Microsatellites or Single Sequence Repeats (SSRs) are extensively employed in plant genetics studies, using both low and high throughput genotyping approaches. Motivated by the importance of these sequences over the last decades this review aims to address some theoretical aspects of SSRs, including definition, characterization and biological function. The methodologies for the development of SSR loci, genotyping and their applications as molecular markers are also reviewed. Finally, two data surveys are presented. The first was conducted using the main database of Web of Science, prospecting for articles published over the period from 2010 to 2015, resulting in approximately 930 records. The second survey was focused on papers that aimed at SSR marker development, published in the American Journal of Botany's Primer Notes and Protocols in Plant Sciences (over 2013 up to 2015), resulting in a total of 87 publications. This scenario confirms the current relevance of SSRs and indicates their continuous utilization in plant science.

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Microsatellite markers: what they mean and why they are so useful.

Assessments of population genetic structure have become an increasing focus as they can provide valuable insight into patterns of migration and gene flow structure, the most highly cited of several clustering-based methods, was developed to provide robust estimates without the need for populations to be determined a priori structure introduces the problem of selecting the optimal number of clusters, and as a result, the ΔK method was proposed to assist in the identification of the "true" number of clusters In our review of 1,264 studies using structure to explore population subdivision, studies that used ΔK were more likely to identify K = 2 (54%, 443/822) than studies that did not use ΔK (21%, 82/386) A troubling finding was that very few studies performed the hierarchical analysis recommended by the authors of both ΔK and structure to fully explore population subdivision Furthermore, extensions of earlier simulations indicate that, with a representative number of markers, ΔK frequently identifies K = 2 as the top level of hierarchical structure, even when more subpopulations are present This review suggests that many studies may have been over- or underestimating population genetic structure; both scenarios have serious consequences, particularly with respect to conservation and management We recommend publication standards for population structure results so that readers can assess the implications of the results given their own understanding of the species biology

https://onlinelibrary.wiley.com/doi/pdf/10.1111/mec.14187

The K = 2 conundrum.

Summary 1. Using scripting languages such as R to perform population genetic analyses can improve the reproducibility of research, but using R can be challenging for many researchers due to its steep learning curve. 2. POPGENREPORT is a new R package that simplifies performing population genetics analyses in R, through the use of a new report-generating function. The function POPGENREPORT allows users to perform up to 13 pre-defined and 1 user-defined analyses through the use of a single command line. Each analysis generates figures and tables that are incorporated into a PDF report and are also made available as individual files (figures are provided in multiple formats, table contents are provided as CSV files). 3. The package includes new R functions that simplify the importation of data from a spreadsheet file, examine allele distributions across populations and loci and identify private alleles, determine pairwise individual genetic distances using the methods of Smouse and Peakall (1999) and Kosman and Leonard (2005), respectively, detect the presence of null alleles, calculate allelic richness, and test for spatial autocorrelation in genotypes using the methods of Smouse and Peakall (1999). 4. The package has a modular structure that makes the process of adding new functionality straightforward. To facilitate the addition of user-designed functions, the package includes a fully customizable module that can be automatically included in the PDF report. 5. To support users not experienced in R, the website (www.popgenreport.org) has a tutorial for the package and a downloadable, portable version of the package with LaTeX pre-configured for the Windows operating system.

https://besjournals.onlinelibrary.wiley.com/doi/pdf/10.1111/2041-210X.12158

PopGenReport: simplifying basic population genetic analyses in R

Directly labelling locus-specific primers for microsatellite analysis is expensive and a common limitation to small-budget molecular ecology projects. More cost-effective end-labelling of PCR products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus-specific primers with 5' universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co-amplifying large numbers of size overlapping loci and without requiring locus-specific PCR conditions to be modified. In this study, we report a suite of four high-performance universal primers that can be employed in a three primer PCR approach for efficient and cost-effective fluorescent end-labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co-amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.

Universal primers for fluorescent labelling of PCR fragments--an efficient and cost-effective approach to genotyping by fluorescence.

Despite the importance of polyploidy and the increasing availability of new genomic data, there remain important gaps in our knowledge of polyploid population genetics. These gaps arise from the complex nature of polyploid data (e.g. multiple alleles and loci, mixed inheritance patterns, association between ploidy and mating system variation). Furthermore, many of the standard tools for population genetics that have been developed for diploids are often not feasible for polyploids. This review aims to provide an overview of the state-of-the-art in polyploid population genetics and to identify the main areas where further development of molecular techniques and statistical theory is required. We review commonly used molecular tools (amplified fragment length polymorphism, microsatellites, Sanger sequencing, next-generation sequencing and derived technologies) and their challenges associated with their use in polyploid populations: that is, allele dosage determination, null alleles, difficulty of distinguishing orthologues from paralogues and copy number variation. In addition, we review the approaches that have been used for population genetic analysis in polyploids and their specific problems. These problems are in most cases directly associated with dosage uncertainty and the problem of inferring allele frequencies and assumptions regarding inheritance. This leads us to conclude that for advancing the field of polyploid population genetics, most priority should be given to development of new molecular approaches that allow efficient dosage determination, and to further development of analytical approaches to circumvent dosage uncertainty and to accommodate ‘flexible’ modes of inheritance. In addition, there is a need for more simulation-based studies that test what kinds of biases could result from both existing and novel approaches.

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Recent progress and challenges in population genetics of polyploid organisms: an overview of current state-of-the-art molecular and statistical tools.

Microsatellites have been popular molecular markers ever since their advent in the late eighties. Despite growing competition from new genotyping and sequencing techniques, the use of these versatile and cost-effective markers continues to increase, boosted by successive technical advances. First, methods for multiplexing PCR have considerably improved over the last years, thereby decreasing genotyping costs and increasing throughput. Second, next-generation sequencing technologies allow the identification of large numbers of microsatellite loci at reduced cost in non-model species. As a consequence, more stringent selection of loci is possible, thereby further enhancing multiplex quality and efficiency. However, current practices are lagging behind. By surveying recently published population genetic studies relying on simple sequence repeats, we show that more than half of the studies lack appropriate quality controls and do not make use of multiplex PCR. To make the most of the latest technical developments, we outline the need for a well-established strategy including standardized high-throughput bench protocols and specific bioinformatic tools, from primer design to allele calling.

/pdf/current-trends-in-microsatellite-genotyping-wmvq1xapkj.pdf

Current trends in microsatellite genotyping.

Angelica heterocarpa (Apiaceae) is a wild endemic French species with special conservation interest in the European Union. It belongs to Angelica complex genus which is widespread throughout the north temperate zone, and is sympatric with other congeneric species. The objective of this work is to develop and characterize microsatellite markers as a new tool for understanding the ecology and evolution of Angelica species complex.We identified simple sequence repeat (SSR) regions in a microsatellite-enriched library from A. heterocarpa and A. sylvestris. All 16 selected SSR regions were found to amplify in these species and were highly polymorphic. Marker transferability was validated in A. razulii and A. archangelica. These markers will help us to better understand the evolutionary dynamic between rare endemics and widespread sister species, and be useful for conservation of the endemic species. Moreover, they can provide new tools for studying the numerous traditional medicinal herbs of the Angelica genus. 

/pdf/development-and-characterization-of-microsatellite-markers-n4c4do5q.pdf

Development and characterization of microsatellite markers for the French endemic Angelica heterocarpa (Apiaceae) and congeneric sympatric species

/pdf/development-and-characterization-of-microsatellite-markers-2svgndib.pdf

E. Revardel

Papers

Current trends in microsatellite genotyping.

Development and characterization of microsatellite markers for the French endemic Angelica heterocarpa (Apiaceae) and congeneric sympatric species

Development and characterization of microsatellite markers for the French endemic Angelica heterocarpa (Apiaceae) and congeneric sympatric species