Many of the applications noted above select microsatellites for their presumably neutral nature. Many microsatellite primers will work in species closely related to the one for which they were originally designed, allowing for multispecies studies. Because they are PCR-based markers, microsatellite loci can be successfully amplified from poor-quality or low quantities of DNA, making them useful markers for studies involving ancient DNA or museum specimens ( Wandeler et al., 2007). Additionally, unlike with NGS data, the relatively small number of loci used in an SSR study means that each locus can be manually genotyped, reducing errors. They are inexpensive when compared to the cost of using next-generation sequencing (NGS) techniques to generate sufficient data to differentiate among closely related individuals ( Davey et al., 2011). Their abundance in the genome, high levels of polymorphism, and cost effectiveness have contributed to the attractiveness of these markers. Microsatellites have been used for a wide variety of applications, including genome mapping, forensics, parentage analysis, conservation genetics, identification of the parentage of polyploids, phylogeography, and population genetics ( Ellegren, 2000 Esselink et al., 2004 Kalia et al., 2011). Since developing microsatellite loci (see Appendix 1 for a glossary of terms used in this paper) became cost-effective in the late 1990s, researchers have used them frequently in studies requiring high levels of polymorphism, generating approximately 225,000 published articles (search of Web of Science performed April 2016, term: microsatellite* OR “simple sequence repeat*”). The high level of polymorphism in microsatellites makes these markers powerful tools for assessing genetic similarity between individuals or closely related taxa ( Guichoux et al., 2011 Kalia et al., 2011). Microsatellites exhibit high levels of polymorphism and have a high mutation rate-between 10 −3 and 10 −4 per locus per generation, compared to approximately 10 −9 nucleotides per generation for nucleotide substitutions across the entire genome in eukaryotes ( Li et al., 2002). Replication slippage is generally considered the mechanism that creates variation in the number of repeats ( Ellegren, 2004). Within microsatellite regions, these motifs are repeated several to dozens of times, although the number of repeats is highly variable ( Selkoe and Toonen, 2006). Microsatellites, or simple sequence repeats (SSRs), are short repeated DNA motifs (typically one to six nucleotides) located throughout eukaryotic genomes ( Li et al., 2002 Zane et al., 2002). We argue that microsatellites still have an important place in the genomic age as they remain effective and cost-efficient markers. Finally, we discuss the future of microsatellites and make recommendations for researchers preparing to use microsatellites.
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Additionally, we review software for microsatellite development and report on an experiment to assess the utility of currently available software for SSR development. We also review the variety of techniques currently used for identifying microsatellite loci and developing primers, with a particular focus on those that make use of next-generation sequencing (NGS). We compare SSRs and newer methods, such as genotyping by sequencing (GBS) and restriction site associated DNA sequencing (RAD-Seq), and offer recommendations for researchers considering which genetic markers to use. They have diverse applications, including genome mapping, forensics, ascertaining parentage, population and conservation genetics, identification of the parentage of polyploids, and phylogeography. Microsatellites, or simple sequence repeats (SSRs), have long played a major role in genetic studies due to their typically high polymorphism.
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