cenetic analysis with random amplified polymorphic dna markers · to identify rapd markers linked...

Plant Physiol. (1993) 101: 349-352

Cenetic Analysis with Random Amplified Polymorphic DNA Markers

Scott V. Tingey* and Joseph P. de1 Tufo

Du Pont Agricultura1 Products, E.I. du Pont de Nemours & Company, Wilmington, Delaware 19880-0402

For many years, the principles of genetics have been applied to crop variety improvement with great success. Severa1 crop species, notably corn, wheat, and tomato, have been used as model genetic systems because of their central im- portance to food production. Until recently, virtually a11 pro- gress in both breeding and model genetic systems has relied on a phenotypic assay of genotype. Because the efficiency of a selection scheme or genetic analysis based on phenotype is a function of the heritability of the trait, factors like the environment, multigenic and quantitative inheritance, or par- tia1 and complete dominance often confound the expression of a genetic trait. Many of the complications of a phenotype- based assay can be mitigated through direct identification of genotype with a DNA-based diagnostic assay. For this reason, DNA-based genetic markers are being integrated into several plant systems and are expected to play an important role in the future of plant breeding.

The utility of DNA-based diagnostic markers is determined to a large extent by the technology that is used to reveal DNA-based polymorphisms. Currently, the technology of choice for many species is the RFLP assay. RFLP assays detect DNA polymorphisms through restriction endonuclease diges- tions, coupled with DNA blot hybridizations, and are, in general, time consuming and labor intensive. Over the last few years, polymerase chain reaction technology has led to the development of several nove1 genetic assays based on selective DNA amplification (Krawetz, 1989; Innis et al., 1990). One of the strengths of these new assays is that they are more amenable to automation 'than conventional tech- niques. They are also simple to perform and are preferable in experiments where the genotype of a large number of individuals is to be determined at a few genetic loci. Unfor- tunately, because of a prerequisite for DNA sequence information, these assays are limited in their application.

Nearly 2 years ago, a new genetic assay was developed independently by two different laboratories (Welsh and McClelland, 1990; Williams et al., 1990). This procedure, which we have called the RAPD assay, detects nucleotide sequence polymorphisms in a DNA amplification-based assay using only a single primer of arbitrary nucleotide sequence. In this reaction, a single species of primer binds to the genomic DNA at two different sites on opposite strands of the DNA template. If these priming sites are within an amplifiable distance of each other, a discrete DNA product

* Corresponding author; fax 1-302-695-4296.

is produced through thermocyclic amplification (Fig. 1). The presence of each amplification product identifies complete or partia1 nucleotide sequence homology, between the genomic DNA and the oligonucleotide primer, at each end of the amplified product. On average, each primer will direct the amplification of several discrete loci in the genome, making the assay an efficient way to screen for nucleotide sequence polymorphism between individuals. For example, the fre- quency of finding RAPD polymorphisms has been shown to be 0.3 per primer in Arabidopsis thaliana, 0.5 per primer in soybean, 1 per primer in corn, and 2.5 per primer in Neuro- spora crassa. The major advantage of this assay is that there is no requirement for DNA sequence information. The pro- toco1 is also relatively quick and easy to perform and uses fluorescence in lieu of radioactivity (Williams et al., 1992). Because the RAPD technique is an amplification-based assay, only nanogram quantities of DNA are required, and automation is feasible.

APPLICATIONS OF THE RAPD ASSAY

There are several applications of the RAPD assay that have been developed over the past 2 years. Each of these tech- niques exploits the efficiency of detection of DNA sequence- based polymorphisms in the RAPD assay. The RAPD technology has quickly gained widespread acceptance and application because it has provided a tool for genetic analysis in biological systems that have not previously benefitted from the use of molecular markers.

Development of Genetic Maps

One of the first practical uses of RAPD markers was in the creation of high-density genetic maps. By using a more efficient assay, Reiter et al. (1992) were able to place over 250 new genetic markers on a recombinant inbred population of A. thaliana in only 4 person-months, clearly demonstrating the utility of RAPD markers for quickly saturating both a global and local genetic map.

Historically, many important crop systems have suffered from a lack of genetic markers. For example, genetic linkage analysis in conifers has been slow primarily due to the large size of the genome and the inherent difficulty involved in producing a segregating F2 population (Carlson et al., 1991).

Abbreviations: RAPD, random amplified polymorphic DNA; RFLP, restriction fragment-length polymorphism.

349

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350 Tingey and del Tufo Plant Physiol. Vol. 101, 1993

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Figure 1. A, Schematic representation of the amplification of DNA with a single oligodeoxynucleotide primer. B,Amplification products from F2 individuals segregating from a cross between Clycine max cv Bonus and Glycine so/aPI81762. The arrow points to a segregating locus, and the size standards are shown to the right of the gel. The RAPDreaction was performed as described in Williams et al. (1990), and the products of the amplification reaction werevisualized by separation on a 1.4% agarose gel stained with ethidium bromide.

Carlson et al. (1991) and Chaparro et al. (1992) have recentlyshown that the speed and efficiency of RAPD analysis hasmade mapping in conifers a reasonable endeavor. For ex-ample, Chaparro et al. (1992) were able to create a 191-marker RAPD map of loblolly pine in only 6 person-months.Both the A. thaliana and loblolly pine genetic maps weresynthesized with unprecedented speed, taking advantage ofthe unique reproductive biology of each system.

Because RAPD polymorphisms are the result of either anucleotide base change that alters the primer binding site, oran insertion or deletion within the amplified region (Williamset al., 1990; Parks et al., 1991), polymorphisms are usuallynoted by the presence or absence of an amplification productfrom a single locus. This also means that the RAPD techniquetends to provide only dominant markers. Individuals contain-ing two copies of an allele are not distinguished quantitativelyfrom those containing only one copy of the allele. Thedisadvantage of mapping with dominant markers is thatmarkers linked in repulsion, for example markers residing onseparate chromatids, such as could be found in an F2 popu-lation, provide little information for the estimate of geneticdistance (Allard, 1956). Therefore, when mapping with dom-inant markers, it is necessary to work with markers that areonly linked in coupling, i.e. markers residing on a singlechromatid as can be found in a backcross or recombinantinbred population, in haploid or gametophytic tissue, oralternatively in an F2 population where only RAPD markersamplified from a single parent are mapped (Williams et al.,1992). Genetic simulations show that dominant markerslinked in coupling are as efficient for mapping as codominantmarkers on a per gamete basis (Hanafey Maize GeneticsMeeting).

Targeting Genetic Markers

Several groups have used the RAPD assay as an efficienttool to identify molecular markers that lie within regions ofa genome introgressed during the development of near iso-genic lines (Klein-Lankhorst et al., 1991; Martin et al., 1991;Paran et al., 1991). By definition, any region of the genomethat is polymorphic between two near-isogenic plants ispotentially linked to the introgressed trait. Thus, Klein-Lank-horst et al. (1991) were able to identify RAPD markers specificto chromosome 6 of tomato by screening a Lycopersiconesculentum substitution line, and Martin et al. (1991) wereable to confirm linkage of RAPD markers to the Pfo locus intomato after screening two near-isogenic lines. Paran et al.(1991) used two different sets of near-isogenic lettuce linesto identify RAPD markers linked to the Dml, Dm3, and Dm 11locus. RAPD markers were 4 to 6 times more efficient, on aper assay basis, than was screening for these polymorphismsusing RFLP technology, and were over 10-fold more efficientin time and labor. Another advantage of this technology isthat a genetic map of the entire genome is not required toidentify markers linked to a trait of interest; instead, specificregions of the genome can be focused on.

There are, however, two disadvantages to using near-isogenic lines to identify markers linked to a genetic trait. Thefirst is that it takes several generations of backcrossing tocreate a near-isogenic line. The second is that frequentlythere are several regions of the donor genome that areinadvertently co-introgressed into the near-isogenic line(Young and Tanksley, 1989). This results in the identificationof marker polymorphisms between near-isogenic lines thatare not necessarily linked to the trait that is being studied.



Random Amplified Polymorphic DNA 351

Population Cenetics

The area of research that has shown the most growth with respect to the use of RAPD technology is that of population genetics (Hedrick, 1992). RAPD markers have been used to create DNA fingerprints for the study of individual identity and taxonomic relationship in both eukaryotic and prokar- yotic organisms (Caetano-Anollés et al., 1991a; Hu and Qui- Tos, 1991; Welsh et al., 1991; Wostemeyer et al., 1991; Hadrys et al., 1992; Kresovich et al., 1992; Lark et al., 1992; Stiles et al., 1992; Wilde et al., 1992). An important question is whether RAPD bands of equal mo1 wt that are shared between individuals are homologous characters (characters in- herited from a common ancestor) or homoplastic characters (characters that arise independently within a population). It seems likely that closely related individuals would co-inherit a shared character state from a common ancestor and unlikely that they would acquire the same character independently. Williams et al. (1992) demonstrated this to be the case by using single RAPD bands as hybridization probes to detect homologous characters on a DNA blot of RAPD products. Within the limits of the resolution of an agarose gel, RAPD bands that were amplified from different species of the genus Glycine, and scored as homologous by relative mobility, were also shown to be homologous by hybridization.

Severa1 groups have reported on the utility of RAPD markers as a source of phylogenetic information. Arnold et al. (1991) were successful in using RAPD markers to test for interspecific nuclear gene flow between lris fulva and 1. hexagona, and to study the presumed hybrid origin of 1. nelsonii. Hu and Quiros (1991) were able to show that the amplification products from only four random primers were sufficient to discriminate between 14 different broccoli and 12 different cauliflower cultivars (Brassica oleracea L.). RAPD markers have also been used effectively to assess the amount of genetic diversity in germplasm collections. Using only 25 different decamer oligonucleotide primers, Kresovich et al. (1992) collected information on 140 different polymorphic characters in a "test array" of individuals representing B. oleracea L. and B. rapa L. They showed the utility of the assay for discriminating between different individuals in a germplasm collection and that the ability to distinguish between closely related individuals was simply a function of the number of RAPD bands that were observed. RAPD markers provided an efficient technology for discovering these polymorphic characters.

Using an arbitrary primer as short as five nucleotides, combined with silver staining to increase the sensitivity of DNA band detection, Caetano-Anollés et al. (1991a) produced a detailed and relatively complex DNA fingerprint for severa1 different species. This approach, termed DNA amplification fingerprinting, has been reviewed recently (Caetano- Anollés et al., 1992b) and promises to generate more genetic information from each amplification.

Pooling Strategies

Recently, another technology has been developed that is designed to identify genetic markers linked to very specific regions of the genome. Arnheim et al. (1985) outlined a genome pooling strategy that allows RFLP markers to be targeted to a region of the genome that is not in linkage equilibrium as a result of selection at a particular locus. The strategy requires pooling genomic DNA from individuals that are known to be genetically fixed at a particular locus. Mark- ers linked to that locus are identified by their linkage disequilibrium with respect to the rest of the population. The limitation of this approach is that it relies on RFLP technology, which is relatively inefficient for identifying polymorphic regions of a genome, and, more importantly, on the premise that linkage disequilibrium exists at the locus of interest within the source population.

Recently, Michelmore et al. (1991) have described the use of RAPD markers to screen efficiently for markers linked to specific regions of the genome. This method, called bulk segregant analysis, uses two bulked DNA samples gathered from individuals segregating in a single population. Each bulk is composed of individuals that differ for a specific phenotype or genotype, or individuals at either extreme of a segregating population. For simple genetic traits, a11 loci in the genome should appear to be in linkage equilibrium except in the region of the genome linked to the selected locus. Markers linked to this locus should appear polymorphic between the pools for alternate parental alleles. Because many segregating individuals are used to generate the pools, there is only a minimal chance that regions of the genome unlinked to the target locus will also be polymorphic between the pools. Random primers can then be used efficiently to amplify loci from each pool and to identify RAPD polymorphisms linked to the trait of interest. Michelmore et al. (1991) have successfully used this technique to target markers to the Dm5/8 locus in lettuce.

The advantage of this technology is that markers are targeted to a much smaller locus within the genome, and the likelihood of identifying false positive markers is small (Mich- elmore et al., 1991) compared with near-isogenic line analysis. Selections made from an FZ population will always be in linkage disequilibrium with respect to selected regions of the genome, and markers can be targeted to any locus where any form of selection can be applied, either phenotypic or geno- typic.

Giovannoni et al. (1991) demonstrated the use of a pooling strategy, based on known RFLP genotypes from existing mapping populations, to create pools of DNA from individuals homozygous for opposite parental alleles in a targeted chromosomal interval. This method was used to target RAPD markers to regions of the tomato genome responsible for fruit ripening and pedicel abscission. Reiter et al. (1992) used this pooling strategy to identify 100 RAPD markers specific to chromosome 1 of A. thaliana. As genetic maps approach saturation, pooling on phenotype or genotype will allow researchers to move away from a random approach to map saturation and focus more efficiently on specific regions of the genome.

CONCLUSION

DNA-based diagnostics are now well established as a means to assay diversity at the locus, chromosome, and whole genome levels. As technology has advanced, DNA sequence-



352 Tingey and de1 Tufo Plant Physiol. Vol. 101, 1993

based assays have become easier to use, more efficient at screening for nucleotide sequence-based polymorphisms, a n d available to a wider cross-section of the genetics research community. The ultimate genetic assay would be based on the determination of the complete DNA sequence at any locus of interest. Increased genetic resolution would be ob- tained simply by sequencing a larger contiguous segment of D N A a t the locus. As a prelude to this, there are now severa1 good examples of D N A sequence-based diagnostic assays that are designed to identify the presence or absence of a specific nucleotide bp at a discrete locus (Landegren et al., 1988; Korner and Livak, 1989; Newton et al., 1989; Wu et al., 1989; Barany, 1991; Dockhorn-Dworniczak et al., 1991; Kuppuswamy et al., 1991; Suzuki et al., 1991). Today these assays are limited in their application only by the high cost of DNA sequence determination. As DNA sequencing technology becomes more cost efficient a n d automated, genetic assays may be based directly on DNA sequence analysis.

Received September 3, 1992; accepted October 11, 1992. Copyright Clearance Center: 0032-0889/93/101/0349/04.

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