Chapter 1

Microsatellites: Evolution and Contribution

Panagiotis Madesis, Ioannis Ganopoulos, and Athanasios Tsaftaris
Abstract
Microsatellites are codominant molecular genetic markers, which are universally dispersed within genomes. These markers are highly popular because of their high level of polymorphism, relatively small size, and rapid detection protocols. They are widely used in a variety of fundamental and applied elds of biological sciences for plants and animal studies. Microsatellites are also extensively used in the eld of agriculture, where they are used in characterizing genetic materials, plant selection, constructing dense linkage maps, mapping economically important quantitative traits, identifying genes responsible for these traits. In addition microsatellites are used for marker-assisted selection in breeding programs, thus speeding up the process. In this chapter, genomic distribution, evolution, and practical applications of microsatellites are considered, with special emphasis on plant breeding and agriculture. Moreover, novel advances in microsatellite technologies are also discussed. Key words Microsatellites, Inter simple sequence repeats, Simple sequence repeats, High-resolution melting analysis

Introduction
Assessing genetic variation is an important parameter in genetic studies, in studying biodiversity, germplasm characterization and generation of genetic variability in plant breeding. Moreover, estimation of genetic variation is important for the selection of desirable genotypes. The understanding that high amounts of genetic variation is not expressed on the phenotype rendered obvious that new methods should be developed to estimate and use this variation in favor of a breeding program. Recently, due to several technical advances made in molecular genetics, genetic variation could be measured at the DNA level by developing different molecular markers. Microsatellites which are the subject of this chapter are based on different repetitive sequences present in the genome. Marker assisted selection could help breeders avoid the traditional phenotype based selections in the eld, thus speeding up the breeding programs and maximizing its progress. By denition, molecular marker is any site (locus) in the genome of an organism where the

Stella K. Kantartzi (ed.), Microsatellites: Methods and Protocols, Methods in Molecular Biology, vol. 1006, DOI 10.1007/978-1-62703-389-3_1, Springer Science+Business Media, LLC 2013

Panagiotis Madesis et al.

DNA base sequence differs among the individuals of a population. The arrival of genetic tools like restriction enzymes and the polymerase chain reaction plus the growing abundance of DNA sequence data, coupled with automated high-throughput assays, have revealed several classes of molecular markers, including restriction fragment length polymorphisms (RFLPs), variable number tandem repeats (VNTRs), microsatellite DNA, and single nucleotide polymorphisms (SNPs) (molecular marker, http://www. encyclopedia.com/doc/1O6-molecularmarker.html). The genome of higher organisms contains three types of simple repetitive DNA sequences (satellite DNAs, minisatellites, and microsatellites), organized in clusters of differing sizes (1, 2). Microsatellites rst described by Litt and Luty (3) can be found under the term simple sequence repeats (SSRs), simple repetitive sequences (SRS), or simple tandem repeats (STRs) (4) (SSLPs) (5).

Microsatellites
Microsatellites are tandem repeats of very short 16 bp patterns which are not repeated many times at a particular locus but are distributed relatively evenly at many different genomic loci (6). Other scientists dene microsatellites as 28 or even 15 bp repeats (7, 8). The most abundant patterns found in the plant genome are (AT)n, (GA)n, and (GAA)n where n refers to the total number of repeats, usually ranging from 10 to 100. In addition, mononucleotide repeats consisting of A/T repeats are also present in the chloroplast genome. Increased intra- and inter-genetic variation is observed when the number of repeats is increasing (9). Between the two types of mononucleotide repeats, A/T was the most abundant in all plant species, while G/C was comparatively limited. In the mononucleotide repeats category, the maximum (99 %) A/T repeats have been found in the Arabidopsis genome and the minimum (78 %) in the Brachypodium genome. In the dinucleotide repeat category, the distribution of SSRs in different motif types was not uniform and the most frequent motif type was different for each plant species. For example, AG/CT repeats were more frequent in Brachypodium and rice, with 50.7 % and 41.9 % frequency respectively, whereas AT/AT repeats were more frequent in Populus (60.5 %) and Medicago (59.9 %). In rice, both AG/CT and AT/ AT repeats were the most abundant among the other dinucleotide repeats. Interestingly, the CG/CG motif contributed less than 0.5 % in dicots, whereas it was 3.17.0 % in all dinucleotide repeats identied in the monocots. The analysis of mononucleotide and dinucleotide repeats concluded that CG-rich motifs were least preferred in both monocot and dicot genomes. However, for trinucleotide repeats the AGC/CGT, AGG/CCT, and CCG/CGG were observed more frequently in all monocot species, whereas A/T-rich

Microsatellites: Evolution and Contribution

repeats, such as AAC/GTT, AAG/CTT, and AAT/ATT, were more frequent in dicots. The frequency of tetranucleotide, pentanucleotide, and hexanucleotide repeats was very low in all the plant genomes (10). Overall, there are 501 possibilities of nonredundant monomeric to hexameric repeats. In plants, the frequency and number of microsatellites have been estimated for a number of species and results indicate that the most frequent microsatellite is (GT)n, while in mammals is (AT)n (10). Microsatellites are sometimes associated with other genomic repeats, especially transposable elements. In humans, microsatellites are associated with repetitive DNA, especially non-LTR retrotransposons (11, 12). In plants, however, including Arabidopsis, rice, soybean, maize, and wheat, microsatellites are preferentially located in nonrepetitive DNA regions, which indicates that they reside in regions predating genome expansion (13, 14). Nevertheless, microsatellites are evenly spaced in the genome, although they are highly variable in number of repeat units among individuals (7). Because unique DNA sequences ank individual microsatellites they could be genotyped via STS (Sequence Tagged Site) PCR (15). In species with low levels of genetic diversity identication of a fast mutating locus would be the optimal resource for the development of markers, markers which would thus be ideal for breeding programs. At the moment there are a vast number of SSR markers publicly available for research concerning the most important agricultural crops (1619).

Generation of Microsatellite Diversity

The mutational rate for unique eukaryotic sequences is of approximately 109 per nucleotide per generation (20). Moreover, the mutation rate differs between species but also differs to a great extent within species (between loci) with long loci mutating more (21). The rate at which its SSR loci mutate varies and depends on repeated motif, GC content in anking DNA, allele size, chromosome position, cell division (mitotic vs. meiotic), sex, age, repeat type, and genotype (2, 8, 22). The differences are mainly observed as changes in the number of SSR repeats. These observations have signicant implications for the development of molecular markers, as these differences can be visualized and can facilitate plant breeding. The main mutation event is gain and loss of entire repeat units, which suggest a specic mutational mechanism called replication slippage. As microsatellites mutate at such a high rate one would expect the microsatellite size to increase over time, yet this does not happen, probably because a point mutation breaks the perfect repeats of a microsatellite and, as has been shown, imperfect repeats have a reduced slippage mutation rate (23). Two mutational mechanisms can be used to explain such high rates of mutation. The rst involves

Panagiotis Madesis et al.

Fig. 1 Replication leads to new alleles with less (deletion) or more (expansion) repeats depending on the strand containing the error

DNA slippage during DNA replication (24), caused by mismatches between DNA strands when they are being replicated during meiosis (25). It has been estimated that replication slippage at each microsatellite occurs about once per 1,000 generations (26). The second involves recombination between DNA strands (27).

Replication Slippage
Replication slippage accounts for many mutations at SSR loci (28). This type of mutation occurs when one DNA strand is mispairing (slip strand) during DNA replication. The mispairing refers to a repeat unit hybridizing to a repeat in such a way where a loop is formed in the nascent strand resulting in the addition of a repeat (22). If the loop occurs in the template strand, then there will be a decrease in the number of units (29). These can lead to gain or loss of certain repeats. The mismatch repair mechanism and exonuclease activity of polymerase corrects a number of errors but many escape and become mutations (Fig. 1).

Recombination
Another mechanism of mutation is the recombination process which could change the SSR length by asymmetrical crossing over or by gene conversion (2, 3033). Asymmetric exchanges, random

Microsatellites: Evolution and Contribution

Fig. 2 (a) Amplication of microsatellites using a pair of SSR markers. PCR products are analyzed on polyacrylamide gels. (b) Amplication of microsatellites using one ISSR markers. PCR products are analyzed on polyacrylamide gels or simple 1.5 % agarose gels

genetic drift and selection can have a signicant effect on the accumulation of tandem-repetitive sequences in the genome (34). Non reciprocal recombination also mutates tandem repeat number (for both microsatellites and minisatellites) (32, 33).

Infrastructure and Methods for the Study of Microsatellites

Molecular markers using microsatellites as targeting sequence polymorphisms can either multiply a DNA region containing the microsatellites, as in the case of SSR markers. This type of markers recognizes the microsatellite anking sequences (using one pair of specic primers) (Fig. 2a). Another type of microsatellite markers (ISSR) bind on the microsatellite using only one primer and multiply the region between two microsatellites (Fig. 2b). PCR fragments are usually separated on polyacrylamide gels in combination with AgNO3 staining (SSR primers) or on simple agarose gels (1.5 %), ISSR respectively. However, the development of microsatellite SSR primers for a new species is difcult, laborious, and expensive, although the genomic era could facilitates this process. Several protocols have been developed so far for the development of SSR markers (5, 35).

Technical Problems and Difculties in Studying Microsatellites

Although microsatellites are extremely useful for genetic analysis, mapping, etc., there are certain difculties concerning their use. They are expensive to develop, as a large number of sequences

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must be cloned and only a small number of these will be useful for the development of the SSR markers. Moreover, only a number of these markers will give informative results, especially for species with large genomes (3638). In addition, problems that might occur are, for instance, as follows: (a) the primer may not amplify any PCR product; (b) the primer may produce very complex, weak, or nonspecic amplication patterns; (c) the amplication product may not be polymorphic. Other possible problems using SSR markers are as follows: the difculty to resolve bands differing only in one or two base pairs, the cost of polyacrylamide gels and labeled primers, and the differences in identifying band size and their calling between laboratories, making comparisons between results very hard. Yet, despite any problems, SSRs are now the marker of choice in many areas of molecular genetics due to their codominant and polymorphic nature, even between closely related lines, their requirement for low amounts of DNA, and the possibility of being automated for high-throughput screening make them attractive. In addition they can be easily exchanged between laboratories, and are highly transferable between populations (39). For example, a total of 18,828 SSR sequences have been detected in the rice genome (40), of which only 1015 % have yet been used, suggesting the high potential available for such marker systems. SSRs are mostly codominant markers and are indeed excellent for studies of population genetics and mapping (31, 41). Another technical development like the use of uorescent primers in combination with automatic capillary or gel-based DNA sequencers has facilitated the detection of bands and their analysis.

Advances in Microsatellites
Although microsatellites mainly occur in noncoding sequences, the development of EST databases revealed that microsatellite repetitive sequences also occur inside coding sequences (4244). The information obtained by EST libraries has been recently used for the development of SSR markers (4549). Microsatellites designed EST are expected to be slightly less polymorphic than genomic library derived SSRs, as there is selection pressure for sequence conservation in coding regions (5053). This also explains why the most abundant microsatellites in genes are trinucleotides and hexanucleotides and the less frequent are mononucleotides and dinucleotides, as these types cause frame shift and most probably premature stop codons. While technology progresses and new genomes and EST libraries become available with the help of bioinformatics approaches, the development of SSR markers based on ESTs through data mining has become a fast, efcient and relatively inexpensive, compared to development of

Microsatellites: Evolution and Contribution

genomic SSRs (54). However, these approaches require the existence of sequence information.

The Advances of High-Resolution Melting Analysis in Microsatellite Studies

Generally, laborious polyacrylamide gels followed by silver staining or, for better resolution, uorescently labeled PCR products and automated sequencers are needed for microsatellite analysis. Moreover, post-PCR handling and dilution steps as well as uorescently labeled primers for each microsatellite, are required by this method, resulting in increases in time and cost of the analysis. Over the last few years, Real-time PCR is often used to analyze amplied DNA and identify viruses and pathogens. In addition, it can also be used as an extremely quick analysis for reactions that do not require subsequent use of the amplied DNA. Lately, high-resolution melting (HRM), a sensitive mutation detecting method has been introduced, extending the possibilities of analyzing the DNA melting curves which was a standard diagnostic feature in qPCR (55). HRM analysis is rapidly gaining in popularity as a cost-effective and faster alternative to traditional post-PCR genotyping methods such as single-stranded conformation polymorphism, denaturing high-performance liquid chromatography, and restriction fragment length polymorphism. The determination of the Tm values distance can be used for identifying the targeted amplicon among the nonspecic products (55) (Fig. 3a). The HRM curves obtained are highly specic for each amplicon and depend on the GC content, amplicon length and sequence (56).

Fig. 3 (a) A double stranded DNA fragment melts at a specic temperature (Tm) which is specic for each DNA fragment. The highest rate of uorescence decrease is generally at the melting temperature of the DNA sample (Tm). The Tm is dened as the temperature at which 50 % of the DNA sample is double stranded and 50 % is single stranded. (b) Different PCR products in size have different melting curves and can be distinguished having even one single point mutation

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HRM analysis is based on the fact that although DNA melting curves are used primarily for the determination of the melting temperature (Tm) of amplied double-stranded DNA, the precise shape of a melting curve is typical of each DNA sequence (57). By making very small temperature size steps, accurate melting curves can be produced, while normalization and comparison of the melting curves can clarify whether different amplicons have the same or different sequences (58). Different amplicons can have the same Tm values but the advantage of the HRM method is that these different amplicons can be distinguished by the shape of HRM melting curves (59) (Fig. 3b). Even not being strictly a banding pattern-based method, HRM analysis is categorized as such because it relies on PCR amplication and detection of sequence variants without sequencing or hybridization procedures (60). HRM could be used as an alternative method to detect microsatellites, especially for those laboratories that do not have immediate access to capillary sequencers (61). Its main advantage is the fast, accurate, and closed-tube determination of SNPs and sequence variations (62). The sensitivity of HRM analysis has already been broadly veried (63). Besides numerous applications in clinical mutation screening, HRM was suggested as another population genetics genotyping system (64) and has been used to discriminate closely related plant cultivars (61, 6569). Mader et al. (69) proved the ability of HRM application to SSR analysis in principle, but also its limitations in comparison to CE (Capillary Electrophoresis). Specically, only low-complexity SSRs with a few alleles in a population can be fully detected with HRM. The need for production of unknown PCR products articial mixtures with already genotyped standards makes the procedure more complex and labor intensive. It may therefore be unlikely that HRM will replace CE for genotyping the highly complex SSRs typically used in population genetics. Ganopoulos et al. (67) suggested that HRM is able to detect and screen single locus markers without the need of labeled primers, product fractionation, DNA restriction or individual sequence analyses. This makes the technique ideal for cultivar identication studies where large populations are to be scored with numerous SSR loci. There are numerous advantages of the HRM method of scoring SNPs/microsatellites comparing to existing systems that are based on high-resolution gel or CE (70). First, there is no a priori requirement to identify the position or identity of the SNP/microsatellite; any SNP or length polymorphism giving rise to a melt polymorphism can be scored without characterization. Second, there are no additional reagent costs for labeled primers. Third, the capacity to perform HRM directly after PCR makes the need for further handling of samples unnecessary. The capacity of HRM analysis instruments to perform more assays in the same

Microsatellites: Evolution and Contribution

time means that more data points can be generated within the 1520 min required to perform an HRM following the end of PCR, thus increasing the overall throughput. Finally, the fact that melting curves shapes depends not only to amplied size fragments but also to base composition and SNP position, is more sensitive to distinguish closely related genotypes such as cultivars of the same species.

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Applications of Microsatellites
Microsatellites have become a marker of choice for a huge range of applications in plants with a vast literature; refer all this literature is beyond the scope of this article. SSR markers are useful for a variety of applications in plant genetics and breeding because of their reproducibility, multiallelic nature, codominant inheritance, relative abundance, and good genome coverage (71). Furthermore, SSR markers have been useful for integrating the genetic physical and sequenced based physical maps in plant species and at the same time they have provided breeders and geneticists with an efcient tool to link phenotypic and genotypic variation (72). Microsatellites are also used in order to estimate genetic variation at molecular level in a germplasm collection which will help towards the correct choice of parents for crosses in a breeding program (i.e., hybrid breeding), mapping and tagging of genes or QTLs (quantitative trait loci) for agronomic and disease resistance traits, genome mapping, MAS of promising lines and Marker Assisted Backcrossing (MAB) during breeding programs, gender identication, studying the population structure and taxonomic and phylogenetic relationships. In addition, the knowledge of genetic variation is mostly useful for characterization of accessions in plant germplasm collections and taxonomic studies and phylogenetic studies (73). In phylogenetic studies organelle specic markers (i.e., cpSSR and mtSSR) have also been used making great impact on the determination of structure and variation within a natural population too. Organelle microsatellites are attractive targets for phylogenetic studies or evolution studies and even migration histories due to uniparental mode of inheritance, conserved gene order and lack of heteroplasmy and recombination of organelle (74). Microsatellites have also been used for hybrid determination and characterization of allelic contribution of each parent (71). Moreover, microsatellites have been used for mapping of specic genomic regions responsible for agronomic traits or mapping of specic genes (75).

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Conclusions
Ever since their development, microsatellite markers are constantly being isolated and characterized in a wide range of plants including cereals, legumes, vegetables, forest trees, fruit plants, conifers, and other economically important plant species. Arrival of new technologies did not eliminate the use of microsatellites instead they have rendered microsatellites a useful multi-tool in plant breeding. Microsatellites are still the method of choice for marker assisted selection, population genetics, estimation of genetic diversity, ngerprinting, mapping, and gene association studies. SSR based association mapping holds a great promise for exploiting genetic diversity, characterizing accumulated phenotypic variation, and associating markers with traits in plant germplasm especially with the progress made in the genome programs. They owe their broad use to their cost-effectiveness easy to use and their excellent results. Microsatellite markers not only are involved in genetic diversity studies, and evolutionary studies, but are also being used in fundamental research like genome analysis, gene mapping, markerassisted selection, etc., yet there are several limitations limiting their use like the need to isolate them de novo although genome projects are expected to solve this problem, the presence of stutter bands, null alleles, and heterologous amplicons (76, 77). In conclusion, genomic progress and advancement in microsatellites markers will make their use even more attractive for molecular breeding and plant genetics and eventually they will have great contribution in major crop improvement.

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