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Critical Reviews in Plant Sciences, 20(3):251–275 (2001)

0735-2689/01/$.50© 2001 by CRC Press LLC

Gene Tagging with Random Amplified PolymorphicDNA (RAPD) Markers for Molecular Breeding inPlants

S. A. Ranade,* Nuzhat Farooqui, Esha Bhattacharya, and Anjali VermaPlant Molecular Biology Division, National Botanical Research Institute, Rana Pratap Marg, Lucknow226 001 (U.P.) India

* Corresponding author. Plant Molecular Biology Division, National Botanical Research, Institute, Rana Pratap Marg, Lucknow226001. (U.P.) India. Fax. no.: (91) 522 205836, 205839 E-mail: shirishranade@yahoo.com.

ABSTRACT: Markers are of interest to plant breeders as a source of genetic information on crops and for use inindirect selection of traits to which the markers are linked. In the classic breeding approach, the markers wereinvariably the visible morphological and other phenotypic characters, and the breeders expended considerableeffort and time in refining the crosses as the tight linkage or association of the desired characters with the obviousphenotypic characters was never unequivocally established. Furthermore, indirect selection for a trait using suchmorphological markers was not practical due to (1) a paucity of suitable markers, (2) the undesirable pleiotropiceffects of many morphological markers on plant phenotype, and (3) the inability to score multiple morphologicalmutant traits in a single segregating population. With the advancement in molecular biology, the use of molecularmarkers in plant breeding has become very commonplace and has given rise to “molecular breeding”. Molecularbreeding involves primarily “gene tagging”, followed by “marker-assisted selection” of desired genes or genomes.Gene tagging refers to the identification of existing DNA or the introduction of new DNA that can function as atag or label for the gene of interest. In order for the DNA sequences to be conserved as a tag, important prerequisitesexist. This review also summarizes the achievements in gene tagging that have been made over the last 7 to 8 years.

KEY WORDS: RAPD, gene tagging.

I. INTRODUCTION

Markers are of interest to plant breeders as asource of genetic information on crops and foruse in indirect selection of traits to which themarkers are linked. In the classic breeding ap-proach, the markers were invariably the visiblemorphological and other phenotypic characters.The inheritance of these characters was scored forin a cross. Further, the success of the selection ofother desirable traits to which these (the visible,morphological characters) were apparently closelyassociated with was also invariably evaluated.This resulted in an indirect selection of the de-sired characters. In all of these processes, the tightlinkage or association of the desired characterswith the obvious phenotypic characters was neverestablished unequivocally. Consequently, the

breeders expended considerable effort and time inrefining the crosses. Therefore, until recently suchindirect selection for a trait was not practical dueto several reasons, such as a paucity of suitablemarkers, the undesirable pleiotropic effects ofmany morphological markers on plant phenotype,and the inability to score multiple morphologicalmutant traits in a single segregating population(Paterson et al., 1991). With the advancement inmolecular biology, the use of molecular markersin plant breeding has become very commonplaceand has given rise to molecular breeding.

Molecular breeding involves primarily genetagging, followed by marker-assisted selection ofdesired genes or genomes. Gene tagging refers toidentification of existing DNA or introduction ofnew DNA that can function as a tag or label forthe gene of interest. In order for the DNA se-

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quences to be conserved as a tag, important pre-requisites have been identified (see box in Figure1). A number of molecular tags have now beendetermined for many genes in most of the impor-tant plants. These molecular tags include clonedrestriction fragment length polymorphism (RFLP)probes, oligonucleotide RFLP probes, variablenumber tandem repeats (VNTR), microsatellite,minisatellite, and other DNA fingerprint loci, andspecific as well as arbitrary sequence primers. Forany or all of these to be used as tags, these mustsatisfy the given criteria.

Gene tagging and marker-assisted selection isan essential component of molecular breedingand is based on saturation mapping of the ge-nomes. This has opened up the possibility of iden-tifying, mapping, tagging, and even isolating ortransferring quantitative trait loci (QTLs). Thus,the most powerful application of DNA markers inplant breeding may be the ability to clone geneshitherto known only by phenotype. In the past,cloning such genes was difficult or impossible.With the advent of DNA marker technology andtransposon tagging, important genes have nowbecome accessible to molecular cloning. DNAmarkers provide the essential starting point forphysical isolation of genomic regions containingthe gene of interest (positional cloning). The ef-forts that are involved in tagging a gene can beused further as a part of marker-assisted selectionprogram. As the economically important genesare tagged, they can even be transferred to unre-lated species.

Molecular breeding has an important role incrop-improvement programs. However, in the caseof the “difficult” plants, molecular breeding canhave an even more profound impact. Forest treesare the dominant plant life covering millions ofhectares on the Earth and form vital plant com-munities that sustain a great diversity of life forms.Tree breeding programs have become the mostimportant part of intensive forestry practices. Thetrees, however, due to large genome sizes andlack of any or substantial genetic linkage data, areconsidered to be among the “difficult” subjectsfor genetic studies (Lehner et al., 1995). Simi-larly, for all other plants where genetic data arescanty or crosses are difficult to achieve or in thecase of the long-lived perennials, genetic linkage

and mapping work is never easy to carry out.Consequently, in all such “difficult” cases, it isexpected that complex trait dissection and mo-lecular breeding will be better achieved throughthe use of gene tags or molecular markers (Landerand Schork, 1994) than through conventionalbreeding. The utility of molecular markers in treebreeding and improvement programs has beenreviewed previously (Strauss et al., 1992; Taueret al., 1992; Kremer et al., 1994).

II. MARKERS USED FOR GENETAGGING

Markers based on variation in length of DNAfragments obtained by digestion with restrictionendonuclease (RFLPs, Botstein et al., 1980) werethe earliest to be developed for molecular breed-ing work. Such markers have several advantagesover other markers. They can detect more numberof loci and alleles, are phenotypically neutral, andcan be scored at any stage of plant development.RFLP markers have been employed extensivelyto tag useful genes in several crop plants andtrees. To list all of these is beyond the scope ofthis review. The trend in the recent years is, how-ever, to combine RFLP markers with RAPD andother PCR-based markers to carry out saturationmapping and even marker-assisted selection forpyramiding desirable genes (Huang et al., 1997).RFLP technology has been reviewed previously(Paterson et al., 1991; Young, 1992; Lee, 1995;Winter and Kahl 1995). Despite the demonstratedusefulness of RFLP markers, the development ofthese markers involves a tedious, expensive, andmultistep process that requires considerable in-vestment in personnel, equipment, and chemicaland safety concerns if radioactive probes are used.Furthermore, only one of several markers screenedis polymorphic, and this can be of serious concernin cases of crosses involving closely related plants(Winter and Kahl, 1995). Finally, the RFLP tech-nique requires repeated application and a largeamount of DNA for each application. This makesthe RFLP analysis a cost- and effort-intensivetechnology.

Isozymes have been used as markers andgenetic characters. However, the numbers of

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FIGURE 1. The important prerequisites for the use of DNA sequences as gene tags are listed in the textbox. It isonly when these prerequisites are fulfilled that gene tagging with DNA sequences will succeed.

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isozymes that could be reliably assayed were lim-ited by their assay conditions, and at best only100 or so of the isozyme loci were detected. Theselow numbers of isozyme loci relative to the enor-mously large size of the plant genome were thusinadequate to help in saturation mapping, andtherefore in gene tagging. Furthermore, in manycases the detection and assay of the enzyme wereinfluenced by temporal and spatial factors, as aresult of which the isozyme assay of genetic lociwere ineffective. Thus, the isozymes have foundlesser applications in gene tagging and marker-assisted selection programs, despite being amongthe earliest to be developed for molecular breed-ing work (Tanksley and Orton, 1983).

Recently, a molecular marker based on PCRhas been developed that overcomes many of theselimitations of RFLP and isozyme markers. Thebasic PCR was modified to develop a new form ofmolecular marker, the RAPD marker (Welsh andMcClelland, 1990; Williams et al., 1990). Essen-tially, a single short primer of arbitrary sequenceis selected at random to be used singly in PCR.This primer is expected to anneal to one or moresequence sites on both strands of the templateDNA. Every time the primer anneals to at leasttwo sites on the opposite strands such that themaximum distance between the two sites is lessthan 5 kbp and such that the 3'-OH ends of theprimer at the two sites face each other, a discreteproduct is formed. A schematic description of theRAPD strategy is described in Figure 2. Further,as the starting plant DNA used as the template ismuch larger than both the primer and this maxi-mum size of 5 kbp, there can be several suchdiscrete products formed. Given the enormouslylarge number of such short arbitrary sequenceprimers possible, this technique offers an excel-lent prospect for generating several polymorphicprofiles that can be useful for gene tagging, MAS,and related techniques.

RAPD technique has revolutionized geneticanalyses in many plants and animals in the decadesince it was first discovered. The polymorphismis identified as presence or absence of discretebands and thus is of dominant nature. This wasinitially considered as being detrimental to carry-ing out detailed genetic analyses. Despite theselimitations, however, several notable achievements

have been made in tagging genes with RAPDprofiles wholly or partially. The entire range ofgenetic populations, simple segregants and bulksamples have been elegantly used to tag diversegenes such as the genes for resistance to pest andpathogens, the genes encoding yield and growthfunction, and the genes encoding sex determina-tion.

Gene tagging using RAPD markers has atleast three major advantages over other methods.First, a universal set of primers can be used andscreened in a short period, second, isolation ofcloned DNA probes or preparation of hybridiza-tion filters is not required; and third only a smallquantity of genomic DNA is needed for eachanalysis. The genomic DNA can be easily ob-tained using simple and rapid methods (Dellaportaet al., 1983; Edward et al., 1991; Weing andCulter, 1993). Ragot and Hoisington (1993) haveshown in their study that the cost per data pointfor RFLPs is less for large populations, whileRAPDs are preferred in the case of small popula-tions. This difference is, however, immaterial inmany of the self-pollinated crops where RFLPslack a sufficient level of polymorphism within aspecies or between related breeding material.RAPDs under these conditions appear to detecthigher levels of polymorphism and are more valu-able in gene tagging too, an area where breedersare focusing their attention currently.

III. GENETIC POPULATIONS USED FORTAGGING

The actual applications of RAPD in gene tag-ging programs have generally involved the use ofspecific genetic populations. These include popu-lations derived from or consisting of recurrentback cross selection progenies, recombinant in-bred lines, near isogenic lines, and in a few casessingle segregants of defined crosses. In those plantswhere such genetic populations are not availableor easily obtainable, gene tagging has beenachieved using bulked samples and segregantssuch that the contrasting characters to be tagged isanalyzed in separate bulks.

The advancement in gene tagging over thelast 5 to 6 years is summarized in Tables 1 to 4.

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FIGURE 2. RAPD strategy. The strategy for carrying out RAPD PCR in case of say two trees is diagrammaticallyrepresented in this figure. The gel photograph in the lower most panel is part of an actual experimental result in caseof several neem variety DNAs, using primer OP-D18 (from Operon Technologies Inc., Alameda, CA, USA). Just twolanes are selected for display here to illustrate the RAPD strategy for gene tagging. The two trees are shown to differin branching character and the strategy aims at the identification of a RAPD pattern that wholly or partly seems tocorrelate with the differences in branching patterns between the trees.

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