PerspectivesAre you interested in submitting a Perspective Article? Be sure to read The Science Advisory Board's Editorial Guides for Perspective Articles. Click here. Molecular Genetic Markers: What? Why? Which one for Exploring Genetic Diversity? by Madhugiri Nageswara-Rao & Jaya R. Soneji University of Florida, IFAS, Citrus Research and Education Centre, 700 Experiment Station Road, Lake Alfred, FL 33850, USA Correspondence: E-mail: mnrao@crec.ifas.ufl.edu or jrs@crec.ifas.ufl.edu Measuring genetic variation and patterns of population/species (intra-/inter-specific level) genetic diversity among biota helps us in drawing appropriate methodology for its selective breeding, rapid domestication and/or conservation. Availability of a wide array of molecular genetic markers offers tools for quick detection and characterization of genetic variation. However, the major constraint is the selection of the precise or appropriate molecular genetic marker. The selection of the marker may depend on the number of gene loci to be studied, the levels of polymorphism to be detected and/or the cost to be involved in it. Molecular genetic markers are regions in the genome that are heritable as simple Mendelian traits, are easy to document (Schulman et al., 2004) and can be used to quickly detect genetic variation. Genetic variation results from differences in the DNA sequences which may have arisen due to mutations caused by either an insertion, deletion, duplication or inversion of DNA fragments. They may be 'functional' causing changes in metabolic or phenotypic traits or 'neutral' when not subjected to positive, negative or balancing selection (Marsjan and Oldenbroek, 2007). Variants arising due to mutations may have an increased or decreased metabolic efficiency or may have various levels and/or patterns of gene expression at different developmental and/or physiological stages or may lose their functionality completely or gain a novel function. However, the challenge lies in making the correct choice of molecular genetic markers for the identification of these variations in populations. Molecular genetic markers can be divided into two classes a) biochemical markers which detect variation at the gene product level such as changes in proteins and amino acids and b) molecular markers which detect variation at the DNA level such as nucleotide changes: deletion, duplication, inversion and/or insertion. Markers can exhibit two modes of inheritance, i.e. dominant/recessive or co-dominant. If the genetic pattern of homozygotes can be distinguished from that of heterozygotes, then a marker is said to be co-dominant. Generally co-dominant markers are more informative than the dominant markers. Biochemical markers Variations are detected by biochemical markers as electrophoretic protein polymorphisms (isozymes). Isozymes are able to detect the diversity at the functional gene level and have simple inheritance, co-dominant expression, complete penetrance and no pleiotropic and epistatic interactions. They are relatively inexpensive, rapid, and technically easy to apply as compared to DNA techniques. They show lower levels of diversity or polymorphism in a population as they assay relatively small numbers of genetic loci. They are unsuitable when a very high resolution of diversity is required. They may also be affected by the environmental factors such as age, plant part sampled, etc. However, these markers may be the choice when the cost involved for the routine testing is lower than using molecular markers. Molecular markers The advent of DNA-based molecular markers have circumvented the limitations encountered by protein-based markers and are revolutionizing the area of molecular genetic analysis. These technologies detect and analyze the variation at the DNA level. A large number of such marker systems are now available which can directly evaluate the genome, ensure genome wide coverage and are neutral to the environmental factors and developmental stages and are not influenced by other genes and factors. Molecular markers can be classified into two major groups a) based on DNA-DNA hybridization (eg. RFLP) and b) based on Polymerase Chain Reaction (PCR) amplification of genomic DNA fragments (RAPD, ISSR, SSR, SCAR, AFLP, SNP, CAPS, etc.). DNA-DNA hybridization marker Restriction Fragment Length Polymorphism (RFLP): Detection of RFLPs involves the fragmentation of genomic DNA by restriction enzymes that recognize specific DNA sequences/motifs (generally frequent cutters 4-10bp in length). The restricted DNA fragments are separated by gel electrophoresis and transferred onto a membrane by Southern blotting (Southern 1979). Hybridization of the membrane to a labeled DNA probe (a piece of known DNA sequence) then determines the size of the fragments that are complementary to the probes. The polymorphism arises from sequence changes in the restriction sites as well as from the detection of insertion/deletion in the restriction fragments detected by the probe. Analysis of RFLP variation is an important tool in genome mapping, identification of disease resistance genes, paternity testing, genetic fingerprinting, etc. PCR Markers PCR is less technically demanding than other DNA-based markers and requires only a small amount of DNA. In addition, PCR provides flexibility in detecting genetic variation as a variety of primers can be designed and used to reveal particular types of polymorphism. PCR-based markers include RAPD, AFLP, SSR, ISSR, SCAR, SNP, CAPS, etc. Random Amplification of Polymorphic DNA (RAPD): In this, primers of short length (~10 nucleotides) are used to amplify random locations in/across the genome (Williams et al., 1990). Due to their short length, they have the possibility of annealing at a number of locations in the genome. The number of PCR amplification products is directly related to the number and orientation of the sequences that are complementary to the primer in the genome. RAPDs require no prior knowledge of sequence information for primer designing. It is simple, fast, relatively cheap and widely used for population diversity studies, construction of genetic maps, tagging desirable traits for marker assisted selection, etc. However, reproducibility can be affected by low stringency PCR amplifications, the source of the tissue used and the protocol used for DNA extraction. It being a dominant marker, the recessive allele expression may not be found. Amplified Fragment Length Polymorphism (AFLP): AFLP combines restriction digestion and PCR amplification. Restriction fragments have adapters ligated to their ends allowing PCR amplification from primers derived from the adapters (Vos et al., 1995). Polymorphisms arise from sequence changes in the restriction sites or the selective bases used by the primers and amplifies 50-100 PCR fragments at a time. AFLP is generally a dominant molecular marker and requires no prior sequence information. It is highly sensitive and reproducible. It is widely used in the identification of genetic variation in populations, hybrids, closely related species, criminal and paternity tests, to determine linkage studies, creating genetic maps for QTL analysis, etc. However, it involves many steps, is labor intensive and time consuming. Problems may also arise due to homoplasy (the co-migration of non-related PCR products). Simple Sequence Repeats (SSR): SSRs, also known as microsatellites, are a group of tandem repeated sequences of mono-, di-, tri-, tetra-, penta-, or hexa-nucleotide units such as (A)10, (GA)8, (CAC)6, (GATA)4 or (GATAG)4. For PCR amplification, two unique primers that flank the microsatellite locus are used to amplify the genomic DNA. The PCR amplification products can be resolved on agarose gel or analyzed by an automated process. SSRs are co-dominant markers, reliable and highly reproducible (Morgante and Olivieri, 1993) having a wide range of applications such as in population genetic studies, determination of paternity, genotyping and genetic mapping, systematic taxonomy, molecular evolution, hybrid selection, etc. Isolation of useful SSR loci can be a time consuming, laborious and expensive process. However, SSR mining from nucleotide sequences is comparatively less laborious, fast and cheap. Inter Simple Sequence Repeats (ISSR): ISSRs are semi-arbitrary markers amplified by PCR in the presence of one primer complementary to a target microsatellite. Such amplification does not require genome sequence information and leads to multilocus and highly polymorphous patterns (Nagaoka et al., 1997). Each band corresponds to a DNA sequence delimited by two inverted microsatellites. Like RAPDs, ISSR markers are quick, cheap, easy to handle and are applicable to routine genetic screening of populations and other diversity analysis. Sequence Characterized Amplified Region (SCAR): To overcome the problems associated with RAPDs and/or AFLPs and improve their utility, they are converted to SCAR markers. This is done by cloning and sequencing the RAPD and/or AFLP products, designing long specific primers based on that sequence and amplifying the DNA under stringent conditions. SCARs are co-dominant markers. Cleaved Amplified Polymorphic Sequence (CAPS): The technique involves PCR amplification of a target DNA followed by restriction digestion of the PCR products. They amplify a short genomic sequence around the polymorphic endonuclease restriction site. They are co-dominant markers and useful for phylogenetic studies, mapping and genotyping in positional or map-based cloning. Single Nucleotide Polymorphism (SNP): SNP is just a single base change in a DNA sequence. Being a biallelic marker, it has an alternative of two possible nucleotides at a given position. They can be generated by sequencing, single-stranded conformational polymorphism or denaturing high-performance liquid chromatography or in silico, aligning and comparing multiple sequences of the same region from public genome and expressed sequence (EST) databases. They can be genotyped either by allele specific hybridization, primer extension, oligonucleotide ligation or invasive cleavage. They are co-dominant markers. Due to their abundance and distribution throughout the genome, they are preferred for mapping, marker-assisted breeding and map-based cloning. Conclusion As every marker has advantages and disadvantages (Table 1), the use of a marker system in one species does not necessarily indicate its applicability in another species. The choice of the marker usually depends on the purpose for which the marker system has to be used. RFLPs, though still used extensively, require large amounts of high quality DNA and information on probe to be used. AFLPs are highly reproducible but are labor intensive and costly. RAPDs and ISSRs are preferred when no sequence information is available and the cost involved is low. SCAR cannot be directly used unless one has developed it by sequencing fragments of important traits. CAPS can be used for those where prior sequence information is available. SSRs are preferred as they generate a high number of alleles as compared to isozymes. SNPs seem very promising; however, they require expensive equipment and are labor intensive.  References:
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