Author: Smita N. Shingane
Microsatellites, or simple sequence repeats (SSRs), are molecular markers widely used for DNA fingerprinting, linkage map construction and population genetic studies. Based on tandem repeats of short (1â€"6 bp) DNA sequences, these markers are highly polymorphic due to variation in the number of repeat units. The repeat length at specific SSR loci is easily analysed by polymerase chain reaction using primers specific to conserved regions flanking the repeat array. The hyper variability and co-dominance of SSRs, their dispersion throughout genomes and suitability for automation are the principle reasons for their wide utility SSR markers are the most widely used markers for genetic map development and also have provided plant breeders with an efficient tool to correlate phenotypic and genotypic variation for its use in marker assisted selection.
Recently, with the establishment of large scale sequencing of expressed sequence tag (EST) many projects for gene discovery programs in crop species, wealth of DNA sequence information has been generated and deposited in online databases. In addition, sequence data from many fully characterized genes and cDNA clones have been generated in many model species such as rice. For quick generation of the EST based makers, using suitable computer programs, the sequence data for ESTs, genes and cDNA clones can be downloaded from public sequence databases and scanned for identification of SSRs, and they are generally referred to as EST-SSRs or genic microsatellites Locus-specific primers flanking EST- or genic SSRs are designed to amplify the microsatellite loci present in the genes. Thus, from publicly available database, generation of (genic) SSR markers is relatively easy and inexpensive.
Generation of genic SSR markers is largely limited to species or close relatives for which there is a sufficiently large number of ESTs available. Genic SSRs have some intrinsic advantages over genomic SSRs because they are quickly obtained by electronic sorting, and are present in expressed regions of the genome. The usefulness of these genic SSRs also lies in their expected transferability because the primers are designed from the more conserved coding regions of the genome. Because of the advantages of genic SSR markers over genomic SSR markers and the public availability of large quantities of sequence data, genic SSRs have been identified, developed and used in a variety of studies, for several plant species for its genetic improvement.
Comparative advantages of genic SSRs over genomic SSRs
| Limitations (Genomic SSRs) | Advantages ( genic SSRs) | |
| High Time and cost | Quickly obtained by electronic sorting â€" public databases | |
| Transcribed or non transcribed region of genome | Transcribed region of genome | |
| Function not known | Putative function known | |
| Clustered near centromeres | Mostly concentrated in gene rich regions | |
| Less transferable | More transferable | |
Applications of microsatellite markers
Diversity analysis
EST SSRs can be used for studying the variation in transcribed and known functional genes and thereby for assaying the functional diversity among the genes. Presence of SSR in the transcripts of genes might have a role in gene expression or function.
Linkage disequilibrium mapping
EST SSRs can be used for non-random association between marker, genes or QTLs in a population. Association mapping or (LD) mapping is a population based survey to identity marker trait relationship based on linkage disequilibrium
Functional genomics
SSR markers can provide opportunity for gene discovery and enhance the role of marker for analysis variation in transcribed and known function genes. These functional genomics in true- gene can be linked or associated with plant breeding for crop improvement
Genome mapping
EST SSR - have been integrated in several plant species. EST SSRs have been mapped as a part of transcript of Barley and in many crop species.
Transferability and comparative mapping
Significant genomic co-linearity has been reported in many cereal species. Comparative genomics using EST SSRs and bioinformatics approaches might provide an efficient transfer of information from model species to minor crops. Genomic information from model cereal species such as Rice, maize and wheat can be potentially exploited in improvement of orphan or minor crops like pearl millet and small millets.
Marker assisted selection
EST-SSR markers are used for indirect selection of difficult traits at an early stage of crop growth which is devoid of environmental stress. This speeds up process of conventional plant breeding.
LIMITATIONS OF EST- SSRs
1. Standard cDNA library represent mRNA present within specific tissue under exact conditions at the time of sampling. Poorly expressed genes will be poorly represented in libraries.
3. A major limitation of SSRs is the time and cost required to isolate and characterize each locus when pre-existing DNA sequence is not available. Typically, this process requires the construction and screening of a genomic library of size-selected DNA fragments with SSR-specific probes, followed by DNA sequencing of isolated positive clones, PCR primer synthesis and testing.
REFERENCES:
• Varshney, R. K., Graner, A. and Sorrells, M. E., 2005, Genomics-assisted breeding for crop improvement. Trends Plant sci.,10: 621-630
• Varshney, R. K., Graner, A., Sorrells, M. E., 2006, Genic microsatellite markers in plants: features and applications. Trends in biotechnol. 23: 48 â€" 55.
• Varshney, R. K., Grosse, I., Hahnel, U., Siefken , R., Prasad, M., Stein, N., Langridge, P., Altschmied, L. and Graner, A. 2006, Genetic mapping and BAC assignment of EST-derived SSR markers shows non-uniform distribution of genes in the barley genome. Theor. Appl. Genet.,113 (2):239 â€" 250.
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