PREFACE REPETITIVE DNA: AN IMPORTANT SOURCE OF VARIATION IN EUKARYOTIC GENOMES Categories of DNA Sequence Mutations Tandem-Repetitive DNA: The Biology of Mini- and Microsatellites Minisatellites Microsatellites Transposable Elements Class I Transposons Class II Transposons Unclassified Transposons Transposons and Genome Evolution Transposons as Molecular Markers DETECTING DNA VARIATION BY MOLECULAR MARKERS Properties of Molecular Markers Traditional Marker Systems Protein Markers and Allozymes DNA Sequencing Restriction Fragment Length Polymorphism (RFLP) Analysis The PCR Generation: Molecular Markers Based on In Vitro DNA Amplification Principle of the PCR Cleaved Amplified Polymorphic Sequences PCR with Arbitrary Primers: RAPD and Its Variants Microsatellites Inter-Repeat PCR DNA Profiling of Genic Regions: Resistance Gene Analog Polymorphism, Sequence-Related Amplified Polymorphism, and Target Region Amplification Polymorphism Hybridization of Microsatellites to RAPD and MP-PCR Products AFLP Analysis and Its Variants Single-Strand Conformation Polymorphism Analysis and Related Techniques Miscellaneous Techniques LABORATORY EQUIPMENT METHODOLOGY Safety Precautions Isolation, Purification, and Quantitation of Plant DNA Collection and Preservation of Plant Tissue in the Field Plant DNA Extraction: General Considerations CTAB Protocol I CTAB Protocol II SDS-Potassium Acetate Protocol DNA Preparation via Nuclei Quantitation of DNA Basic Molecular Techniques Restriction of DNA Polymerase Chain Reaction DNA Sequencing Agarose Gel Electrophoresis PAA Gel Electrophoresis Detection of DNA in Gels Gel Drying Southern Blotting Generation of Radiolabeled Probes, Primers, and PCR Products Blot Hybridization Signal Detection PCR with Arbitrary Primers Standard RAPD Protocol Influence of Reaction Conditions and Components Modifications Microsatellite-Primed PCR Standard Protocol of Microsatellite-Primed PCR Influence of Reaction Conditions and Components Modifications PCR and Hybridization: Combinatory Techniques Assessing the Genomic Copy Number of PCR Amplicons Testing the Homology of Comigrating Bands Random Amplified Polymorphic Microsatellites Amplified Fragment Length Polymorphism Standard AFLP Protocol Using Radioisotopes AFLP Protocol Using Fluorescence-Labeled Primers Selective Amplification of Microsatellite Polymorphic Loci and Microsatellite AFLP Protocols Technical Aspects and Modifications Robustness and Reproducibility Generation and Analysis of Microsatellite Markers Microsatellite Analysis Using Radioisotopes Microsatellite Analysis Using Fluorochromes Technical Aspects and Modifications Generating Microsatellite Markers Without Cloning Microsatellite Cloning CAPS Analysis of cpDNA and mtDNA Standard CAPS Protocol Choice of CAPS Primers EVALUATION OF MOLECULAR MARKER DATA Robustness and Reproducibility Reliability Band Homology Band Linkage and Neutrality Fragment Sizing and Matching General Precautions Equipment Multilocus vs. Single-Locus Approaches Multilocus Markers Single-Locus Markers and Polyploids Band Sharing and Genetic Distances Coefficients of Similarity Dissimilarity Coefficients and Genetic Distances Identity and Uniqueness Clonal Structure Ordination, Clustering, and Dendrograms Ordination Techniques Construction of Dendrograms Population Genetic Analysis Measures of Variation Genetic Differentiation between Populations Genetic Distances between Populations Inbreeding Coefficient and Mating Systems Estimation of Relatedness and Paternity Testing Migration and Hybridization Gene Flow, Isolation-by-Distance, and Spatial Structure Phylogeography and Nested Clade Analysis Statistical Testing of Hypotheses: Analytical and Computational Methods APPLICATIONS OF DNA FINGERPRINTING IN PLANT SCIENCES A Brief History of DNA Fingerprinting Minisatellite and Oligonucleotide DNA Probes Detect Genetic Variation PCR-Based Methods Enter the Stage Microsatellite DNA Analyses Yield Codominant Markers Universal Organellar DNA Primers Produce Uniparental Markers Genotype Identification Individual-Specific DNA Fingerprints Cultivar Identification In Vitro-Propagated Plant Material and Somaclonal Variation Sports and Other Mutants Genetic Diversity Variation and Relatedness among Cultivars Analysis of Population Genetic Diversity and Its Distribution Hybridization and Introgression Plant Conservation Germplasm Characterization and Preservation Plant Taxonomy and Systematics Taxonomic Relationships Revealed by Multilocus DNA Methods Microsatellite Markers in Taxonomic Studies Taxonomic Consequences from DNA Profiling Data Phylogeography Phylogeography Based on cpDNA Phylogeography Based on Nuclear Genes LINKAGE ANALYSIS AND GENETIC MAPS Generating High-Density Genetic Maps Selection of Parent Plants Mapping Population Linkage Analysis The Genetic Map Cytogenetic Maps Genetic vs. Physical Maps Synteny: The Comparative Analysis of Genomes Marker-Assisted Selection Molecular Markers and Positional Cloning WHICH MARKER FOR WHAT PURPOSE: A COMPARISON Morphological Characters and Allozymes vs. DNA Markers Different Kinds of DNA Markers Discriminatory Power Genetic Distances Within- and Among-Population Variation Gene Tagging and Genetic Linkage Mapping Costs Conclusions FUTURE PROSPECTS: SNIPS AND CHIPS FOR DNA AND RNA PROFILING Single-Nucleotide Polymorphisms What Is a SNiP SNP Discovery DNA Microarrays Expression Profiling and Expression Markers APPENDIX 1: PLANT DNA ISOLATION PROTOCOLS APPENDIX 2: SUPPLIERS AND SELLERS OF REAGENTS AND EQUIPMENT APPENDIX 3: COMPUTER PROGRAMS DEALING WITH THE EVALUATION OF DNA SEQUENCE VARIATION AND MOLECULAR MARKER DATA APPENDIX 4: WEB PAGES OF INTEREST REFERENCES INDEX

DNA fingerprinting in plants: principles, methods, and applications

More than 9000 papers using the random amplified polymorphic DNA (RAPD) or related techniques (e.g. the arbitrarily primed polymerase chain reaction (AP-PCR)) have been published from 1990 to 2005. The RAPD method has been initially used to detect polymorphism in genetic mapping, taxonomy and phylogenetic studies and later in genotoxicity and carcinogenesis studies. Despite their extensive use, these techniques have also attracted some criticisms, mainly for lack of reproducibility. In the light of their widespread applications, the objectives of this review are to (1) identify the potential factors affecting the optimisation of the RAPD and AP-PCR assays, (2) critically describe and analyse these techniques in genotoxicity and carcinogenesis studies, (3) compare the RAPD assay with other well used methodologies, (4) further elucidate the impact of DNA damage and mutations on the RAPD profiles, and finally (5) provide some recommendations/guidelines to further improve the applications of the assays and to help the identification of the factors responsible for the RAPD changes. It is suggested that after proper optimisation, the RAPD is a reliable, sensitive and reproducible assay, has the potential to detect a wide range of DNA damage (e.g. DNA adducts, DNA breakage) as well as mutations (point mutations and large rearrangements) and therefore can be applied to genotoxicity and carcinogenesis studies. Nevertheless, the interpretation of the changes in RAPD profiles is difficult since many factors can affect the generation of RAPD profiles. It is therefore important that these factors are identified and taken into account while using these assays. On the other hand, further analyses of the relevant bands generated in RAPD profile allow not only to identify some of the molecular events implicated in the genomic instability but also to discover genes playing key roles, particularly in the initiation and development of malignancy. Finally, to elucidate the potential genotoxic effects of environmental contaminants, a powerful strategy could be firstly to use the RAPD assay as a screening method and secondly to apply more specific methods measuring for instance DNA adducts, gene mutations or cytogenetic effects. It is also envisaged that these assays (i.e. RAPD and related techniques), which reflect effects at whole genome level, would continue to complement the use of emerging technologies (e.g. microarrays which aim to quantify expression of individual genes).

The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: a critical review.

Screening of an apple genomic library with (GA)15 and (GT)15 probes demonstrated that these repeats are abundant, occurring about every 120 and 190 kb, respectively. Microsatellites isolated from a small insert library enriched for (GA) repeats contained numbers of repeats ranging from 7 to 39. Primers to these microsatellite loci were able to direct the amplification of the repeats in 21 different cultivars. The majority of markers were highly polymorphic, diploid, and showed simple Mendelian inheritance, although about 25% of markers generated complex banding patterns consistent with the amplification of more than one locus. As few as three microsatellite markers were sufficient to differentiate between all 21 cultivars.

Microsatellites in Malus X domestica (apple): abundance, polymorphism and cultivar identification

Linkage maps for two apple clones, White Angel and Rome Beauty, were constructed using isozyme and DNA polymorphisms segregating in a population produced from a Rome Beauty x White Angel cross. The linkage map for White Angel consists of 253 markers arranged in 24 linkage groups and extends over 950 cM. The Rome Beauty map contains 156 markers on 21 linkage groups. The White Angel map was taken as the standard, and we were able to identify linkage groups in Rome Beauty homologous to 13 White Angel linkage groups. The location of several genes not segregating in the Rome Beauty x White Angel population could be determined on the basis of known linkages with segregating markers. Hence, the standard map for apple now contains about 360 markers, with most linkage groups saturated at 10-15 cM. The double pseudotestcross format of the mapping population permitted the comparison of recombination frequencies in male and female parents in certain regions of the genome where appropriate markers were available. The recombination frequencies observed for the approximately 170 cM that were comparable gave no indication that a sex-related difference in recombination rate was characteristic of apple.

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Molecular Marker Linkage Map for Apple

AFLP markers were evaluated for determining the phylogenetic relationships Lactuca spp Genetic distances based on AFLP data were estimated for 44 morphologically diverse lines of cultivated L sativa and 13 accessions of the wild species L serriola, L saligna, L virosa, L perennis, and L indica The same genotypes were analyzed as in a previous study that had utilized RFLP markers The phenetic tree based on AFLP data was consistent with known taxonomic relationships and similar to a tree developed with RFLP data The genetic distance matrices derived from AFLP and RFLP data were compared using least squares regression analysis and, for the cultivar data, by principal component analysis There was also a positive linear relationship between distance estimates based on AFLP data and kinship coefficients calculated from pedigree data AFLPs represent reliable PCR-based markers for studies of genetic relationships at a variety of taxonomic levels

PCR-based fingerprinting using AFLPs as a tool for studying genetic relationships in Lactuca spp

Eleven apple cultivars were differentiated using randomly amplified polymorphic DNA (RAPD) markers obtained by the polymerase chain reaction (PCR). The variability of the technique and of the origin of the DNA extract was analyzed. A set of bands consistent in their presence or absence was chosen to create a differentiating band pattern. A key is proposed by which one can differentiate apple cultivars using commercially available prime.

A Lehmann

Papers

Identification of apple cultivars using RAPD markers.