GENETICS | Molecular Markers

GENETICS | Molecular Markers

334 GENETICS/Molecular Markers Of the many methods available today for genomic analysis, genetic mapping has been used to construct three separate p...

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GENETICS/Molecular Markers

Of the many methods available today for genomic analysis, genetic mapping has been used to construct three separate pairs of medium-density maps providing random coverage of the rose genome. Physical mapping has been employed to study a single gene region. Comparative mapping has been explored only on a limited basis. Despite having one of the small genomes among the ¯owering plants, no method has been adequately employed to obtain a deep understanding of the rose genome. Consequently, our knowledge of important rose genes and their chromosomal localization remains sketchy at best. However, in the near future, fairly saturated marker maps will become available and we will have a consolidated genetic map covering the seven haploid chromosomes. While it is not anticipated that the entire rose genome will be sequenced soon, a number of gene regions will be studied at the physical level towards map-based cloning of valuable rose genes. What is most practical for understanding the genomic organization and identi®cation of rose genes in the future is comparative mapping with related species, mainly with peach. Comparative analysis with related species can also shed light on the chromosomal rearrangements, duplications and other genomic changes associated with the evolution of the family Rosaceae. Furthermore, comparative analysis with Arabidopsis and other species such as lettuce (Lactuca sativa) and tomato, in which disease resistance genes have been studied thoroughly, would also provide the means for more ef®cient isolation of rose disease-resistance genes. Hence, comparative mapping methods hold the most promise for detailed rose genomics research in the future. See also: Fragrance and Pigments: Functional Genomics. Genetics: Karyology; Molecular Markers; DNA Fingerprinting; Cloned Genes; Marker-Assisted Selection.

Further Reading Baird WV, Ballard RE, Rajapakse S and Abbott AG (1997) Progress in Prunus mapping and application of molecular markers to germplasm improvement. HortScience 31(7): 1099ÿ1106. Crespel L, Chirollet M, Durel CE et al. (2002) Mapping of qualitative and quantitative phenotypic traits in Rosa using AFLP markers. Theoretical and Applied Genetics 105: 1207ÿ1214. Debener T and Mattiesch L (1999) Construction of a genetic linkage map for roses using RAPD and AFLP markers. Theoretical and Applied Genetics 99: 891ÿ899. Debener T, Mattiesch L and Vosman B (2001) A molecular marker map for roses. Acta Horticulturae 547: 283ÿ287. Delseny M, Salses J, Cooke R et al. (2001) Rice genomics: present and future. Plant Physiology and Biochemistry 39: 323ÿ334.

Gale MD and Devos KM (1998) Comparative genetics in the grasses. Proceedings of the National Academy of Sciences USA 95: 1971ÿ1974. Gillham NW (1994) Organelle Genes and Genomes. New York: Oxford University Press. Jauhar PP (1996) Methods of Genome Analysis in Plants. Boca Raton, FL: CRC Press. Rajapakse S, Byrne DH, Zhang L et al. (2001) Two genetic linkage maps of tetraploid roses. Theoretical and Applied Genetics 103: 575ÿ583. Zhang H-B, Woo S-S and Wing RA (1996) BAC, YAC and cosmid library construction. In: Foster G and Twell D (eds.) Plant Gene Isolation: Principles and Practice, pp. 75ÿ99. London: John Wiley.

Molecular Markers S Rajapakse, Clemson University, Clemson, SC, USA # 2003, Elsevier Ltd. All Rights Reserved.

Introduction Molecular markers are short segments of DNA that usually comprise 100ÿ1000 basepairs, and are located somewhere along the long stretches of the genome of an organism that typically consists of several billions of basepairs of DNA. Because of the vast amount of resources needed to study the entire genomes of various organisms, more often, markers are used as `windows to the genome' to extract various kinds of information regarding the structure and the composition of a genome. For example, two molecular markers mapped close to and on either side of a particular gene of interest indicate that the gene is located between the two markers. These markers linked to the gene provide valuable information about the gene's location because it is more dif®cult to pinpoint the precise location of a gene initially. Location of all kinds of markers are not known unless they have been mapped and not all markers represent genes since they can be located in nongene regions of the genome as well. Other kinds of markers can provide information regarding the genetic composition. Those markers revealing speci®c basepair sequences unique to one or a group of organisms are valuable in their identi®cation. This kind of marker can also be used to study genetic variation among individuals, offspring, populations, species and other taxonomic groups. Molecular markers are broadly applicable to all organisms having DNA as their genetic material, including animals, plants, bacteria, fungi and roses are no

GENETICS/Molecular Markers

exception. The advent of methods to generate DNAbased markers in plants during the early 1980s transformed the way scientists have been studying genetic differences in plants. Originally spun off from human DNA ®ngerprinting methods and later expanded by the development of the polymerase chain reaction (PCR), molecular markers have made rapid advancements possible in a wide range of applications including plant identi®cation, taxonomic analysis, ecology, breeding, genetics, genome analysis, gene identi®cation and gene cloning. Prior to the development of methods to generate DNA markers, genetic analyses of plants relied heavily on morphology. Morphological characters that can be used to distinguish plants are limited in number. Their expression can also vary depending on the environmental conditions in which they grow. In contrast, molecular markers offer many advantages over conventional phenotypic markers. Molecular markers are developmentally stable, detectable in all tissues and unaffected by environmental conditions. Characterization of isozymes and secondary metabolites such as ¯avonoids has also been used for plant genetic analyses. While these methods have proven useful to some degree, they too suffer from the low number of markers available for generation and hence have a limited discriminatory power. In contrast, a practically unlimited number of markers can be generated with the wide range of DNA-based marker types now available. Molecular markers serve as powerful research tools for a broad spectrum of scientists analysing plants at the genetic level, from providing more ef®cient methods of evaluating progeny of crosses for desirable genotypes for the interest of plant breeders, to discovering and cloning new genes for the interest of biologists. This article will provide a brief review of the main types of DNA markers and their potential applications, along with their limitations. Speci®c applications of molecular markers in roses for cultivar identi®cation, DNA ®ngerprinting, marker-assisted selection and genome analysis are discussed in separate entries.

Types of Molecular Markers RFLP

Following their successful application in humans and animals, restriction fragment length polymorphisms (RFLPs) were the ®rst DNA-based markers applied to plants. These markers detect variation in the length of DNA segments after digestion of DNA with a restriction endonuclease enzyme. A large number of these enzymes is available, each recognizing a speci®c short (4ÿ8) DNA sequence and cleaving the DNA

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wherever its target sequence occurs. DNA sequence alterations in a restriction endonuclease site or insertions and deletions between sites can give rise to detectable differences in the length of DNA fragments. Therefore, these genetic markers are referred to as RFLPs. To develop these markers, DNA is cut with a restriction enzyme and subjected to size separation by agarose gel electrophoresis. Due to the enormous amount and complexity of the DNA in any higher organism, this separation results in a more or less uniform smear of fragments, large and small. But due to the fact that DNA is composed of two complementary strands, distinct segments of DNA can be highlighted in this smear by using a method known as Southern hybridization. In order to perform this procedure, alkaline solutions are used to denature the doublestranded DNA in the gel. The resulting single-stranded DNA in the gel is then transferred to a nylon membrane and hybridized with a DNA fragment labelled using either radioactive or nonradioactive methods. The most common method of labelling the probe DNA is with radioactive 32P. In this method, the hybridized membrane is placed on X-ray ®lm to detect the areas of the membrane hybridized with the labelled DNA. Probes for hybridization can be selected from a variety of sources, including cloned DNA of the same species or from another species. The probe could also be a synthetic oligonucleotide. Human and bacteriophage-derived minisatellite probes (e.g. M13) have also proven quite useful in detecting RFLPs in plants. Minisatellite probes contain a repeated core sequence and were ®rst used to detect highly polymorphic bands in humans. The complexity of the resulting band pattern depends mainly on the probe. Probes speci®c to a single locus of the genome are preferred in genetic mapping. On the other hand, probes giving rise to RFLP patterns consisting of about 5ÿ20 bands could constitute a universal product code (UPC)-type `barcode' pro®le that would be useful in DNA ®ngerprinting. RFLPs generated with minisatellite probes or moderately repeated genomic clones are usually of this type. Over the past 20 years, RFLP markers have brought tremendous progress to the ®eld, such as genetic characterization of plant germplasm, genetic mapping and gene cloning. In roses, RFLPs have been developed as a means of cultivar identi®cation by DNA ®ngerprinting and classi®cation of wild roses. The major disadvantage of these widely used markers is the long and laborious procedure that is not well amenable to automation. Consequently, the entire process usually takes several days to accomplish. RFLPs also require fairly high-quality, undegraded DNA in amounts usually measured in micrograms, about 5ÿ10 mg. However, once made, membranes carrying bound

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denatured DNA could be probed, stripped of the probe and reprobed. RFLP marker management and dissemination are also tedious. Because of these drawbacks, this method has largely been replaced by new classes of ef®cient PCR-based molecular markers. However, RFLPs remain one of the most reliable molecular markers. RAPD, AP-PCR and DAF

A major leap in the development of molecular markers occurred with the discovery of PCR. PCR is a process for generating many copies of a speci®c target DNA sequence in vitro, using DNA polymerase enzyme and two short (typically 524 base) synthetic DNA fragments, called primers, that anneal to either end of the target sequence and enable the polymerase to copy the DNA between them. Subsequent heating separates the templates and the newly synthesized DNA strands, both of which can anneal to fresh primers when brought to a lower temperature, to begin a new cycle of DNA synthesis. After each cycle, the number of copies of the target region doubles and these copies serve as templates for the next cycle. Due to the exponential increase in the number of copies in each cycle, millions of copies are produced at the end of the PCR process that typically consist of 35ÿ40 cycles. The use of thermostable DNA polymerase permits multiple cycles of primer annealing, DNA copying and strand separation to be performed using automated heating blocks, called thermal cyclers. Several kinds of molecular markers based on PCR were developed initially for the analysis of plant genomes. These methods, termed random ampli®ed polymorphic DNA (RAPD), arbitrary-primed PCR (AP-PCR) or DNA ampli®cation ®ngerprinting (DAF), employ a single short (e.g. 10 base) oligonucleotide as a primer to amplify multiple regions in the genome. They produce a DNA banding pattern that depends on the substrate DNA and the primer used. Because of the use of a short primer, simply by chance it can anneal to sites in the DNA that are close enough together in the right orientation to permit PCR ampli®cation of the intervening DNA segment. For all three methods, no prior knowledge of the target DNA sequence is necessary. Theoretically, variations in primer recognition sites and insertions and deletions between the priming sites provide the basis for polymorphisms. However, in practice, the formation of secondary structures of initial PCR products is also known to produce variations in band pro®les. Among these three types, RAPD and AP-PCR are very similar methods developed simultaneously by two independent laboratories. In these two methods, primers consist of 10 random

bases. In RAPDs, PCR products are separated by size in agarose gels and visualized by staining with ethidium bromide. AP-PCR uses polyacrylamide gels coupled with autoradiography to analyse products. In the DAF method, the primers consist of only 5ÿ8 bases and the PCR products are separated by polyacrylamide gel electrophoresis and visualized by silver staining. Due to the use of short primers, the DAF method gives rise to more variation than RAPDs and is more suitable for analysis of genetically very similar organisms. These initial PCR-based methods offered many advantages over RFLPs and soon became the markers of choice for plant scientists. Among the three types, RAPD markers were used extensively in germplasm characterization, taxonomic assessments and gene mapping. The technical simplicity and the fast rate at which the markers can be developed are the most attractive features of these methods. These markers can be generated within just a few hours as opposed to the many days that it used to take for RFLPs. Another important advantage was the elimination of the requirement of a probe and associated steps in probe labelling and hybridization. Being PCR-based, only a small amount of DNA, usually a few nanograms per reaction, was needed for these methods. Therefore, mini DNA extraction methods from plant tissue often suf®ced. Moreover, the procedures were amenable to automation to increase the throughput. Despite these attractions, initially, questions were raised regarding the reproducibility of band pro®les between different DNA extraction methods, PCR runs or laboratories. Ampli®cation of genomic DNA using short random primers is sensitive to reaction conditions. Therefore, PCR reactions and temperature cycles had to be optimized carefully to obtain consistent results. However, with time, RAPDs became widely used and most scientists using this method were able to separate artifacts from true ampli®cation products and relied only on bands that could be well reproduced. Informative RAPD markers were often converted to speci®c markers such as sequence characterized or ampli®ed regions or SCARs to make screening for polymorphisms more ef®cient. RAPD markers generally detect fairly high levels of variation, making them very useful in genetic analyses. But because of the high level of polymorphism revealed by this method, they are most useful in studies at or below the species level, even though at times they have been used at higher taxonomic levels. In roses, RAPDs have been developed to study phylogenetic relationships between species and sections, identify and ®ngerprint cultivars, and analyse wild populations. RAPDs have also been used in constructing genetic maps and

GENETICS/Molecular Markers

marker-assisted screening of rose germplasm for resistance to black spot disease. In addition, they have been employed to test for apomictic origin of dog rose progeny and triparental origin of Damask roses. AP-PCR markers have been employed to study the domestication process of the modern roses. A major drawback of these PCR-based markers is that they are dominant markers scored only as present or absent. In other words, the dominant allele, which gives rise to the PCR product, masks the presence of the recessive allele that does not give rise to a PCR product. Therefore, homozygotes of the genotype with two dominant alleles are not distinguishable from heterozygotes having one dominant allele and one recessive allele. However, a small percentage of RAPD markers show codominance at some loci, where different alleles of a marker can be seen. Development of RAPDs also accelerated the process of identifying markers linked to phenotypic characters through bulked segregant analysis (BSA). Instead of screening individual plants of a segregating progeny in search of markers linked to a trait, BSA allowed creation of bulks consisting of plants with similar phenotype and the rapid screening of the bulks with a large number of primers. Many markers linked to economically important traits have been discovered by this now-widespread method, including markers linked to a gene conferring resistance to black spot in roses. AFLP

More recently, a novel marker system that scans the genome more rapidly has been developed. These markers, known as ampli®ed fragment length polymorphism (AFLP), are generated by PCR ampli®cation of a selection of restriction-digested genomic DNA. First, genomic DNA is cut with two restriction enzymes and short synthetic DNA adapters are ligated to the restriction fragments. PCR ampli®cation is performed on the restriction fragments with primers based on adapter sequence along with two to four additional bases chosen at random. These additional bases select fragments to be ampli®ed in each run from the initial restriction fragment pool, reducing the number of ampli®ed fragments to allow them to be clearly resolved by electrophoresis. Then, denaturing polyacrylamide gel electrophoresis is used to separate ampli®ed products. The AFLP marker system combines both PCR and RFLP technology, involves many steps in development and is technically fairly sophisticated. The patented AFLP procedure is a powerful method that allows simultaneous identi®cation of polymorphism at a large number of places in the genome. The method is easily adapted for bulked segregant analysis. Informative markers are routinely converted

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to sequence tagged sites (STS) or SCARs. As most markers are dominant, AFLPs share the drawbacks of the PCR-based methods described above. Because of the large number of markers that can be developed in a relatively short period of time, marker scoring is perhaps the most tedious aspect of developing AFLPs. Software for automated scoring is available when ¯uorescent dye-labelled primers are used in conjunction with sequencing machines. AFLP has been the primary marker system used to construct two tetraploid and two diploid rose maps. Other rose maps used both RAPD and AFLP markers. An example of AFLP markers developed by denaturing polyacrylamide gel electrophoresis for map construction is given in Figure 1. AFLP markers linked to single-gene rose traits and quantitative trait loci have also been identi®ed. STS and SCAR

Once an informative marker has been detected among the bands in any one of the PCR-based methods described above, that particular marker is separated from the rest for effective genotype scoring. The speci®c band is cut from the gel and DNA-cloned. Then its DNA sequence is determined. Based on the information of the DNA sequence, primers speci®c to this product are designed and used in the ampli®cation of that particular region in the genome. These markers, termed STS or SCAR, use a pair of unique primers, each about 20 bases long, to amplify speci®c short target sites that have a single occurrence in the genome. Conversion from random primers to speci®c primers makes screening of the DNA samples ef®cient. Another advantage of this is the conversion of dominant markers such as RAPD and AFLP to codominant types where multiple alleles can be visualized. In practice, when RAPD or AFLP markers are converted to STS, sometimes the original polymorphism is lost. In this situation, additional steps such as restriction digestion of the PCR products are necessary to visualize polymorphisms again. Markers developed by cleaving the speci®c PCR products with restriction enzymes are termed cleaved ampli®ed products (CAPs). STS markers derived from expressed genes are known as expressed sequence tags (ESTs). SSR (STMS)

A special class of molecular markers based on simple sequence repeats (SSRs) has lately become very useful in observing genetic variability in plants. Tandem arrays of short nucleotide repeats, e.g. (AT)n, (CAT)n or (GATA)n, are ubiquitous in regions known as microsatellites in eukaryotic genomes. The copy number (n) of these repeats can vary among alleles and provides the

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GENETICS/Molecular Markers

GP1 P1 P2 F1 ........................................... F2 .................................................................

Figure 1 Denaturing polyacrylamide gel exhibiting ampli®ed fragment length polymorphism (AFLP) markers generated in rose for genetic map construction. DNA from a tetraploid test progeny set identi®ed as 90ÿ69 was cut with restriction enzymes EcoRI and MseI. Bases used for further selection of cut pieces were ACC and CAT, for EcoRI and MseI, respectively (see text for details of the method). Lane representations are as below. Lane 1, GP1, one of the grandparents of the progeny, Rosa wichurana CreÂp.cv. `Basye's Thornless'; lane 2, P1, the pollen parent of the progeny, identi®ed as 86ÿ7; lane 3, P2, the seed parent of the progeny, known as `Basye's Blueberry'; lane 4, the F1, 90ÿ69. The rest of the lanes represent various F2 progeny. Of all roses that produced these markers, only GP1 is a diploid; all others are tetraploids. Note the large number of bands ampli®ed from multiple loci in the genome. No allelic relationships can be directly inferred from these markers. Compare with Figure 2, that shows allelic variation arising from a single locus.

basis for polymorphism. In the classical method of generating these markers, a random collection of short cloned DNA fragments (or library) is screened with a number of microsatellite probes to identify clones containing SSRs. The DNA sequence is determined for SSR-containing clones and primers are designed based on unique sequences ¯anking SSR. The primers are used in amplifying genomic DNA to reveal polymor-

phisms based on number of repetitions present in each copy of the SSR. When a speci®c primer pair is used to amplify a single SSR-containing region, the marker is known as a sequence tagged microsatellite site (STMS). SSRs are abundant throughout plant genomes and exhibit high levels of polymorphism. The codominant markers allow all alleles in a given locus to be observed. This is an advantage of microsatellite markers, especially in genetic analysis and mapping of polyploid species, where multiple alleles are present. SSRs are currently being used to map tetraploid roses. A polyacrylamide gel displaying SSR markers developed from a rose-mapping progeny set is shown in Figure 2. The main shortcoming of microsatellite markers is the large amount of resources needed to generate the markers. Once established, they are simple PCR-based markers that are highly informative. While being effective in the species in which they were developed, SSRs can also be effective in species that are closely related to it. Indeed, several SSR primers derived from peach, apple and sour cherry, all of which are in the family Rosaceae, have been effective in amplifying rose DNA. Some of the peach SSR markers have been placed on rose maps. Both nuclear and chloroplast-derived SSRs have also been developed in rose. Nuclear-derived SSR markers have been employed in the genetic analysis of dog rose progenies and to elucidate the genomic composition in the polyploids. SSR markers are also being used to consolidate various genetic maps now available into one core map for rose. These markers will be extensively used in the future for diverse genetic studies of roses, including tracking of gene ¯ow, population analysis and establishment of a core marker set for rose cultivar identi®cation. ISSR

Because of the initial expenses in developing SSR markers, variations of the above-mentioned original SSR method have come into use. In one such modi®cation, single primers consisting of simple sequence repetitions are used in the PCR to amplify the region in between matching primer sites. These intersimple sequence repeats (ISSRs) are quite similar to RAPDs in the overall method of marker generation and visualization, simultaneous ampli®cation of multiple loci and dominance of markers at most loci. However, ISSR primers are typically longer, consisting of about 15ÿ25 bases, and stringent PCR reaction conditions are used. These differences result in more reproducible markers compared to RAPDs. ISSR primers have been claimed to be more likely to amplify unique regions in the genome compared to short random primers that tend to amplify repeated regions. Long RAPD primers (15ÿ20 bases) are also known to amplify a low amount of repetitive

GENETICS/Molecular Markers GP1 P1 P2 F1...........................................F2..................................................................

Figure 2 Denaturing polyacrylamide gel exhibiting microsatellite markers generated in rose for genetic map construction. Genomic DNA of a tetraploid test progeny set 90ÿ69 was polymerase chain reaction (PCR)-ampli®ed with speci®c primers to amplify DNA from a single locus (Rw5D11). Lane representations are as below. Lane 1, GP1, one of the grandparents of the progeny Rosa wichurana CreÂp.cv. `Basye's Thornless'; lane 2, P1, the pollen parent of the progeny, identi®ed as 86ÿ7; lane 3, P2, the seed parent of the progeny, known as `Basye's Blueberry'; lane 4, the F1, 90ÿ69. The rest of the lanes represent various F2 progeny. Of all roses that were used to produce these markers, only GP1 is a diploid; all others are tetraploids. Note that the few bands produced show variation of the four alleles of a single locus in the tetraploid genomes. Compare with Figure 1, that shows genetic variation at many loci.

DNA. There are no reports available on the use of ISSR markers in roses.

Applications of Molecular Markers The main types of markers used in plants are described above. A plethora of terms are available to describe slight variations in these different kinds of markers. Hence, terms not widely used have been omitted from the above discussion. These markers are applicable in diverse ®elds, from plant breeding to gene cloning. In each ®eld, speci®c features of markers become important for successful application. Some of the important features of various types of markers are summarized in Table 1. Marker-assisted Selection

By using DNA-based markers, many problems associated with the phenotypic evaluation of traits can be avoided in breeding programmes. DNA markers are especially useful in selecting for polygenic traits that prove dif®cult to select based on phenotypic assessment alone. When breeding programmes are assisted by the use of molecular markers, the process is commonly known as marker-assisted breeding or molecular breeding. For assessing genetic variation in the germplasm, RAPD and AFLP markers that scan the genome quickly would be useful, although technical sophistication of the AFLP method limits its use with

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the breeder. Markers that can be effectively applied in selecting progeny should be technically simple methods that can be performed in breeders' setting as opposed to methods used in a research laboratory. Markers should also be cost-effective to be performed. Data handling and management should be easy. For screening large numbers of progeny for markerassisted selections, simple PCR-based markers such as STS or SCARS are most appropriate. Allele-speci®c markers that would allow PCR-based allele identi®cation are also quite useful for the breeder. Markers tightly linked to single-gene traits as well as quantitative trait loci regions controlling polygenic traits will ®rst have to be established and then applied in the selection of progeny in breeding programmes. To track gene introgression, in addition to markers linked to the trait of interest in the nonrecurrent parent, markers distributed throughout the genome in the recurrent parent, in the form of a genetic map, are useful. The breeder and the molecular technologist must cooperate for the effective use of molecular markers as breeders trained in traditional ways alone are unable to use these methods due to the lack of skill and experience. Ecological Studies

Molecular markers can provide highly detailed assessments of the dynamics of genetic variation in natural populations in relation to ecological, geographical and temporal distributions. They could also be used to identify clonal populations, hybrids and offspring of apomictic origin. Molecular markers can shed light on the origin of domesticated plants from wild relatives. RFLP and RAPD markers have been used in diverse ecological studies, but AFLPs are now more ef®ciently employed in whole-genome scanning while SSR and STS markers are used in the detailed analysis of selected regions in the genomes. Genetic marker loci could be followed through in dispersal and gene-¯ow studies. Seed and pollen dispersal could be distinguished by using maternally derived (cytoplasmic) and paternally derived (pollen parent-speci®c) genetic markers. Markers derived from the chloroplast, which are often maternally inherited with exceptions in some plant species, are used to trace the cytoplasmic lineage. Molecular markers could also be used to trace the presence and ¯ow of transgenes arising from genetically modi®ed plants. For this purpose, STS markers with speci®c primers designed for the transgene can be used. Taxonomic/Phylogenetic Studies

Mutations in DNA provide a window to the evolutionary changes over millennia. Thus, molecular markers could be highly informative in uncovering

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Table 1 Comparison of some of the key features of main types of DNA markers Marker type

Development based on

Primer speci®city

RFLPa

Restriction digestion

(Not applied)

Codominant

RAPD AP-PCR DAF AFLP

PCR

Random primer

SSR (STMS)

Restriction digestion and PCR PCR

ISSR STS EST a

Mode of marker action

Loci speci®city

Ability to screen across progenies Direct

Dominant

Single locus to few loci Multiple loci

Random primer

Dominant

Multiple loci

Speci®c primer

Codominant

PCR

Random primer

Dominant

Single locus to few loci Multiple loci

After conversion to speci®c markers Direct

PCR

Speci®c primer

Codominant

Single locus

After conversion to speci®c markers

After conversion to speci®c markers Direct

See text for the de®nition of abbreviations.

the changes in genetic blueprint not visible in the phenotype. Markers shared among plant species and genera can provide a measure of their genetic distance which could be used to determine their phylogenetic relationships. Chloroplast genes are thought to evolve slower than the nuclear-genes. Therefore, chloroplast and nuclear-derived markers are used when studying evolution at different taxonomic levels. Genetic Mapping

In genetic mapping, molecular markers consist of short segments of DNA that provide landmarks along the chromosomes. The markers provide a scaffold of the entire genome where genes are located. In order to appreciate the contribution of genetic markers to mapping, it is interesting to note the progress of mapping over the decades. Initial maps of plants were constructed based on morphology and were dependent on ®nding phenotypic mutations. Therefore, the process was extremely slow and only a few traits were mapped by following this method. Then, the discovery of RFLPs enabled the use of genetic markers that are not visible to the naked eye, to be used in constructing maps, while it was still a time-consuming process. With the eventual development of RAPD markers, it was possible to construct molecular maps of many plants in a few months ÿ a relatively short time compared to what it used to be with the RFLPs. AFLPs further expedited the process, now routinely used, and maps developed in weeks. However, compared to codominant markers such as RFLP, STS and SSR, dominant markers such as RAPD and AFLP have a limited use in genetic analysis and mapping. Linkage maps speci®c to one parent are often constructed when using RAPDs or AFLPs with progeny derived from highly heterozygous parents, because a large number of progeny are required to ®nd linkage relationships

between markers arising from the two parents, which are said to be in repulsion. The ability to utilize markers in different progenies is best when using STS or SSR markers. RAPD and AFLP markers have to be converted to STS markers before they could be tested effectively in progeny sets other than the one from which they were ®rst derived. STS and SSR markers are also useful in combining maps constructed from different progenies. They also serve as landmarks on physical maps. Certain regions in the genomes of related plants are conserved in the course of evolution. Therefore, some of the markers that are developed from species related to rose, such as peach, apple and strawberry, can be useful in comparing the genomic structure between these species. Due to the similarity in the composition and order of genes among the related species, marker and map information derived from economically important crops in Rosaceae that have been studied extensively, such as apple and peach, can aid in rose map construction and saturation. Gene Identi®cation and Cloning

RAPD and AFLP markers are often used to identify markers linked to phenotypic traits using the bulked segregant analysis. These markers are very ef®cient when scanning the genome and identifying many markers linked to a gene. This is usually the ®rst step in many of the gene identi®cation and cloning experiments. The speci®c markers found linked to traits of interest will then be converted to PCR-based markers for ef®cient screening.

Conclusion All genetic markers represent a variation in DNA sequence. The usefulness of a marker depends on

GENETICS/Cloned Genes

its characteristics. Some markers detect variation at a single locus while others detect variation at multiple loci. Those that detect variation at a single locus are more descriptive and informative, while the latter kind examines more places in the genome for variation, albeit not providing as much genetic information as the single locus types. There are many other tradeoffs among the various markers in terms of simplicity of the method, ef®ciency of screening, cost and transferability to other studies. The choice of markers depends mainly on the purpose for which they will be used. Not all genetic markers de®ne a gene. Only those that have been developed from expressed genes or demonstrated to be located in a gene would. ESTs and RFLPs developed with cDNA probes are such gene markers. Molecular markers are a rapidly developing ®eld and simpler, less expensive and more ef®cient methods are constantly being developed. New transposon-based markers known as miniature inverted repeat transposable elements (MITES) for large genome species are currently available, though not widely identi®ed among plant species. A powerful new technique to detect single nucleotide polymorphisms (SNPs) has also been developed in humans and is now being applied to plants. While many studies have already been aided by the use of molecular markers, the full potential of various molecular markers has yet to be realized in roses. Undoubtedly, the future will bring the application of molecular markers to more studies in diverse ®elds to understand the world of roses. See also: Classi®cation: Cultivar Identi®cation by Image Analysis. Fragrance and Pigments: Functional Genomics. Genetics: DNA Fingerprinting; Gene Mapping; Molecular Markers; Cloned Genes; Marker-Assisted Selection.

Further Reading Bachmann K (1994) Tansley review no. 63. Molecular markers in plant ecology. New Phytologist 126: 403ÿ418. Beyermann B, Nurnberg P, Weihe A et al. (1992) Fingerprinting plant genomes with oligonucleotide probes speci®c for simple repetitive DNA sequences. Theoretical and Applied Genetics 83: 691ÿ694. Botstein D, White R, Skolnick M and Davis R (1980) Construction of a genetic map in man using restriction fragment length polymorphisms. American Journal of Human Genetics 32: 314ÿ331. Caetano-Anolles G, Bassam BJ and Gresshoff PM (1991) DNA ampli®cation ®ngerprinting using very short

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arbitrary oligonucleotide primers. BioTechnology 9: 553ÿ557. Jefferys AJ, Wilson V and Thein SL (1985) Individual speci®c `®ngerprints' of human DNA. Nature 316: 76ÿ79. Lander ES and Botstein D (1989) Mapping Mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 121: 185ÿ199. Michelmore RW, Paran I and Kesseli RV (1991) Identi®cation of DNA markers linked to disease resistance genes by bulked segregant analysis: a rapid method to detect markers in speci®c genomic regions using segregation populations. Proceedings of the National Academy of Sciences of the USA 88: 9828ÿ9832. Mullis KB and Faloona FA (1987) Speci®c synthesis of DNA in vitro via a polymerase catalysed reaction. Methods in Enzymology 255: 335ÿ350. Mullis KB, Faloona FA, Scharf S et al. (1986) Speci®c enzymatic ampli®cation of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symposia on Quantitative Biology 51: 263ÿ273. Paran I and Michelmore RW (1993) Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theoretical and Applied Genetics 85: 985ÿ993. Phillips RL and Vasil IK (2001) DNA-based Markers in Plants. Dordrecht: Kluwer Academic. Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Plainview, NY: Cold Spring Harbor Laboratory Press. Vos P, Hogers R, Bleeker M et al. (1995) AFLP: a new technique for DNA ®ngerprinting. Nucleic Acids Research 23: 4407ÿ4414. Welsh J and McClelland M (1991) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Research 18: 7213ÿ7218. Williams JGK, Kubelik AR, Livak KJ, Rafalski JA and Tingy SV (1990) DNA polymorphism ampli®ed by arbitrary primers as useful genetic markers. Nucleic Acids Research 18: 6531ÿ6535.

Cloned Genes Y Tanaka and M Fukuchi-Mizutani, Institute for Fundamental Research, Suntory Ltd, Osaka, Japan J G Mason, Florigene Ltd, Collingwood, Victoria, Australia # 2003, Elsevier Ltd. All Rights Reserved.

Introduction Recent rapid progress in genomics has made it possible to understand and discuss plant species in term of genes.