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.     

Multiplexed microsatellite markers for the genetic analysis of Eucalyptus leucoxylon (Myrtaceae) and their utility for ecological and breeding studies in other eucalyptus species

Eucalyptus leucoxylon is a widespread woodland tree species found in southeastern Australia that has suffered from, and continues to be, threatened by the impacts of habitat clearance and degradation. Populations now consist predominantly of scattered individuals, and their conservation status is of increasing concern. We report the development and characterization of a set of eight highly polymorphic microsatellite loci for E. leucoxylon. The loci can be amplified in three PCR multiplexes and electrophoresed in a single lane, allowing rapid throughput of large numbers of samples. A total of 111 alleles were detected in 68 individuals with an average of 12.3 alleles per locus, a mean expected heterozygosity of 0.83, and a mean observed heterozygosity of 0.72. The combined probabilities of identity and probabilities of paternity exclusion allow an extremely precise level of individual identification, indicating that these microsatellite markers will be ideal for population genetic and parentage-type studies in E. leucoxylon. The markers also exhibited an average of 76% conservation within the subgenus Symphyomyrtus, to which E. leucoxylon belongs, and 53% conservation across other subgenera of Eucalyptus, demonstrating the potential of these markers in ecological and breeding studies in a wide range of Eucalyptus species.     

Lessons learned from microsatellite development for nonmodel organisms using 454 pyrosequencing

Microsatellites, also known as simple sequence repeats (SSRs), are among the most commonly used marker types in evolutionary and ecological studies. Next Generation Sequencing techniques such as 454 pyrosequencing allow the rapid development of microsatellite markers in nonmodel organisms. 454 pyrosequencing is a straightforward approach to develop a high number of microsatellite markers. Therefore, developing microsatellites using 454 pyrosequencing has become the method of choice for marker development. Here, we describe a user friendly way of microsatellite development from 454 pyrosequencing data and analyse data sets of 17 nonmodel species (plants, fungi, invertebrates, birds and a mammal) for microsatellite repeats and flanking regions suitable for primer development. We then compare the numbers of successfully lab‐tested microsatellite markers for the various species and furthermore describe diverse challenges that might arise in different study species, for example, large genome size or nonpure extraction of genomic DNA. Successful primer identification was feasible for all species. We found that in species for which large repeat numbers are uncommon, such as fungi, polymorphic markers can nevertheless be developed from 454 pyrosequencing reads containing small repeat numbers (five to six repeats). Furthermore, the development of microsatellite markers for species with large genomes was also with Next Generation Sequencing techniques more cost and time‐consuming than for species with smaller genomes. In this study, we showed that depending on the species, a different amount of 454 pyrosequencing data might be required for successful identification of a sufficient number of microsatellite markers for ecological genetic studies.     

New methods to identify conserved microsatellite loci and develop primer sets of high cross-species utility - as demonstrated for birds

We have developed a new approach to create microsatellite primer sets that have high utility across a wide range of species. The success of this method was demonstrated using birds. We selected 35 avian EST microsatellite loci that had a high degree of sequence homology between the zebra finch Taeniopygia guttata and the chicken Gallus gallus and designed primer sets in which the primer bind sites were identical in both species. For 33 conserved primer sets, on average, 100% of loci amplified in each of 17 passerine species and 99% of loci in five non-passerine species. The genotyping of four individuals per species revealed that 24-76% (mean 48%) of loci were polymorphic in the passerines and 18-26% (mean 21%) in the non-passerines. When at least 17 individuals were genotyped per species for four Fringillidae finch species, 71-85% of loci were polymorphic, observed heterozygosity was above 0.50 for most loci and no locus deviated significantly from Hardy-Weinberg proportions. This new set of microsatellite markers is of higher cross-species utility than any set previously designed. The loci described are suitable for a range of applications that require polymorphic avian markers, including paternity and population studies. They will facilitate comparisons of bird genome organization, including genome mapping and studies of recombination, and allow comparisons of genetic variability between species whilst avoiding ascertainment bias. The costs and time to develop new loci can now be avoided for many applications in numerous species. Furthermore, our method can be readily used to develop microsatellite markers of high utility across other taxa.     

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.     

Thousands of microsatellite loci from the venomous coralsnake Micrurus fulvius and variability of select loci across populations and related species

Studies of population genetics increasingly use next‐generation DNA sequencing to identify microsatellite loci in nonmodel organisms. There are, however, relatively few studies that validate the feasibility of transitioning from marker development to experimental application across populations and species. North American coralsnakes of the Micrurus fulvius species complex occur in the United States and Mexico, and little is known about their population structure and phylogenetic relationships. This absence of information and population genetics markers is particularly concerning because they are highly venomous and have important implications on human health. To alleviate this problem in coralsnakes, we investigated the feasibility of using 454 shotgun sequences for microsatellite marker development. First, a genomic shotgun library from a single individual was sequenced (approximately 7.74 megabases; 26 831 reads) to identify potentially amplifiable microsatellite loci (PALs). We then hierarchically sampled 76 individuals from throughout the geographic distribution of the species complex and examined whether PALs were amplifiable and polymorphic. Approximately half of the loci tested were readily amplifiable from all individuals, and 80% of the loci tested for variation were variable and thus informative as population genetic markers. To evaluate the repetitive landscape characteristics across multiple snakes, we also compared microsatellite content between the coralsnake and two other previously sampled snakes, the venomous copperhead (Agkistrodon contortrix) and Burmese python (Python molurus).     

Design and implementation of degenerate microsatellite primers for the mammalian clade

Microsatellites are popular genetic markers in molecular ecology, genetic mapping and forensics. Unfortunately, despite recent advances, the isolation of de novo polymorphic microsatellite loci often requires expensive and intensive groundwork. Primers developed for a focal species are commonly tested in a related, non-focal species of interest for the amplification of orthologous polymorphic loci; when successful, this approach significantly reduces cost and time of microsatellite development. However, transferability of polymorphic microsatellite loci decreases rapidly with increasing evolutionary distance, and this approach has shown its limits. Whole genome sequences represent an under-exploited resource to develop cross-species primers for microsatellites. Here we describe a three-step method that combines a novel in silico pipeline that we use to (1) identify conserved microsatellite loci from a multiple genome alignments, (2) design degenerate primer pairs, with (3) a simple PCR protocol used to implement these primers across species. Using this approach we developed a set of primers for the mammalian clade. We found 126,306 human microsatellites conserved in mammalian aligned sequences, and isolated 5,596 loci using criteria based on wide conservation. From a random subset of ~1000 dinucleotide repeats, we designed degenerate primer pairs for 19 loci, of which five produced polymorphic fragments in up to 18 mammalian species, including the distinctly related marsupials and monotremes, groups that diverged from other mammals 120-160 million years ago. Using our method, many more cross-clade microsatellite loci can be harvested from the currently available genomic data, and this ability is set to improve exponentially as further genomes are sequenced.     

High degree of transferability of 86 newly developed zebra finch EST-linked microsatellite markers in 8 bird species

High-resolution analysis for population genetic and functional studies requires the use of large numbers of polymorphic markers. The recent increase of available genetic tools is facilitated by the use of publicly available expressed sequence tag (EST) sequence databases that are a valuable resource for identifying gene-linked markers. In the present study, we applied bioinformatics analyses to identify microsatellite markers present in EST sequences from a zebra finch (Taeniopgia guttata) EST database and we explore the success of cross-species amplification of EST-linked microsatellite markers in 7 passerine and 1 nonpasserine species. Eighty-six zebra finch EST-linked microsatellite loci were screened for polymorphism revealing a high amplification success rate and adequate levels of polymorphism (33.3-51%) for relatively closely related species, whereas success decreased in the most distantly related species to zebra finch. EST-linked microsatellites appear to be more highly transferable between taxa than anonymous microsatellites as they revealed higher amplification and polymorphism success between different families indicating that they will be a useful source of gene-linked polymorphic markers in a broad range of avian species.     

Nine novel microsatellite markers for the hog deer (Axis porcinus)

Hog deer (Axis porcinus) is listed as an endangered species in many countries and its taxonomic status remains ambiguous. In this study, we developed nine novel microsatellite markers using enrichment methods. These polymorphic microsatellite loci showed allele numbers of 2–3 with an average of 2.22 in a group of 26 individuals and observed and expected heterozygosity ranging from 0.044 to 0.619 (average 0.397) and 0.194 to 0.632 (average 0.433) respectively. Linkage disequilibrium was not detected. These markers could be used in the genetic study and taxonomic identification of this species.     

Transferability of microsatellite and sequence tagged site markers in Oryza species

The genus Oryza comprises 22 species which are potentially useful as a source of genetic variability that can be introgressed into the worldwide cultivated rice, Oryza sativa. Molecular markers are useful tools for monitoring gene introgressions and for detecting polymorphism among species. In this study, cross-amplification was estimated among 28 accessions of 16 Oryza species, representing the genomes AA, BB, CC, BBCC and CCDD, using 59 microsatellite (OG, OS and RM series) and 15 STS (Sequence Tagged Sites) markers. All markers amplified at least one Oryza species, indicating different levels of transferability across species. Markers based on microsatellite sequences amplified 37 % of the accessions, with an average of 6.58 alleles per locus and an average polymorphism information content (PIC) of 70 %. For STS markers, the amplification level was 53.3 %, and the average number of alleles and PIC values were 1.6 and 10 %, respectively. These Results showed that although the STS markers detected a reduced level of genetic diversity, the transferability was higher, indicating that they can be used for genetic analysis when evaluating less genetically related species of Oryza. Among the microsatellite markers, an analysis of species with an AA genome showed that the OG markers produced the highest level of polymorphic loci (54.6 %), followed by RM markers (48 %). Highly polymorphic and transferable molecular markers in Oryza can be useful for exploiting the genetic resources of this genus, for detecting allelic variants in loci associated with important agronomic traits, and for monitoring alleles introgressed from wild relatives to cultivated rice.     

Microsatellite markers for the Indian golden silkmoth, Antheraea assama (Saturniidae: Lepidoptera)

Antheraea assama, an economically important and scientifically unexplored Indian wild silkmoth, is unique among saturniid moths. For this species, a total of 87 microsatellite markers was derived from 35 000 expressed sequence tags and a microsatellite‐enriched sub‐genomic library. Forty individuals collected from Tura and West Garo Hills region of Northeast India were screened for each of these loci. Ten loci from expressed sequence tags and one from genomic library were found to be polymorphic. These microsatellite markers will be useful resources for population genetic studies of A. assama and other closely related species of saturniids. This is the first report on development of microsatellite markers for any saturniid species.     

A panel of 20 highly variable microsatellite polymorphisms in rhesus macaques (Macaca mulatta) selected for pedigree or population genetic analysis

This paper reports 20 new microsatellite loci that are highly polymorphic in rhesus macaques (Macaca mulatta). We screened known human microsatellite loci to identify markers that are polymorphic in rhesus macaques, and then selected specific loci that show substantial levels of heterozygosity and robust, reliable amplification. The 20 loci reported here were chosen to include one highly informative microsatellite from each rhesus monkey autosomal chromosome. Fourteen of the 20 polymorphisms are tetranucleotide repeats, and all can be analyzed using standard PCR and electrophoresis procedures. These new rhesus markers have an average of 15.5 alleles per locus and average heterozygosity of 0.83. This panel of DNA polymorphisms will be useful for a variety of different genetic analyses, including pedigree testing, paternity analysis, and population genetic studies. Many of these loci are also likely to be informative in other closely related Old World monkey species.     

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.     

Conserved Microsatellites in Ants Enable Population Genetic and Colony Pedigree Studies across a Wide Range of Species

Broadly applicable polymorphic genetic markers are essential tools for population genetics, and different types of markers have been developed for this purpose. Microsatellites have been employed as particularly polymorphic markers for over 20 years. However, PCR primers for microsatellite loci are often not useful outside the species for which they were designed. This implies that a new set of loci has to be identified and primers developed for every new study species. To overcome this constraint, we identified 45 conserved microsatellite loci based on the eight currently available ant genomes and designed primers for PCR amplification. Among these loci, we chose 24 for in-depth study in six species covering six different ant subfamilies. On average, 11.16 of these 24 loci were polymorphic and in Hardy-Weinberg equilibrium in any given species. The average number of alleles for these polymorphic loci within single populations of the different species was 4.59. This set of genetic markers will thus be useful for population genetic and colony pedigree studies across a wide range of ant species, supplementing the markers available for previously studied species and greatly facilitating the study of the many ant species lacking genetic markers. Our study shows that it is possible to develop microsatellite loci that are both conserved over a broad range of taxa, yet polymorphic within species. This should encourage researchers to develop similar tools for other large taxonomic groups.     

Isolation and characterization of 12 microsatellites from the black surfperch, Embiotoca jacksoni, a reef fish that lacks a pelagic larval phase

A set of 12 microsatellite markers was developed from Embiotoca jacksoni genomic DNA and tested for polymorphism using 64 individuals from two populations. All loci were polymorphic with a number of alleles ranging from two to 19 with expected heterozygosities ranging from 0.17 to 0.89. There was no evidence of linkage disequilibrium and all loci were in Hardy-Weinberg equilibrium, except for four loci in one population. High numbers of private alleles were consistent with strong population structure, and very limited dispersal. Six microsatellite markers successfully cross amplified and were polymorphic in closely related species, Embiotoca lateralis and Hyspurus caryi.     

Evolution of microsatellites in Arabis petraea and Arabis lyrata, outcrossing relatives of Arabidopsis thaliana

We examined microsatellite variation in two diploid, outcrossing relatives of Arabidopsis thaliana, Arabis petraea and Arabis lyrata. The primer sequences were derived from A. thaliana. About 50% (14 loci) of the A. thaliana primers could successfully amplify microsatellites in the related species. Analysis of microsatellite structure in the related species showed that there had been large changes in the microsatellites: there were large differences in repeat numbers and many of the A. thaliana simple repeats were shorter in the related species. For the loci we compared, the related species had a much lower level of variability at the microsatellites than Japanese wild populations of A. thaliana. This is presumably related to the different microsatellite structures, because allozyme data showed that the outcrossing relatives were highly polymorphic compared to other outcrossing herbaceous species. Use of microsatellites in assessing variability or phylogenetic relationships between different species requires caution, because changes in microsatellite structure may alter evolutionary rates.      

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.     

Microsatellite DNA markers for Australian geckos

Nine highly polymorphic microsatellite markers were developed for Gehyra variegata from an enriched library screened for tetranucleotide repeats. An average of 19.44 alleles per locus and an average observed heterozygosity of 0.86 were found in 81 samples from fragmented populations in the Western Australian wheatbelt. Different gecko species exhibit varying rates of extinction in fragmented landscapes and the genetic markers were developed to test whether these difference are due to different rates of dispersal. The loci were tested for their ability to cross amplify in 12 species of Australian Gekkonidae and Pygopodidae, in an attempt to identify species which might be amenable to population genetic analysis using these microsatellite markers.     

A panel of polymorphic microsatellite markers in Himalayan monal Lophophorus impejanus developed by cross-species amplification and their applicability in other Galliformes

Isolation and development of new microsatellite markers for any species is still labour-intensive and requires substantial inputs of time, money and expertise. Therefore, cross-species microsatellite amplification can be an effective way in obtaining microsatellite loci for closely related taxa in bird species. We have reported microsatellite loci for Himalayan monal for the first time. Fifteen microsatellite markers developed for chicken were cross-amplified in Himalayan monal. All the tested 15 microsatellite markers were polymorphic, with mean (± s.e.) allelic number of 4 ± 1.51, ranging 2–7 per locus. The observed heterozygosity in the population ranged between 0.285 and 0.714, with mean (± s.e.) of 0.499 ± 0.125, indicating considerable genetic variation in this population. While 12 loci conformed to Hardy–Weinberg equilibrium (P > 0.05), 3 loci, i.e. MCW0295, MCW0081, MCW0330 deviated from it (P < 0.05). No evidence for linkage disequilibrium was observed among pair of loci. Our study show that these 15 microsatellites loci could be employed in population genetic studies for Himalayan monal and their applicability in Jungle Bush Quail, Grey francolin and Kalij pheasant.