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      SSR‐seq: Genotyping of microsatellites using next‐generation sequencing reveals higher level of polymorphism as compared to traditional fragment size scoring

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          Abstract

          Microsatellites (or simple sequence repeats, SSR) are widely used markers in population genetics. Traditionally, genotyping was and still is carried out through recording fragment length. Now, next‐generation sequencing (NGS) makes it easy to obtain also sequence information for the loci of interest. This avoids misinterpretations that otherwise could arise due to size homoplasy. Here, an NGS strategy is described that allows to genotype hundreds of individuals at many custom‐designed SSR loci simultaneously, combining multiplex PCR, barcoding, and Illumina sequencing. We created three different datasets for which alleles were coded according to (a) length of the repetitive region, (b) total fragment length, and (c) sequence identity, in order to evaluate the eventual benefits from having sequence data at hand, not only fragment length data. For each dataset, genetic diversity statistics, as well as F ST and R ST values, were calculated. The number of alleles per locus, as well as observed and expected heterozygosity, was highest in the sequence identity dataset, because of single‐nucleotide polymorphisms and insertions/deletions in the flanking regions of the SSR motif. Size homoplasy was found to be very common, amounting to 44.7%–63.5% (mean over all loci) in the three study species. Thus, the information obtained by next‐generation sequencing offers a better resolution than the traditional way of SSR genotyping and allows for more accurate evolutionary interpretations.

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          Most cited references 32

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          Estimation of average heterozygosity and genetic distance from a small number of individuals.

           M Nei (1978)
          The magnitudes of the systematic biases involved in sample heterozygosity and sample genetic distances are evaluated, and formulae for obtaining unbiased estimates of average heterozygosity and genetic distance are developed. It is also shown that the number of individuals to be used for estimating average heterozygosity can be very small if a large number of loci are studied and the average heterozygosity is low. The number of individuals to be used for estimating genetic distance can also be very small if the genetic distance is large and the average heterozygosity of the two species compared is low.
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            The rate and molecular spectrum of spontaneous mutations in Arabidopsis thaliana.

            To take complete advantage of information on within-species polymorphism and divergence from close relatives, one needs to know the rate and the molecular spectrum of spontaneous mutations. To this end, we have searched for de novo spontaneous mutations in the complete nuclear genomes of five Arabidopsis thaliana mutation accumulation lines that had been maintained by single-seed descent for 30 generations. We identified and validated 99 base substitutions and 17 small and large insertions and deletions. Our results imply a spontaneous mutation rate of 7 x 10(-9) base substitutions per site per generation, the majority of which are G:C-->A:T transitions. We explain this very biased spectrum of base substitution mutations as a result of two main processes: deamination of methylated cytosines and ultraviolet light-induced mutagenesis.
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              THE NUMBER OF ALLELES THAT CAN BE MAINTAINED IN A FINITE POPULATION.

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                Author and article information

                Contributors
                sarhanova@gmail.com
                Journal
                Ecol Evol
                Ecol Evol
                10.1002/(ISSN)2045-7758
                ECE3
                Ecology and Evolution
                John Wiley and Sons Inc. (Hoboken )
                2045-7758
                25 October 2018
                November 2018
                : 8
                : 22 ( doiID: 10.1002/ece3.2018.8.issue-22 )
                : 10817-10833
                Affiliations
                [ 1 ] Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben Germany
                [ 2 ] Institute of Biology and Environmental Sciences Carl von Ossietzky University Oldenburg Oldenburg Germany
                [ 3 ]Present address: Department of Botany and Biodiversity Research University of Vienna Vienna Austria
                [ 4 ]Present address: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben Germany
                [ 5 ]Present address: Max Planck Genome Centre Cologne Cologne Germany
                Author notes
                [* ] Correspondence

                Petra Šarhanová, Department of Botany and Biodiversity Research, University of Vienna, Vienna, Austria.

                Email: sarhanova@ 123456gmail.com

                [†]

                These authors contributed equally to this work.

                Article
                ECE34533
                10.1002/ece3.4533
                6262739
                © 2018 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 3, Tables: 5, Pages: 17, Words: 13292
                Product
                Funding
                Funded by: Deutsche Forschungsgemeinschaft
                Award ID: AL632/7‐1
                Award ID: BL462/11
                Funded by: Dr. Karl Wamsler Stiftung
                Funded by: Deutscher Akademischer Austauschdienst
                Award ID: D/10/49464
                Categories
                Original Research
                Original Research
                Custom metadata
                2.0
                ece34533
                November 2018
                Converter:WILEY_ML3GV2_TO_NLMPMC version:version=5.5.3 mode:remove_FC converted:29.11.2018

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