PSMD: An extensive database for pan‐species microsatellite investigation and marker development

Microsatellites are widely distributed throughout nearly all genomes which have been extensively exploited as powerful genetic markers for diverse applications due to their high polymorphisms. Their length variations are involved in gene regulation and implicated in numerous genetic diseases even in cancers. Although much effort has been devoted in microsatellite database construction, the existing microsatellite databases still had some drawbacks, such as limited number of species, unfriendly export format, missing marker development, lack of compound microsatellites and absence of gene annotation, which seriously restricted researchers to perform downstream analysis. In order to overcome the above limitations, we developed PSMD (Pan‐Species Microsatellite Database, http://big.cdu.edu.cn/psmd/ ) as a web‐based database to facilitate researchers to easily identify microsatellites, exploit reliable molecular markers and compare microsatellite distribution pattern on genome‐wide scale. In current release, PSMD comprises 678,106,741 perfect microsatellites and 43,848,943 compound microsatellites from 18,408 organisms, which covered almost all species with available genomic data. In addition to interactive browse interface, PSMD also offers a flexible filter function for users to quickly gain desired microsatellites from large data sets. PSMD allows users to export GFF3 formatted file and CSV formatted statistical file for downstream analysis. We also implemented an online tool for analysing occurrence of microsatellites with user‐defined parameters. Furthermore, Primer3 was embedded to help users to design high‐quality primers with customizable settings. To our knowledge, PSMD is the most extensive resource which is likely to be adopted by scientists engaged in biological, medical, environmental and agricultural research.     

Long tomato microsatellites are predominantly associated with centromeric regions.

Microsatellites as genetic markers are used in many crop plants. Major criteria for their usability as molecular markers include that they are highly polymorphic and evenly spread throughout a genome. In tomato, it has been reported that long arrays of tetranucleotide microsatellites containing the motif GATA are highly clustered around the centromeres of all chromosomes. In this study, we have isolated tomato microsatellites containing long arrays (> 20 repeats) of the dinucleotide motifs GA, GT, AT, as well as GATA, assessed their variability within Lycopersicon esculentum varieties and mapped them onto a genetic map of tomato. The investigated microsatellite markers exhibited between 1 and 5 alleles in a diverse set of L. esculentum lines. Mapping of the microsatellites onto the genetic map of tomato demonstrates that, as previously shown, GATA microsatellites are highly clustered in the regions of the tomato centromeres. Interestingly, the same centromeric location was now found for long dinucleotide microsatellite markers. Because of this uneven distribution, genetic mapping of the entire tomato genome using long dinucleotide microsatellites will be very difficult to achieve and microsatellite markers with shorter arrays of microsatellites could be more suitable for mapping experiments albeit their lower level of polymorphism. Some microsatellite markers described in this study might provide a useful tool to study the molecular structure of tomato centromeric regions and for variety identification.     

New softwares for automated microsatellite marker development

Microsatellites are repeated small sequence motifs that are highly polymorphic and abundant in the genomes of eukaryotes. Often they are the molecular markers of choice. To aid the development of microsatellite markers we have developed a module that integrates a program for the detection of microsatellites (TROLL), with the sequence assembly and analysis software, the Staden Package. The module has easily adjustable parameters for microsatellite lengths and base pair quality control. Starting with large datasets of unassembled sequence data in the form of chromatograms and/or text data, it enables the creation of a compact database consisting of the processed and assembled microsatellite containing sequences. For the final phase of primer design, we developed a program that accepts the multi-sequence ‘experiment file’ format as input and produces a list of primer pairs for amplification of microsatellite markers. The program can take into account the quality values of consensus bases, improving success rate of primer pairs in PCR. The software is freely available and simple to install in both Windows and Unix-based operating systems. Here we demonstrate the software by developing primer pairs for 427 new candidate markers for peanut.     

How much effort is required to isolate nuclear microsatellites from plants?

The attributes of codominance, reproducibility and high resolution have all contributed towards the current popularity of nuclear microsatellites as genetic markers in molecular ecological studies. One of their major drawbacks, however, is the development phase required to obtain working primers for a given study species. To facilitate project planning, we have reviewed the literature to quantify the workload involved in isolating nuclear microsatellites from plants. We highlight the attrition of loci at each stage in the process, and the average effort required to obtain 10 working microsatellite primer pairs.     

Rise of the machines--recommendations for ecologists when using next generation sequencing for microsatellite development

Next generation sequencing is revolutionizing molecular ecology by simplifying the development of molecular genetic markers, including microsatellites. Here, we summarize the results of the large-scale development of microsatellites for 54 nonmodel species using next generation sequencing and show that there are clear differences amongst plants, invertebrates and vertebrates for the number and proportion of motif types recovered that are able to be utilized as markers. We highlight that the heterogeneity within each group is very large. Despite this variation, we provide an indication of what number of sequences and consequent proportion of a 454 run are required for the development of 40 designable, unique microsatellite loci for a typical molecular ecological study. Finally, to address the challenges of choosing loci from the vast array of microsatellite loci typically available from partial genome runs (average for this study, 2341 loci), we provide a microsatellite development flowchart as a procedural guide for application once the results of a partial genome run are obtained.     

Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences

The application of next-generation sequencing (NGS) technologies for the development of simple sequence repeat (SSR) or microsatellite loci for genetic research in the botanical sciences is described. Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been a difficult and costly process. NGS technologies allow the efficient identification of large numbers of microsatellites at a fraction of the cost and effort of traditional approaches. The major advantage of NGS methods is their ability to produce large amounts of sequence data from which to isolate and develop numerous genome-wide and gene-based microsatellite loci. The two major NGS technologies with emergent application in SSR isolation are 454 and Illumina. A review is provided of several recent studies demonstrating the efficient use of 454 and Illumina technologies for the discovery of microsatellites in plants. Additionally, important aspects during NGS isolation and development of microsatellites are discussed, including the use of computational tools and high-throughput genotyping methods. A data set of microsatellite loci in the plastome and mitochondriome of cranberry (Vaccinium macrocarpon Ait.) is provided to illustrate a successful application of 454 sequencing for SSR discovery. In the future, NGS technologies will massively increase the number of SSRs and other genetic markers available to conduct genetic research in understudied but economically important crops such as cranberry.     

An improved enrichment procedure to develop multiple repeat classes of cotton microsatellite markers

The availability of a large number of molecular markers is a prerequisite, especially in cotton, for identifying a sufficient number of informative markers for mapping and genetic analysis. Despite the global importance of the cotton crop, few informative microsatellite markers are available, primarily because of the cost associated with their development. This report describes an improved and cost-effective strategy for developing microsatellite markers. Genomic DNA was randomly sheared with nitrogen gas to obtain unbiased representation of the genome, and the fragments containing microsatellites were captured by using biotinylated oligos and streptavidin-based recovery. Six libraries enriched for 14 microsatellite motifs were constructed and screened. Nearly 4900 simple sequence repeat (SSR)-containing sequences were identified, leading to the development of more than 1200 markers in a small amount of time.     

Microsatellite DNA markers for rice chromosomes

We found 369 complete microsatellites, of which (CGG/GCC)_n was the most frequent, in 11 798 rice sequences in the database. Of these microsatellites, 35 out of 45 could be successfully converted into microsatellite DNA markers using sequence information in their flanking regions. Thus, the time and labor used to develop new microsatellite DNA markers could be saved by using these published sequences. Twenty eight polymorphic markers between Asominori (japonica) and IR24 (indica) have been correctly mapped on the rice genome and microsatellites appear to be randomly distributed in the rice chromosomes. Integration of these markers with the published microsatellite DNA markers showed that about 35% of the rice chromosomes were covered by the 56 microsatellite DNA markers. These microsatellites were hypervariable and were easily to assay by PCR; they were distributed to all chromosomes and therefore, one can easily select plants carrying desired chromosome regions using these microsatellite DNA markers. Thus, microsatellite maps should aid the development of new breeds of rice saving time, labor, and money.     

Challenges of microsatellite isolation in fungi

Although they represent powerful genetic markers in many fields of biology, microsatellites have been isolated in few fungal species. The aim of this study was to assess whether obtaining microsatellite markers with an acceptable level of polymorphism is generally harder from fungi than in other organisms. We therefore surveyed the number, nature and polymorphism level of published microsatellite markers in fungi from the literature and from our own data on seventeen fungal microsatellite-enriched libraries, and in five other phylogroups (angiosperms, insects, fishes, birds and mammals). Fungal microsatellites indeed appeared both harder to isolate and to exhibit lower polymorphism than in other organisms. This appeared to be due, at least in part, to genomic specificities, such as scarcity and shortness of fungal microsatellite loci. A correlation was observed between mean repeat number and mean allele number in the published fungal microsatellite loci. The cross-species transferability of fungal microsatellites also appeared lower than in other phylogroups. However, microsatellites have been useful in some fungal species. Thus, the considerable advantages of these markers make their development worthwhile, and this study provides some guidelines for their isolation.     

Conserved flanking microsatellite sequences (ReFS) differentiate between Lepidoptera species,and provide insight into microsatellite evolution

High rates of mutation and homoplasy mean that microsatellites generally are not considered to be useful molecular markers for inferring systematic relationships between species. However, an earlier pilot study suggested that conserved flanking microsatellite sequences, also known as repetitive flanking sequences (ReFS), may form a basis for a dominant marker that can differentiate between species of Lepidoptera. We present data that demonstrate that ReFS are quick and easy to use, and generate highly repeatable banding patterns from a range of Lepidoptera species. Sequence data from a subset of ReFS‐amplified bands revealed microsatellite families with flanking sequences that are more conserved within than among species: this is probably attributable to recombination‐mediated events, transposition of mobile elements or a combination of the two. Our data support the use of ReFS as dominant interspecific molecular markers, and add to the growing literature on the evolution of microsatellites in Lepidoptera.     

Microsatellite cross-amplification in coccolithophores: application in population diversity studies

The development and isolation of microsatellites entails a significant input of time and money. Therefore there is an interest in using existing microsatellites on species from which markers have not yet been developed. Conservation of six previously identified microsatellite loci in the marine coccolithophorid species Emiliana huxleyi was found in a survey of two bloom forming coccolithophorid species--Gephyrocapsa oceanica and Coccolithus pelagicus. The number of alleles per locus varied from 1 to 8, and half of the microsatellite loci tested showed 4 or more alleles. The microsatellite markers used in this study may be applied to other coccolithophorid species for population analysis, eliminating the time-consuming, costly development of microsatellite markers for other coccolithophorid species.     

Heterologous nuclear and chloroplast microsatellite amplification and variation in tea, Camellia sinensis.

The advantage of the cross transferability of heterologous chloroplast and nuclear microsatellite primers was taken to detect polymorphism among 24 tea (Camellia sinensis (L.) O. Kuntze) genotypes, including both the assamica and the sinensis varieties. Primer information was obtained from the closely related Camellia japonica species for four nuclear microsatellites, and from Nicotiana tabaccum for seven universal chloroplast microsatellites. All of the nuclear microsatellite loci tested generated an expected DNA fragment in tea, revealing between three and five alleles per locus. Four out of the seven chloroplast microsatellites primers amplified positively, and of these only one was polymorphic with three alleles, which is in agreement with the conserved nature of chloroplast microsatellites at the intraspecific level. A factorial correspondence analysis carried out on all genotypes and nuclear microsatellite alleles separated the assamica and sinensis genotypes into two groups, thus demonstrating the value of these markers in establishing the genetic relationship between tea varieties. Genetic diversity measured with nuclear microsatellites was higher than that measured with other types of molecular markers, offering prospects for their use in fingerprinting, mapping, and population genetic studies, whereas polymorphisms detected at a cpSSR locus will allow the determination of plastid inheritance in the species.     

Detection, distribution and selection of microsatellites (SSRs) in the genome of the yeast Saccharomyces cerevisiae as molecular markers

AIMS: The aim of this work was the selection of six polymorphic microsatellite loci for their use as molecular markers in the identification, typification and genetic differentiation of S. cerevisiae strains. METHODS AND RESULTS: The selection was undertaken following a search of the genomic DNA database of Saccharomyces cerevisiae for simple tandem repeat sequences (microsatellites) of di- and trinucleotides. The genetic variability generated by these markers was evaluated in 51 isolates. The discriminatory power produced by combining the information obtained by the six microsatellites was very high. A total of 57 alleles, which generated 44 genotypes, were found. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The multiple analysis of microsatellites proved to be a powerful and agile tool for analysing the genome of S. cerevisiae populations.     

AFLP utility for population assignment studies: analytical investigation and empirical comparison with microsatellites

Individual-based population assignment tests have thus far mainly relied on the use of microsatellite loci. However, the logistic difficulty of screening large numbers of loci required to reach sufficient statistical power hampers the usefulness of microsatellites in situations of weak population structuring. Amplified fragment length polymorphisms (AFLP) represents an alternative for overcoming this logistical issue as the technique allows the user to characterize a much larger number of loci with a comparable analytical effort. In this study, an assignment test based on maximum likelihood for dominant markers was used to investigate the potential usefulness of AFLP for population assignment. We also compared assignment success achieved with AFLP with that obtained using microsatellites in a case study of low population differentiation involving whitefish (Coregonus clupeaformis) sympatric ecotypes. The analytical investigation showed that the minimum number of AFLP loci required to reach an assignment success of 95% stood within values that are easily achievable in many situations. This also showed how assignment success varied according to the number of AFLP loci used, their absolute frequency and their frequency differential and sampling errors, as well as the number of putative source populations. The case study showed that given a comparable analytical effort in the laboratory, AFLP were much more efficient than the microsatellite loci in discriminating the source of an individual among putative populations. AFLP resulted in higher assignment success at all levels of stringency and the log-likelihood differences between populations obtained with AFLP for each individual were much larger than those obtained with microsatellites. These results indicate that research involving individual-based population assignment methods should benefit importantly from the use of AFLP markers, especially in systems characterized by weak population structuring.     

Genome‐wide microsatellite characterisation and marker development in Chenopodium quinoa

Microsatellites, as the tracts of repetitive DNA, are an essential constituent of the plant genome that holds important evolutionary significance, and have been extensively used to develop molecular makers for genetic analysis. To understand the microsatellite dynamics of quinoa genome and its relatives, in this study we performed a genome‐wide analysis of microsatellites in five Amaranthaceae species using available genome sequences. The results demonstrated that the microsatellites of the five Amaranthaceae species were characterised by relatively high proportions of mono‐, di‐ and trinucleotide repeats with A/T rich motifs, implying conservative organisation and composition of microsatellites in this family. Furthermore, a significant negative correlation between microsatellite frequencies and GC contents (r = ?.87) were observed. In total, 533,961 (89.57%) and 542,601 (89.86%) microsatellite loci could be used to develop simple sequence repeat (SSR) molecular markers, of which 7,178 were found to be polymorphic between the two sequenced quinoa cultivars, QQ74 and Real Blanca, through in silico PCR analysis. Finally, 15 SSR markers were randomly selected to validate their polymorphism across 12 quinoa accessions by wet‐lab PCR amplification. The newly developed genome‐wide SSR markers provide a useful resource for population genetics, gene mapping and molecular breeding studies in quinoa and beyond.     

Challenges in analysis and interpretation of microsatellite data for population genetic studies

Advancing technologies have facilitated the ever‐widening application of genetic markers such as microsatellites into new systems and research questions in biology. In light of the data and experience accumulated from several years of using microsatellites, we present here a literature review that synthesizes the limitations of microsatellites in population genetic studies. With a focus on population structure, we review the widely used fixation (F_ST) statistics and Bayesian clustering algorithms and find that the former can be confusing and problematic for microsatellites and that the latter may be confounded by complex population models and lack power in certain cases. Clustering, multivariate analyses, and diversity‐based statistics are increasingly being applied to infer population structure, but in some instances these methods lack formalization with microsatellites. Migration‐specific methods perform well only under narrow constraints. We also examine the use of microsatellites for inferring effective population size, changes in population size, and deeper demographic history, and find that these methods are untested and/or highly context‐dependent. Overall, each method possesses important weaknesses for use with microsatellites, and there are significant constraints on inferences commonly made using microsatellite markers in the areas of population structure, admixture, and effective population size. To ameliorate and better understand these constraints, researchers are encouraged to analyze simulated datasets both prior to and following data collection and analysis, the latter of which is formalized within the approximate Bayesian computation framework. We also examine trends in the literature and show that microsatellites continue to be widely used, especially in non‐human subject areas. This review assists with study design and molecular marker selection, facilitates sound interpretation of microsatellite data while fostering respect for their practical limitations, and identifies lessons that could be applied toward emerging markers and high‐throughput technologies in population genetics.     

Isolation and characterization of nine microsatellite markers from Coffea arabica L., showing wide cross‐species amplifications

Genetic improvement of coffee (Coffea arabica L.) is constrained by low genetic diversity and lack of genetic markers, suitable screening tools, information on the genetic make‐up of available gene pool and long generation time. In this context, use of DNA markers such as microsatellites that provide high genetic‐resolution becomes highly desirable. Here, we report the development of nine new microsatellite markers from partial genomic library of an elite variety of Coffea arabica. The developed microsatellites revealed robust cross‐species amplifications in 17 related species of coffee, and their Polymorphic Information Content varied from 0 to 0.6, 0 to 0.78 and 0.67 to 0.90 for the arabica, robusta genotypes and species representatives, respectively. The data thus suggest their potential use as genetic markers for assessment of germplasm diversity and linkage analysis of coffee.     

Isolation and characterization of microsatellite markers and analysis of genetic variability in Curculigo latifolia Dryand

Curculin, a sweet protein found in Curculigo latifolia fruit has great potential for the pharmaceutical industry. This protein interestingly has been found to have both sweet taste and taste-modifying capacities comparable with other natural sweeteners. According to our knowledge this is the first reported case on the isolation of microsatellite loci in this genus. Hence, the current development of microsatellite markers for C. latifolia will facilitate future population genetic studies and breeding programs for this valuable plant. In this study 11 microsatellite markers were developed using 3' and 5' ISSR markers. The primers were tested on 27 accessions from all states of Peninsular Malaysia. The number of alleles per locus ranged from three to seven, with allele size ranging from 141 to 306?bp. The observed and expected heterozygosity ranged between 0.00-0.65 and 0.38-0.79, respectively. The polymorphic information content ranged from 0.35 to 0.74 and the Shannon's information index ranged from 0.82 to 1.57. These developed polymorphic microsatellites were used for constructing a dendrogram by unweighted pair group method with arithmetic mean cluster analysis using the Dice's similarity coefficient. Accessions association according to their geographical origin was observed. Based on characteristics of isolated microsatellites for C. latifolia accessions all genotype can be distinguished using these 11 microsatellite markers. These polymorphic markers could also be applied to studies on uniformity determination and somaclonal variation of tissue culture plantlets, varieties identification, genetic diversity, analysis of phylogenetic relationship, genetic linkage maps and quantitative trait loci in C. latifolia.     

微卫星DNA在进化中的研究进展

在过去的十年中,微卫星已经变成最流行的基因标记之一。尽管微卫星分析已有广泛的应用,然而关于微卫星DNA变异动力学的完整的画面才刚刚浮现。文章将从微卫星的起源、微卫星进化模式的推断、DNA复制的滑动是微卫星DNA变异的主要机制、影响微卫星突变率的因素、微卫星的长度分布和微卫星的频率分布等方面综述有关微卫星进化动力学方面的研究进展。