A molecular marker is a fragment of DNA that is associated with a part of the genome and can be identified by a simple assay. Molecular markers are based on differences in DNA sequence and, as such, are not subject to environmental influence; they are abundant throughout the entire genome, and tests can be performed at any time during plant development. Molecular markers are detected as differences in DNA fragment size, which arise from differences in DNA sequence. Molecular markers are used widely in cultivar identification, assessment of genetic variability among and within population(s), molecular mapping (i.e., determining the linear order of molecular markers in a genome), and marker-assisted breeding (tagging of an important trait or traits in a breeding program). Thus, they are considered the basic tools of genomic research. The effectiveness of molecular markers depends on their ability to identify variation in the DNA of a population, often known as marker polymorphism. They are genetic components and highly heritable. The genome size of most plant species ranges between 108 to 1010 base pairs, so even a small proportion of variation in DNA can yield a large number of potential markers (Paterson et al., 1991). The ideal marker class should be abundant, stable, easily detectable, and have a high degree of polymorphism. The marker classes commonly used for germplasm characterization and genetic improvement of tall fescue are described below.
Restriction fragment length polymorphisms (Tanksley et al., 1989) were the first set of molecular markers developed that were linked to genes controlling important traits. The RFLPs are codominant and all three morphs (i.e., forms) can be identified, so these are highly informative because homozygotes, in which all copies of a specific gene have the same form, do not provide any information. Major disadvantages of RFLPs are the robust, laborious, and costly protocols needed to generate these markers. In the early 1990s, attempts were undertaken to generate RFLP markers for tall fescue. These were used for the construction of genetic linkage maps (Xu et al., 1995), comparative mapping (Chen et al., 1998), genetic diversity analysis (Xu et al., 1994), phylogenetic analysis (Xu and Sleper, 1994), analyses of Festuca × Lolium hybrids (Chen et al., 1995), cultivar identification, and differentiation of monosomic lines (Eizenga et al., 1998).
The polymerase chain reaction (PCR) amplifies discrete fragments of DNA and can be used to detect polymorphisms quickly. Randomly amplified polymorphic DNA was the first marker of this kind (Williams et al., 1990). However, imperfect reproducibility of RAPD markers was a major problem. Phylogenetic analysis of the Festuca-Lolium complex (Charmet et al., 1997) and detection of Festuca genome introgression into a Lolium background (Wang et al., 2003) have been examined using RAPD markers.
Amplified fragment length polymorphism markers provide an efficient system for molecular mapping because a large number of markers can be generated within a short time (Vos et al., 1995). However, developing AFLP markers is costly and requires skilled personnel. In addition, an AFLP marker linked to a specific trait in one population will rarely be useful for marking the same trait in a different population. As a result, AFLPs are not used commonly in framework mapping (Jones et al., 2002). Hundreds of tall fescue AFLP markers were developed and used for the construction of a genetic linkage map (Saha et al., 2005) and genetic diversity analysis of forage grass species (Mian et al., 2002, 2005).
Microsatellites, also known as simple sequence repeats (SSRs), have become highly useful molecular markers for plant improvement. Microsatellites have the potential advantages of reliability, reproducibility, discrimination, standardization, and cost effectiveness over RFLPs. In addition, SSR markers can be used across a wider range of populations and species than more restrictive markers such as AFLPs. High-throughput detection systems make SSR genotyping much faster, easier, and cost efficient than many other types of markers. Development of SSR markers is costly but, once developed, they can be used for many different purposes.
Microsatellites are composed of short stretches of DNA with a repetitive sequence, for example, CTGCTGCTGCTGCTGCTGCTG. Primers are short stretches of DNA that are designed from the start (forward primer) and end (reverse primer) of sequences that flank such repeat regions. Using PCR, primers can amplify fragments of DNA that vary in length of the repetitive sequences and/or adjacent sequences, which in turn can serve as molecular markers for tall fescue and other grasses (Mian et al., 2005). Thus, sequence information is essential to generate SSR markers. Microsatellites can be identified from genomic DNA, which refers to segments of DNA derived from either expressed or nonexpressed regions. Expressed sequence tags also are considered to be important resources for the development of SSR markers.
Microsatellite markers have been developed from tall fescue sequences (Saha et al., 2004, 2006) and used for the construction of genetic linkage maps of tall fescue (Saha et al., 2005), annual ryegrass (L. multiflorum Lam.) × perennial ryegrass (L. perenne L.) (Warnke et al., 2004), and bentgrass (Agrostis stolonifera L.) (Zhao et al., 2006) and to conduct genetic diversity and phylogenetic analysis of grass species (Saha et al., 2004; Zwonitzer et al., 2004; Hopkins and Saha, 2005; Mian et al., 2005). Cross-species applicability of these microsatellite markers was also evaluated (Saha et al., 2004; Mian et al., 2005). However, the development of molecular markers for these complex polyploid species lags far behind those for major cereal crops [e.g., rice (Oryza sativa L.), maize (Zea mays L.), and wheat (Triticum aestivum L.)].
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