Using Next Generation RAD Sequencing to Isolate Multispecies Microsatellites for Pilosocereus (Cactaceae)

Microsatellite markers (also known as SSRs, Simple Sequence Repeats) are widely used in plant science and are among the most informative molecular markers for population genetic investigations, but the development of such markers presents substantial challenges. In this report, we discuss how next generation sequencing can replace the cloning, Sanger sequencing, identification of polymorphic loci, and testing cross-amplification that were previously required to develop microsatellites. We report the development of a large set of microsatellite markers for five species of the Neotropical cactus genus Pilosocereus using a restriction-site-associated DNA sequencing (RAD-seq) on a Roche 454 platform. We identified an average of 165 microsatellites per individual, with the absolute numbers across individuals proportional to the sequence reads obtained per individual. Frequency distribution of the repeat units was similar in the five species, with shorter motifs such as di- and trinucleotide being the most abundant repeats. In addition, we provide 72 microsatellites that could be potentially amplified in the sampled species and 22 polymorphic microsatellites validated in two populations of the species Pilosocereus machrisii. Although low coverage sequencing among individuals was observed for most of the loci, which we suggest to be more related to the nature of the microsatellite markers and the possible bias inserted by the restriction enzymes than to the genome size, our work demonstrates that an NGS approach is an efficient method to isolate multispecies microsatellites even in non-model organisms.     

Two highly informative dinucleotide SSR multiplexes for the conifer Larix decidua (European larch)

We have designed two highly polymorphic microsatellite multiplexes for Larix decidua Mill (European larch), a coniferous tree species with a fragmented distribution across Europe. The multiplexes combine microsatellites previously designed for the sister species L. kaempferi and newly identified microsatellites obtained by pyrosequencing of an enriched microsatellite library and subsequent marker candidate selection. As we wanted to target highly polymorphic markers, only microsatellite motifs with a high number of repeats (≥ 12) were selected. An important proportion of the marker candidates presented multiple bands, bad amplification or insufficient polymorphism. Such difficulties were expected owing to the large genome size of the studied species. Our strategy for marker validation followed most recent recommendations for microsatellite development, for example verifying marker quality in terms of polymorphism and accurate allele binning before multiplexing. The most promising loci were combined in two multiplexes, a 7-plex and a 6-plex. These were tested on a sample of 413 individuals from 18 populations distributed across the natural range. The 13 loci had from 9 to 36 alleles. Markers were successfully tested in another laboratory, confirming robustness of the marker protocols. We also tested transferability on six other larch species from Asia and North America. Overall, this study shows that, even in species with large genome size and relatively low overall polymorphism, microsatellites can be successfully developed using next-generation sequencing technologies, provided that some additional precautions are taken compared to species lacking these characteristics.     

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.     

Pig microsatellites isolated from cosmids revealing polymorphism and localized on chromosomes

One of the most widely studied simple sequences in the mammalian genome is the (TG)_n dinucleotide sequence. Because these microsatellites are highly polymorphic, we chose to study microsatellites from cosmids to provide genetic markers for the porcine genome. After screening a porcine cosmid library with a (CA)₁₀ probe, 20 cosmids containing microsatellites were subcloned and 17 microsatellites identified by sequencing. Oligonucleotide primers flanking the repeat were designed for seven (TG)_n microsatellites with n > 14. These seven microsatellites revealed polymorphism and were regionally assigned to chromosomes by fluorescent in situ hybridization of initial cosmids. These seven loci will be useful for both the construction of the genetic map and as landmark loci on the physical map of the porcine genome.     

New microsatellite markers in chicken optimized for automated fluorescent genotyping

We have isolated and developed 180 new polymorphic chicken microsatellite markers. In addition, primers have been developed for 91 microsatellites derived from the GenBank sequence database (isolated by the laboratory of Terry Burke, Leicester University), of which 89 were polymorphic, and six existing polymorphic markers (HUJ) have been modified. The primer sequences were designed to allow optimal performance of the markers, in sets containing multiple microsatellites, on ABI sequencers. The average number of alleles for the 275 polymorphic markers described was 4·0. Of these markers, 93% were polymorphic in the Wageningen resource population whereas 57% of the markers were polymorphic in the East Lansing reference population and only 44% could be mapped in the Compton reference population. The microsatellite markers described in this paper, in combination with the microsatellite markers published previously, are particularly well suited for performing a total genome scan for the detection of quantitative trait loci (QTL).     

Development of microsatellite genetic markers in Siberian stone pine (<Emphasis Type="Italic">Pinus sibirica</Emphasis> Du Tour) based on the <Emphasis Type="Italic">de novo</Emphasis> whole genome sequencing

Siberian stone pine, Pinus sibirica Du Tour is one of the most economically and environmentally important forest-forming species of conifers in Russia. To study these forests a large number of highly polymorphic molecular genetic markers, such as microsatellite loci, are required. Prior to the new high-throughput next generation sequencing (NGS) methods, discovery of microsatellite loci and development of micro-satellite markers were very time consuming and laborious. The recently developed draft assembly of the Siberian stone pine genome, sequenced using the NGS methods, allowed us to identify a large number of microsatellite loci in the Siberian stone pine genome and to develop species-specific PCR primers for amplification and genotyping of 70 microsatellite loci. The primers were designed using contigs containing short simple sequence tandem repeats from the Siberian stone pine whole genome draft assembly. Based on the testing of primers for 70 microsatellite loci with tri-, tetra- or pentanucleotide repeats, 18 most promising, reliable and polymorphic loci were selected that can be used further as molecular genetic markers in population genetic studies of Siberian stone pine.     

GinMicrosatDb: a genome-wide microsatellite markers database for sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.)

Molecular breeding in sesame is still at infancy due to limited number of microsatellite markers available and the low level of polymorphism exhibited by them. Therefore, whole genome sequencing was used for development of microsatellite markers so as to ensure availability of substantial number of polymorphic markers for use in marker assisted breeding programs. Whole genome sequencing of sesame variety ‘Swetha’ was done using Illumina paired-end sequencing and Roche 454 shotgun sequencing technologies (GCA_000975565.1 in GenBank). ‘GinMicrosatDb’, a genome-wide microsatellite marker database has been developed using the whole genome sequence data of sesame variety ‘Swetha’. The database consists of microsatellites localized on both linkage groups and scaffolds with their genomic co-ordinates. It provides five sets of forward and reverse primers for each of the microsatellite loci along with the flanking sequences, primer GC content, product size and melting temperature etc. The distribution of microsatellites can be viewed and selected through a genome browser as well as through a physical map. The newly identified microsatellite markers are expected to help sesame breeders in developing marker tags for traits of economic importance thereby bringing about greater efficiency in marker-assisted selection programs.     

MicNeSs: genotyping microsatellite loci from a collection of (NGS) reads

Microsatellites are widely used in population genetics to uncover recent evolutionary events. They are typically genotyped using capillary sequencer, which capacity is usually limited to 9, at most 12 loci for each run, and which analysis is a tedious task that is performed by hand. With the rise of next‐generation sequencing (NGS), a much larger number of loci and individuals are available from sequencing: for example, on a single run of a GS Junior, 28 loci from 96 individuals are sequenced with a 30X cover. We have developed an algorithm to automatically and efficiently genotype microsatellites from a collection of reads sorted by individual (e.g. specific PCR amplifications of a locus or a collection of reads that encompass a locus of interest). As the sequencing and the PCR amplification introduce artefactual insertions or deletions, the set of reads from a single microsatellite allele shows several length variants. The algorithm infers, without alignment, the true unknown allele(s) of each individual from the observed distributions of microsatellites length of all individuals. MicNeSs, a python implementation of the algorithm, can be used to genotype any microsatellite locus from any organism and has been tested on 454 pyrosequencing data of several loci from fruit flies (a model species) and red deers (a nonmodel species). Without any parallelization, it automatically genotypes 22 loci from 441 individuals in 11 hours on a standard computer. The comparison of MicNeSs inferences to the standard method shows an excellent agreement, with some differences illustrating the pros and cons of both methods.     

Quantifying ascertainment bias and species-specific length differences in human and chimpanzee microsatellites using genome sequences

Surveys of variability of homologous microsatellite loci among species reveal an ascertainment bias for microsatellite length where microsatellite loci isolated in one species tend to be longer than homologous loci in related species. Here, we take advantage of the availability of aligned human and chimpanzee genome sequences to compare length difference of homologous microsatellites for loci identified in humans to length difference for loci identified in chimpanzees. We are able to quantify ascertainment bias for a range of motifs and microsatellite lengths. Because ascertainment bias should not exist if a microsatellite selected in one species is as likely to be longer as it is to be shorter than its homologue, we propose that the nature of ascertainment bias can provide evidence for understanding how microsatellites evolve. We show that bias is greater for longer microsatellites but also that many long microsatellites have short homologues. These results are consistent with the notion that growth of long microsatellites is constrained by an upper length boundary that, when reached, sometimes results in large deletions. By evaluating ascertainment bias separately for interrupted and uninterrupted repeats we also show that long microsatellites tend to become interrupted, thereby contributing a second component of ascertainment bias. Having accounted for ascertainment bias, in agreement with results published elsewhere, we find that microsatellites in humans are longer on average than those in chimpanzees. This length difference is similar among repeat motifs but surprisingly comprises two roughly equal components, one associated with the repeats themselves and one with the flanking sequences. The differences we find can only be explained if microsatellites are both evolving directionally under a biased mutation process and are doing so at different rates in different closely related species.     

A Pipeline for the Development of Microsatellite Markers using Next Generation Sequencing Data

Background: Also known as Simple Sequence Repetitions (SSRs), microsatellites are profoundly informative molecular markers and powerful tools in genetics and ecology studies on plants.Objective: This research presents a workflow for developing microsatellite markers using genome skimming.Methods: The pipeline was proposed in several stages that must be performed sequentially: obtaining DNA sequences, identifying microsatellite regions, designing primers, and selecting candidate microsatellite regions to develop the markers. Our pipeline efficiency was analyzed using Illumina sequencing data from the non-model tree species Pterodon emarginatus Vog.Results: The pipeline revealed 4,382 microsatellite regions and drew 7,411 pairs of primers for P. emarginatus. However, a much larger number of microsatellite regions with the potential to develop markers were discovered from our pipeline. We selected 50 microsatellite regions with high potential for developing markers and organized 29 microsatellite regions in sets for multiplex PCR.Conclusion: The proposed pipeline is a powerful tool for fast and efficient development of microsatellite markers on a large scale in several species, especially nonmodel plant species.     

High-throughput microsatellite marker development in two sparid species and verification of their transferability in the family Sparidae

Recently, 454 sequencing has emerged as a popular method for isolating microsatellites owing to cost-effectiveness and time saving. In this study, repeat-enriched libraries from two southern African endemic sparids (Pachymetopon blochii and Lithognathus lithognathus) were 454 GS-FLX sequenced. From these, 7370 sequences containing repeats (SCRs) were identified. A brief survey of 23 studies showed a significant difference between the number of SCRs when enrichment was performed first before 454 sequencing. We designed primers for 302 unique fragments containing more than five repeat units and suitable flanking regions. A fraction (<11%) of these loci were characterized with 18 polymorphic microsatellite loci (nine in each of the focal species) being described. Sanger sequencing of alleles confirmed that size variation was because of differences in the number of tandem repeats. However, a case of homoplasy and sequencing errors in the 454 sequencing were identified. These newly developed and four previously isolated loci were successfully used to identify polymorphic markers in nine other economically important species, representative of sparid diversity. The combination of newly developed markers with data from previous sparid cross-species studies showed a significant negative correlation between genetic divergence to focal species and microsatellite transferability. The high level of transferability we described (48% amplification success and 32% polymorphism) suggests that the 302 microsatellite loci identified represent an excellent resource for future studies on sparids. Microsatellite marker development should commonly include tests of transferability to reduce costs and increase feasibility of population genetics studies in nonmodel organisms.     

MICROSATELLITE EVOLUTION IN VERTEBRATES: INFERENCE FROM AC DINUCLEOTIDE REPEATS

Abstract We analyze published data from 592 AC microsatellite loci from 98 species in five vertebrate classes including fish, reptiles, amphibians, birds, and mammals. We use these data to address nine major questions about microsatellite evolution. First, we find that larger genomes do not have more microsatellite loci and therefore reject the hypothesis that microsatellites function primarily to package DNA into chromosomes. Second, we confirm that microsatellite loci are relatively rare in avian genomes, but reject the hypothesis that this is due to physical constraints imposed by flight. Third, we find that microsatellite variation differs among species within classes, possibly relating to population dynamics. Fourth, we reject the hypothesis that microsatellite structure (length, number of alleles, allele dispersion, range in allele sizes) differs between poikilotherms and homeotherms. The difference is found only in fish, which have longer microsatellites and more alleles than the other classes. Fifth, we find that the range in microsatellite allele size at a locus is largely due to the number of alleles and secondarily to allele dispersion. Sixth, length is a major factor influencing mutation rate. Seventh, there is a directional mutation toward an increase in microsatellite length. Eighth, at the species level, microsatellite and allozyme heterozygosity covary and therefore inferences based on large-scale studies of allozyme variation may also reflect microsatellite genetic diversity. Finally, published microsatellite loci (isolated using conventional hybridization methods) provide a biased estimate of the actual mean repeat length of microsatellites in the genome.     

Development of microsatellite markers for <Emphasis Type="Italic">Manilkara maxima</Emphasis> T.D. Penn. (Sapotaceae) and their use in conservation genetics

Manilkara maxima is an endemic tree species of the Atlantic Forest in southern Bahia, Brazil. It is considered important for forest conservation due to its mutualistic interactions with endemic and endangered animals. Our aim was to develop microsatellite markers to estimate genetic diversity in order to provide information for effectiveness of future conservation programs. We used next generation sequencing technology to develop the first specific microsatellite markers for M. maxima. Seventeen new microsatellite loci were applied in 72 individuals sampled in three natural populations. On average, the number of alleles per loci was 8.8. The expected heterozygosity varied between 0.72 and 0.77, indicating that the developed set of molecular markers is useful for genetic diversity studies. Additionally, the estimated value for the combined probability of exclusion (Q) was greater than 0.999, which indicates the powerful of these molecular tools for paternity and kinship analysis. Our results demonstrate that the set of microsatellites developed in this work is a powerful tool for population genetics, molecular ecology and conservation biology purposes.     

A simple method for isolation of microsatellites from the Japanese squirrel,Sciurus lis,without constructing a genomic library

We developed a simple and easy method to isolate microsatellites without screening genomic libraries by hybridization. The method requires only three basic techniques: polymerase chain reaction, DNA cloning and sequencing. We applied this method to develop microsatellite markers for the Japanese squirrel and isolated 45 clones that contained repetitive sequences. Among the 22 clones that we tested further, we found 11 diagnostic microsatellite loci that are applicable to the molecular ecological study of Japanese squirrels.     

Characterization of novel microsatellites from Drosophila transversa

We investigated a partial genomic library of Drosophila transversa for microsatellites and developed 12 markers for genetic analyses. This is the first time that microsatellite primers from the quinaria species group have been described. Four loci were cross-amplified in D. phalerata. Nine out of the 12 microsatellite markers developed are likely to be on the X chromosome.     

Novel polymorphic microsatellite markers developed in the cotton bollworm Helicoverpa armigera （Lepidoptera：Noctuidae）

A novel set of five polymorphic di- or trinucleofide microsatellite loci suitable for population genetic study were developed from an enriched genomic library for the pest insect cotton bollworm, Helicoverpa armigera, and cross-amplifiability of these and other published loci was tested in a closely related species, the tobacco budworm, H.assulta. The expected heterozygosity at these loci ranges from 0.62 to 0.91 in the cotton bollworm. The observed allele numbers varies from 4 to 12 in the limited number of individuals tested. Although a large proportion of cloned microsatellite sequences are present in multi-copy in the cotton bollworm genome, the overwhelming majority of the finalized polymorphic diallelic loci are tri-nucleotide microsatellites - an unexpected outcome, which should facilitate subsequent genotyping analysis.     

Successful development of microsatellite markers in a challenging species: the horizontal borer Austroplatypus incompertus (Coleoptera: Curculionidae)

The analysis of microsatellite loci has allowed significant advances in evolutionary biology and pest management. However, until very recently, the potential benefits have been compromised by the high costs of developing these neutral markers. High-throughput sequencing provides a solution to this problem. We describe the development of 13 microsatellite markers for the eusocial ambrosia beetle, Austroplatypus incompertus, a significant pest of forests in southeast Australia. The frequency of microsatellite repeats in the genome of A. incompertus was determined to be low, and previous attempts at microsatellite isolation using a traditional genomic library were problematic. Here, we utilised two protocols, microsatellite-enriched genomic library construction and high-throughput 454 sequencing and characterised 13 loci which were polymorphic among 32 individuals. Numbers of alleles per locus ranged from 2 to 17, and observed and expected heterozygosities from 0.344 to 0.767 and from 0.507 to 0.860, respectively. These microsatellites have the resolution required to analyse fine-scale colony and population genetic structure. Our work demonstrates the utility of next-generation 454 sequencing as a method for rapid and cost-effective acquisition of microsatellites where other techniques have failed, or for taxa where marker development has historically been both complicated and expensive.     

Isolation and characterization of variable (GT)n repetitive sequences from Atlantic salmon, Salmo salar L.

Microsatellites are islands of long repeats of mono-, di- or trinucleotides evenly distributed in the eukaryotic genome with an average distance of 50–100 kb. They display a high degree of length polymorphism and heterozygosity at individual loci, making them highly useful as markers in the development of genomic maps of eukaryotes. In the present work, we examined the dinucleotide repeat motif (dG-dT)_n in the Atlantic salmon, Salmo salar L., genome. The frequency of (dG-dT)_n microsatellites in salmon correlates well with earlier published estimations. Cloning and sequencing of 45 salmon microsatellites revealed perfect and imperfect repeats, but no compound microsatellites. The distribution of number of repeat units in salmon microsatellites differ significantly from that of higher vertebrates. Salmon tends to have more long repeat stretches and less intermediate length repeats.     

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.     

Identification of 10 882 porcine microsatellite sequences and virtual mapping of 4528 of these sequences

A total of 10 882 porcine microsatellite repeats were identified in genomic shotgun sequences from the Sino-Danish Pig Genome Sequencing Consortium (http://www.piggenome.dk). Of these, 4528 microsatellites were placed on a pig-human comparative map by blast analysis of porcine sequences against the human genome (blast cut-off threshold =1 x 10(-5)). All microsatellite sequences placed on the comparative map are accessible at http://www.animalgenome.org/QTLdb/pig.html. These sequences increase the number of identified microsatellites in the porcine genome by several orders of magnitude. They are a new resource of microsatellite sequences for generating markers to be used in linkage studies and in fine mapping and positional cloning of quantitative trait loci.