneficial organismscould be used to see how species are favoured orsuppressed by a given treatment in vivo. In the postgenomic era, molecular characterization and detectionof plant pathogens would be better done by the use ofsingle nucleotide polymorphisms (SNPs). Thesemarkers can detect differences at single base pairlevel, which is the ultimate limit of moleculardetection and has been successfully used for thedetection of oomycetous fungi (Levesque et al, 1998)and bacteria (Taylor et al, 2001).

Characterization of Genetic Variability in PlantPathogens using DNA Markers

All the disease management strategies based onhost resistance require the knowledge of variability inpathogens. Traditional markers used to study thevariability in pathogens are based on the use of

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differential hosts, culture characteristics,morphological markers and biochemical tests. Thesemarkers distinguish pathogens on the basis of theirphysiological characters i.e. pathogenecity and growthbehaviours and can group them according to theirsimilarity for these particular characters. However.these markers are highly influenced by the host age,inoculum quality and environmental conditions. Thetechniques are time-consuming and laborious.Moreover, differential hosts are not available in mostof the host-pathogen systems, thus variability can notbe assessed. In such cases, molecular markers areused for the characterization of genetic variability inplant pathogens (Sharma et al, 1999). Using PCR,very closely related strains of a pathogen can bedistinguished without prior knowledge of the natureof polymorphic regions by the use of RAPD(Williams et al, 1990). The DNA fingerprints(banding patterns) generated by RAPD are comparedfor their relatedness using genetic similaritycoefficients and phylogenetic trees are constructed.PCR based DNA fingerprinting, particularly withshort oligonucleotide primers (Williams et al, 1990)has been used for the analysis of genetic variation insome plant pathogens (Table 1).

In India, twenty isolates of A. brassicae collectedfrom geographically distinct regions of the world anddifferent host species with RAPD markers have alsobeen analyzed (Sharma & Tewari, 1995, 1998). Outof the five primers tested, primers OPA 07 and OPA09 could not distinguish variation among theseisolates. However, three primers e.g. OPA 03, OPA04 and OPA 18 were efficient in the detection ofinter- and intra-regional variation among the isolatesof A. brassicae. Using parsimony analysis, 4 groupswere obtained with isolates of mixed geographicalorigins. There was no clear grouping of isolates fromdifferent geographical regions because of theformation of mixed or poorly resolved clusters(Sharma & Tewari, 1995, 1998).

RAPD analysis also distinguished genetic variationamong seven A. brassicicola isolates collected fromFrance, Canada and England with all the primerstested. However, grouping of isolates according totheir geographical origin could not be made becauseof small number of isolates. Two isolates ofA. raphani, one each from Canada and France.produced discrete fingerprints indicating geneticvariation in these isolates (Sharma & Tewari, 1996b.1998).

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Table I-Plant pathogens characterized by molecular markers

Pathogen Disease Marker References

Alternaria brassicae Leaf spot of crucifers RAPD Sharma & Tewari, 1995Xanthomonas campestris pv. vesicatoria Bacterial leaf spot of papper and ERIC Louws et al, 1995

tomato REP-PCRverticillium dahliae Wide host range RAPD KoIke et al, 1996V. alboatrumAlternaria brassicae Leaf spot of crucifers RAPD Sharma & Tewari, 1998A. brassicicolaA. rapluuiiAscochyta rabie Chickpea blight RAPD Udupa et al, 1998Pseudomonas syringae pv, pisi Bacterial blight of peas REP-PCR Koike et al, 1999Leptosphaeria tnaculans Black leg of crucifers AFLP Pongam et al, 1999Rhynchosporium secalis Barley scald RAPD Sharma et al. 2000Xanthonionas campastris pv. oryzae Bacterial blight of rice Finckh & Nelson, 2000Magnaporthe grisea Rice blast RFLP Viji et al, 2000Leptosphaeria niaculans Black leg of crucifers RAPD Sharma et al, 2001Phaesariopsis griseola Angular leaf spot of beans RAPD Busogero et al, 200 IPhytophthora infestans Late blight of potato AFLPIRFLP Purvis et al, 200 1venturia inaequalis Apple scab Microsatellite Tenzer et al. 200 iTilletita indica Kamal bunt ITS Levy et al, 2001Magnaporthe grisea Rice blast RAPD Sharma et al, 2002Phomopsis helianthi Brown stem canker of sunflower AFLP Says-alesage et al, 2002

RFLP = restriction fragment length polymorphism; Rep-PCR = repetitive element based PCR; RAPD = random amplifiedpolymorphic DNA; REP = repetitive extragenic palindromic sequences; ERIC = enterobacterial repetitive intergenic consensus; AFLP=amplified fragment length polymorph is; ITS= Internal transcribed spacer.

In Magnaporthe grisea, 14 MGR-DNA fingerprintlineages have been reported from 55 isolates fromSouth India (Sivaraj et al, 1994). Seventy-six isolatesof M. grisea from Central India, have beencharacterized into 7 lineages (Sridhar et al, 1996).These studies could not establish definite relationshipbetween clonal lineages and pathotype diversity in M.grisea. PCR based DNA fingerprinting using RAPDmarkers has revealed high genetic variability among250 isolates of M. grisea populations of HimachalPradesh (Sharma et al, 2002b). The groups reported inthis study were not region specific probably becauseM. grisea isolates were collected from differentlocations of the geographically distinct regions, wherewide genetic diversity exists among the rice cultivarsgrown in those areas. Neeraja et al (2002) reportedcharacterization of 18 Indian isolates of Rhizoctoniasolani (Sheath blight of rice) by using RAPD markers.This fingerprint data could distinguish R. solaniisolates virulent and avirulent on rice lines. There area number of instances where genetic variability inplant pathogen has been characterized by using PCRbased DNA fingerprinting (Table 2). However, it isimportant to study the relationship between RAPD

and AFLP groups and the pathotypes. If there is adirect relationship between DNA fingerprintinggroups and pathotypes/races of pathogen, it will haveimportant implications in the improvement of diseaseresistance breeding programmes.

Molecular Mapping of Disease Resistance GenesThe process of locating genes of interest via

linkage to markers is referred as gene tagging. Thetagging of disease resistance genes with molecularmarkers involves the evaluation of classicalphenotype for resistance and molecular markergenotype on the same individuals and the data isanalysed to determine, if any of the markers cosegregates with the target phenotype (resistantphenotype). A molecular marker closely linked to aresistance gene can be used for indirect selection ofthe genes in breeding programme. There are variousmethods of developing DNA markers linked to theR-genes. The basic requirement of tagging plantdisease resistance genes with molecular markers is thedevelopment of a mapping population. NILs differingfor single gene for resistance are considered a verygood material for use in R-gene mapping experiments

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Table 2-·Partiallist of plant disease resistance genes tagged with DNA markers

Disease Pathogen R-gene tagged Marker References

Loose smut of wheat Ustllago segatium tritici T10 SCAR Procunier et aI, 1997

Powdery mildew of tomato Leveillula tau rica Lv RAPD Chunwongse et al. 1997

Potato Globodera pallida Gpa2 AFLP Rouppe et al, 1997

Apple scab Venturia inaequalis Vf RAPD Tartarini et al, 1998Pepper Bs2 Tai et al, 1999Rice blast Magnaporthe grisea Pi 44(t) Chen et al, 1999Bean anthracnose Colletotrichum lindemuthianum Co4 RAPD Arruda et al, 2000Leaf rust of barley Puccinia hordei Rph7 RFLP/CAPs Graner et al, 2000Powdery mildew of wheat Erysiphe graminis tritici Pm24 Huang et al, 2000Leaf rust of wheat Puccinia graminis tritici Lr47 RAPD Helgurea et al. 2000Rice blast M. grisea Pik" RAPD Shanker, 2002Rice blast M. grisea Pi k" AFLP Singh,2002

RAPD = Random amplified polymorphic DNA; S1'S = Sequence tagged sites; SSR = Simple sequence repeats; SCAR = Sequencecharacterized amplified regions; AFLP = Amplified fragment length polymorphism; C Ps = cleaved amplified polymorphism.

(Muehlbauer et al, 1988), NILs are developed bycrossing resistant individuals with universallysusceptible plants and back crossed for 6-7generations with the susceptible recurrent parent toget homozygous resistant lines, which differ only forone gene for resistance. The NILs which differ by thepresence or absence of a specific R-gene are crossed.The F2 segregating population is analysed fordetecting DNA polymorphism using one of the DNAmarkers technology. Polymorphic bands producedbetween resistant and susceptible lines are analysedfor their linkages with the R-gene(s). In those caseswhere NILs are not available, RILs are used, RILs canbe produced by making crosses between resistant andsusceptible lines by using single seed descent methodin seven or more generations. Plant disease resistanceis many times controlled by multiple genes. Thesegenes are difficult to detect in F2 populations of NILs.Therefore, for such analysis of resistance, fixedpopulations of doubled haploids are produced byusing anther culture technique, and thus plants with100% homozygocity can be produced after a singlegeneration.

In those cases where NILs are not available, bulksegregant analysis (BSA) is the method of choice(Michelmore et al, 1991). In this method,polymorphisms survey is conducted in the DNA bulksmade from resistant and susceptible F2 plants. Eachresistant and susceptible bulk consists of individualshomozygous for all the characters, which differgenetically for the regions cointaining Rvgenes. The

R-genes tightly linked to the markers producedpolymorphic band between R-bulk and S-bulk(Fig. lA & B). While, unlinked regions give rise tomonomorphic banding pattern. There are manyreports available on molecular mapping of R-gene indifferent host- pathogen interactions (Mohan et al,1997; Sharma et al, 1999). A partial list of R-genestagged with DNA markers is given in Table 2.

Investigation on molecular tagging and cloning ofblast resistance genes and resistance gene analoguesfrom rice line Tetep, which possesses durableresistance to the M. grisea population of north-western Himalayan region of India are in progress(Sharma et al, 2002 unpublished). A mappingpopulation by crossing blast resistant line Tetep withsusceptible rice line HP2216 has been developed. Fcpopulation was uniformly inoculated at seedling stagewith an isolate of M. grisea collected from north-western Himalayan region. A total of 205 F2 plants ofmapping population have been maintained and furtherscreened with M. grisea in F, plant progeny rows toidentify the heterzygotes. RAPD marker, S129700 hasbeen mapped at a distance of 2.1 cM (Shanker, 2002)and an AFLP marker, AFLP75 mapped at 15.1 cM onthe linkage map (Singh, 2002). RAPD and AFLPmarkers showing linkage to R- gene are shown in Fig.2 A and B. Another mapping population derived fromdoubled haploids of rice is being used for themolecular tagging of Pi k"gene (Sharma et al, 2002c).Five blast resistance gene analogues (RGAs) havebeen isolated, cloned and sequenced from rice blast

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A

INDIAN J BIOTECHNOL, JANUARY 2003

Kb M R S RB SB R R R R R R R R R R S S S S S S S SSM

BA

III III IV

BIII III IV

CIII III IV

oIII III IV

EIII III IV

Fig. l--(A) Molecular tagging of blast resistance gene in mapping population derived from HP2216 X Tetep. A) RAPD band linked toR-gene ( ~). M = molecular weight marker, PI = resistant (Tetep), P2 = susceptible (HP2216), Reresistant, S= susceptible. RB =resistant bulk. SB = susceptible bulk. (B) Bulked segregant analysis of mapping population derived from HP2216 X Tetep with fiveprimers (A-E), I = resistant, II = susceptible, III = resistant bulk, IV = susceptible bulk, ~ DNA band linked to resistance gene.

SHARMA: MOLECULAR MARKERS AND PLANT DISEASE MANAGEMENT

resistance line Tetep. These RGAs were mapped ondifferent rice chromosomes by in silico analysis(Sharma et al, 2002d).

Gene PyramidingGene pyramiding is the technique of combining

more than one gene for resistance in a commongenetic background by the repeated back crossing andselection with the virulent races of the pathogen. Thetechnique is highly effective in getting durableresistance to the target pathogen. Using conventionalmethods, it is really very difficult to pyramid 2-3genes in a cultivar through crossing and normalscreening procedures, since, presence of one gene inthe plant masks the effect of others. Secondly, thereshould be specific pathogen races (isolates) todiscriminate both the R-genes separately so thatscreening of both the genes can be done insegregating populations. In such cases, DNA markershighly linked to the R-gene can be used for detectingthe presence or absence of both the genes in eachplant after molecular analysis.

Gene pyramiding is being used in many host-pathogen systems. Recently, Hittalmani et al (2000)pyramided three rice blast resistance genes by usingMAS. First, they performed fine mapping of threemajor blast resistance genes Pi-I, Piz-S and Pita onchromosomes 11, 6 and 12, respectively by RFLPmarkers. These RFLP markers were then used forgene pyramiding. Plants carrying 2 and 3 R-genecombinations were also identified from the resistantphenotypes by inoculation with M. grisea. They haveused the isogenic lines containing Pi], Piz-S and Pi tagenes for pyramiding in the background of susceptiblecultivar Co 39, which is not being used forcommercial cultivation. They are now transferringthem to the commercial cultivars of rice. Pyramidingof 5 bacterial blight resistance genes Xal, Xa3, Xa4,xa5 and xalO have also been achieved by MAS(Yoshimura et al, 1995). Huang et al (1997)pyramided four bacterial blight resistance genes Xa4,xa5, xa13 and Xa21 using RFLP and PCR basedmarkers.

Map based Cloning of R-genesVarious strategies have been reported for the

cloning of disease resistance genes in differentsystems (Ellis et al, 1988). Among these, map basedcloning and transposon tagging is being used forcloning disease resistance genes where gene productis not known (Sharma et al, 2002e). Transposon

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tagging was first demonstrated in maize byFedoroft et al (1984). Since then, modifiedtransposons from maize have been used forintroduction in other plant species to facilitate genetagging (Ellis et al, 1988).

Map-based cloning approach (Tanksley et al, 1989)is preferred over other alternatives because, it doesnot require any information regarding the geneproduct and thus each step can be monitored verysystematically. Since it is labour-intensive and timeconsuming, therefore, cannot be used to clone a generesiding in the complex locus of the genome. Varioussteps involved in map based cloning are brieflyexplained as follows.

Fine Mapping of R-genesFine mapping refers to the identification of markers

genetically tightly linked to the target gene. Asexplained earlier, molecular tagging of R-genesrequires a number of different approaches startingfrom the identification of different R-genes effectiveagainst a pathogen population of a particulargeographical region, developing mapping populationand using molecular markers for the tagging of R-genes. Once the DNA markers tightly linked to thetarget gene are identified, a fine saturated genetic mapis often made. Fine mapping is very important togenerate information regarding the orientation oftightly linked markers related to each other. Such typeof information is important for physical mapping andchromosome walking. Therefore, a large segregatingpopulation (more than 1000 individuals) is analyzedwith the DNA markers and recombinant individualsare identified at targeted site.

Physical Mapping of R-genesPhysical mapping is the determination of

relationship between genetic and physical distances ofDNA markers in the target region of a chromosome.Physical mapping helps in determining the distancebetween two markers flanking to R-genes in terms ofnumber of nucleotides. It is basically performed byseparating large DNA molecules with PFGE. Variouselectrophoretical techniques like CHEF and fieldinversion gel electrophoresis (Carle et al. 1986) arecapable of separating large DNA molecules rangingfrom 100kb to 10 Mb in size (Chu et al, ]988). Afterthis, genomic libraries of large inserts are constructedin various cloning vector systems. However, vectorslike plasmids, phages and cosmids can only be used

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primarily in case of prokaryotes where genome size issmall. In case of plants, different vector systems likeYACs (Burke et al, 1987) and BACs (Shizuya et al,1992) have been used for preparing genomic librariesof large DNA segments.

Integration of a physical map with the genetic mapis necessary to place some BAC clones onto a DNAmarker map (Yang et al, 1997). These BAC clonesthen become landmarks, which can be generatedeither by S1'S mapping or colony hybridization withRFLP markers. If the STS and RFLP markers arelinked to disease resistance genes, the landmarksprovide the initial points for chromosome walking andeventual cloning of target genes (Yang et al, 1997).

Chromosome WalkingOnce a physical map of the genome near a target

gene is constructed, this region can then be cloned bychromosome walking. It begins by identifyinggenomic clones that overlap the initial RFLPs, whichis accomplished by probing a complete genomiclibrary with radiolabelled nucleic acid probessynthesized from the initial RFLPs. The newlyidentified genomic clones are then isolated and endsof these clones become the starting points for nextstep in the chromosome walk. It is repeated manytimes until clones covering a large segment ofcontiguous genomic DNA are obtained. It stretchesfrom one of the tlanking RFLP markers to the otherwith the target gene located at one of the clones inbetween the RFLPs. Once the insert with target geneis cloned it can be transferred to the appropriatesusceptible plants along with a suitable promoter.There are many examples of molecular cloning ofdisease resistance genes in different host-pathogen

systems by using map based cloning (Table 3). Itshows the power of this technique and its futureimplications for isolating more number of R-genes inplants.

EpilogueLarge number of DNA markers developed during

the past few years have shown their importance andutilities in biological sciences. These numbers havespecifically increased after PCR techniques have beenused for designing such markers. Researcher canexpect much more advances on the automation ofDNA marker technologies, cost effective and up tothe reach of small laboratories.

Uses of DNA markers in plant disease resistancebreeding programme have considerably increased inthe country. Since, the R-genes have already beentagged in the simplest genome like Arabidopsis to theplant of complex genome and difficult to breed likeapple. It is further expedited with the discovery ofSNPs and decoding of whole genome sequences ofplants. Disease resistance characters being controlledby mostly single and dominant gene(s), would havebeen the most widely studied character as for as genetagging is concerned. Due to their practicalsignificance tagged markers are now being used forthe introgression of more than one gene inagronomically superior cultivars. DNA markers havebeen successfully used for the molecular cloning ofR-genes in various host-pathogen systems and theirmechanism of resistance has been studied in a greaterdetail. Many of these genes are now being mobilizedin the agriculturally important crops. However, DNAmarker technology is not the ultimate replacement ofthe classical breeding approaches, these are only the

R-gene Host-pathogen system

Table 3--Disease resistance genes cloned by map based cloning in different host-pathogen systems

ReferencePredicted gene product

PtoRPS2Xa2JRPMJPrfXa-JRPP812PibBs2RPP13Pi-ta

Tomato- PseudomonasArabidopsis-PseudomonasRice-XanthomonasArabidopsis-PseudornonasPomato- PseudomonasRice - XanthomonasA rabidopsis- Pe ronosporaTomato -FusariumRice- MagnaporthePepper- XanthomonasArabidopsis-PeronosporaRice- Magnaporthe

Ser lThr Protein KinaseLZ,NBS, LRRLRR, Ser/Thr Protein KinaseNBS,LRRLZ,NBS, LRRNBS,LRRNBS,LRRNBS, LRRNBS,LRRNBS,LRRNBS, LRRNBS, LRR

Martin et al, 1993Bent et al, 1994Song et al, 1995Grant et al, 1995Salmeron et al, )996Yoshimura et al, 1998McDowell et al. 1998Simons et al, 1998Wang et al, 1999Tai et al, 1999Bittner-Eddy et a/,2000Bryan et al, 2000

NBS = nucleotide binding site, LRR = leucin rich repeat, LZ = leucin zipper,

SHARMA: MOLECULAR MARKERS AND PLANT DISEASE MANAGEMENT

facilitators, which can expedite the disease resistance-breeding programme and would be helpful in thecloning of R-genes and production of diseaseresistance transgenics.

AcknowledgementAuthor is thankful to the Indian Council of

Agricultural Research, New Delhi for financialassistance in the form of ICAR Young ScientistAward in crop sciences.

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