In the last few years microsatellites have become one of the most popular molecular markers used with applications in many different fields. High polymorphism and the relative ease of scoring represent the two major features that make microsatellites of large interest for many genetic studies. The major drawback of microsatellites is that they need to be isolated de novo from species that are being examined for the first time. The aim of the present paper is to review the various methods of microsatellite isolation described in the literature with the purpose of providing useful guidelines in making appropriate choices among the large number of currently available options. In addition, we propose a fast and easy protocol which is a combination of different published methods.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.0962-1083.2001.01418.x

Strategies for microsatellite isolation: a review.

Genotyping errors occur when the genotype determined after molecular analysis does not correspond to the real genotype of the individual under consideration. Virtually every genetic data set includes some erroneous genotypes, but genotyping errors remain a taboo subject in population genetics, even though they might greatly bias the final conclusions, especially for studies based on individual identification. Here, we consider four case studies representing a large variety of population genetics investigations differing in their sampling strategies (noninvasive or traditional), in the type of organism studied (plant or animal) and the molecular markers used [microsatellites or amplified fragment length polymorphisms (AFLPs)]. In these data sets, the estimated genotyping error rate ranges from 0.8% for microsatellite loci from bear tissues to 2.6% for AFLP loci from dwarf birch leaves. Main sources of errors were allelic dropouts for microsatellites and differences in peak intensities for AFLPs, but in both cases human factors were non-negligible error generators. Therefore, tracking genotyping errors and identifying their causes are necessary to clean up the data sets and validate the final results according to the precision required. In addition, we propose the outline of a protocol designed to limit and quantify genotyping errors at each step of the genotyping process. In particular, we recommend (i) several efficient precautions to prevent contaminations and technical artefacts; (ii) systematic use of blind samples and automation; (iii) experience and rigor for laboratory work and scoring; and (iv) systematic reporting of the error rate in population genetics studies.

/pdf/how-to-track-and-assess-genotyping-errors-in-population-1ccjofq7qv.pdf

How to track and assess genotyping errors in population genetics studies.

Although genotyping errors affect most data and can markedly influence the biological conclusions of a study, they are too often neglected. Errors have various causes, but their occurrence and effect can be limited by considering these causes in the production and analysis of the data. Procedures that have been developed for dealing with errors in linkage studies, forensic analyses and non-invasive genotyping should be applied more broadly to any genetic study. We propose a protocol for estimating error rates and recommend that these measures be systemically reported to attest the reliability of published genotyping studies.

Genotyping errors: causes, consequences and solutions.

Amplified fragment length polymorphisms (AFLPs) are polymerase chain reaction (PCR)-based markers for the rapid screening of genetic diversity. AFLP methods rapidly generate hundreds of highly replicable markers from DNA of any organism; thus, they allow high-resolution genotyping of fingerprinting quality. The time and cost efficiency, replicability and resolution of AFLPs are superior or equal to those of other markers [allozymes, random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), microsatellites], except that AFLP methods primarily generate dominant rather than co-dominant markers. Because of their high replicability and ease of use, AFLP markers have emerged as a major new type of genetic marker with broad application in systematics, pathotyping, population genetics, DNA fingerprinting and quantitative trait loci (QTL) mapping.

/pdf/aflp-genotyping-and-fingerprinting-4sw6vfeake.pdf

AFLP genotyping and fingerprinting

In recent years, a variety of molecular markers, based on microsatellites or simple sequence repeats (SSRs) have become the markers of choice, thus necessitating their development and use in a variety of plant systems. In this review, the basic principles underlying different hybridization-based (oligonucleotide fingerprinting) and PCR based approaches (STMS, MP-PCR, AMP-PCR/ ISSR/ ASSR, RAMPs/ dRAMPs, SAMPL), making use of microsatellites, have been outlined. Different methods for enrichment of genomic libraries for microsatellites have also been outlined. Relevant literature on the subject, giving a summary of results obtained using each approach, has been reviewed and critically discussed. The review also includes a discussion on literature, which deals with the use of microsatellites in genome mapping, gene tagging, DNA fingerprinting, characterization of germplasm and cytogenetics research. Special emphasis has been laid on the genome of bread wheat, where the work done in the authors' own laboratory has also been briefly reviewed.

The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat

A number of PCR-based techniques can be used to detect polymorphisms in plants. For their wide-scale usage in germplasm characterisation and breeding it is important that these marker technologies can be exchanged between laboratories, which in turn requires that they can be standardised to yield reproducible results, so that direct collation and comparison of the data are possible. This article describes a network experiment involving several European laboratories, in which the reproducibility of three popular molecular marker techniques was examined: random-amplified fragment length polymorphism (RAPD), amplified fragment length polymorphism (AFLP) and sequence-tagged microsatellites (SSR). For each technique, an optimal system was chosen, which had been standardised and routinely used by one laboratory. This system (genetic screening package) was distributed to different participating laboratories in the network and the results obtained compared with those of the original sender. Different experiences were gained in this exchange experiment with the different techniques. RAPDs proved difficult to reproduce. For AFLPs, a single-band difference was observed in one track, whilst SSR alleles were amplified by all laboratories, but small differences in their sizing were obtained.

Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories

Simple sequence repeats (SSRs), or microsatellites, have been isolated and characterized from numerous animal and plant species (6,7). These sequences have been shown to be highly polymorphic between individuals within populations or closely related genotypes. This, together with the fact that they can be converted into a simple polymerase chain reaction (PCR)based assay, has led to their widespread use as molecular markers of genetic diversity (6). The use of microsatellites has been limited in plants, however, by the costs involved in isolating large numbers from the target species. Recently, a number of papers have reported the isolation of microsatellite clones by an enrichment procedure (1,2,4,5). These reports have all resulted in the isolation of a single type of microsatellite. However, as little is known about the level of polymorphism of individual microsatellite sequences, it is possible that this approach will result in markers that do not detect the level of polymorphism often required in diversity studies. Furthermore, the targeting of a single microsatellite species may produce only a small number of clones necessitating the construction of further libraries in the future. To eliminate these problems, we have significantly modified the enrichment technique (2,5) to yield clones that contain a variety of microsatellites. These clones can then be characterized by either direct sequencing or colony hybridization using specific microsatellite oligonucleotides. Interestingly, the technique also yields a high percentage of clones containing more than one type of microsatellite. Results to date suggest that the clones produced by this procedure are sufficiently polymorphic to be used in population genetics and/or breeding studies. Preparation of reagents for microsatellite enrichment. The following oligonucleotides can be used to enrich for DNA fragments containing microsatellites: [GA]15, [GT]15, [AT]15, [GC]15, [CAA]10, [CATA]10, [ATT]10, [GATA]10, [GCC]10 and [ATAG]10. Fifty nanograms of each oligonucleotide in 3× SSC (45 mM sodium citrate, pH 7.0 and 450 mM NaCl) are pooled in a total volume of 80 μL, spotted onto a 0.5-cm2 piece of Hybond N+ (Amersham, Arlington Heights, IL, USA) and air-dried for 1 h. The dry membrane is UV-treated for 30 s using a 260-nm transilluminator. Weakly bound oligonucleotides are washed off the membrane by washing twice in 10 mL of hybridization buffer (50% formamide, 3× standard saline citrate [SSC], 25 mM Na-phosphate, pH 7.0, and 0.5% sodium dodecyl sulfate [SDS]) at 45°C for 2 days. The membranes are then stored at -20°C until required. Preparation of genomic DNA. One microgram of genomic DNA is digested with 2 units of RsaI in a volume of 20 μL for 1 h at 37°C. Fifty nanograms

https://www.future-science.com/doi/pdf/10.2144/96205bm04

Microsatellite libraries enriched for several microsatellite sequences in plants.

For the application of molecular techniques to wide-scale diversity screening it is important that the different screening techniques employed can be standardized to yield reproducible results across laboratories in order that direct collation and comparison of the data are possible (1). RAPDs (Random Amplified Polymorphic DNA) involve the use of a single ‘arbitrary’ primer (purchasable from commercial companies) in a PCR reaction and results in the amplification of several discrete DNA products (see Chapter 9). It is now widely recognized that to obtain reproducible band profiles on the gels it is absolutely essential to maintain consistent reaction conditions. Numerous studies have reported the separate effects of altering different parameters, such as ratio of template DNA/primers, concentration of Taq polymerase and Mg concentration, on the bands obtained (2–5). A network reproducibility test was carried by nine European laboratories (L1-L9) to determine whether RAPD profiles could be reproduced by different laboratories if all details of the reaction conditions were standardized (1).

A. Daly

Papers

Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories

Microsatellite libraries enriched for several microsatellite sequences in plants.

Reproducibility testing of RAPDs by a network of European laboratories