Characterization of 42 polymorphic microsatellite loci in Mimulus ringens (Phrymaceae) using Illumina sequencing

• Premise of the study: Microsatellite markers were isolated and characterized in Mimulus ringens (Phrymaceae), a herbaceous wetland perennial, to facilitate studies of mating patterns and population genetic structure. • Methods and Results: A total of 42 polymorphic loci were identified from a sample of 24 individuals from a single population in Ohio, USA. The number of alleles per locus ranged from two to nine, and median observed heterozygosity was 0.435. • Conclusions: This large number of polymorphic loci will enable researchers to quantify male fitness, patterns of multiple paternity, selfing, and biparental inbreeding in large natural populations of this species. These markers will also permit detailed study of fine-scale patterns of genetic structure.     

Isolation and characterization of Ligustrum micranthum (Oleaceae) microsatellite loci using paired‐end Illumina reads

Twenty‐six microsatellite loci were developed and characterized for Ligustrum micranthum, a species endemic to the Ogasawara Islands, Japan. The genetic structure of this species must be clarified in order to restore the island's ecosystem. A total of 8511 primer pairs were designed from de novo sequencing. Of the 48 primer pairs selected, amplification and polymorphisms were tested using one population each from the Chichijima and Hahajima Islands of the Ogasawara Islands. Twenty‐six microsatellite loci were successfully amplified and the number of alleles for these loci ranged from five to 31 per locus, and the mean expected heterozygosities were 0.858 and 0.849, respectively. No significant deviation from the Hardy–Weinberg equilibrium was observed in either population, and no significant linkage disequilibrium was detected between any locus pair. The microsatellite loci reported in this study can be used in future studies to evaluate the genetic structure and mating system of L. micranthum.     

Characterization of novel microsatellite loci for red clover (Trifolium pratense L.) from enriched genomic libraries

Twenty‐seven polymorphic microsatellite markers were isolated from red clover (Trifolium pratense). Allelic variability and cross‐species amplification were assessed on 24 red clover and eight white clover (Trifolium repens) genotypes. The number of alleles detected in red clover ranged from two to 25. Observed and expected heterozygosities were high with average values of 0.71 and 0.88, respectively. Five of the 27 loci were also successfully amplified from white clover, where two to 13 alleles were detected. These highly polymorphic microsatellite loci provide powerful tools for population genetic studies as well as for marker‐assisted selection in this important forage legume species.     

Development and characterization of 29 microsatellite markers for Ligumia nasuta (Bivalvia: Unionidae) using an Illumina sequencing approach

Ligumia nasuta (Say, 1817; Eastern Pondmussel) is an imperiled freshwater mussel (Unionidae) in eastern North America. Population declines in the Laurentian Great Lakes resulting from the introduction of dreissenid mussels and habitat destruction in the 20th Century have greatly reduced and limited its distribution. To properly inform restoration and management efforts for L. nasuta, fine-scale genetic analyses must be performed on the remnant populations. This study used Illumina paired-end shotgun sequencing to identify potential microsatellite loci for L. nasuta, utilizing two samples to develop the Illumina paired-end shotgun library. Forty-eight primer pairs were tested on the remaining 24 samples. Twenty-nine of the 48 microsatellite primer sets screened were successfully amplified using 24 L. nasuta samples collected from the Great Lakes watershed. The estimated fragment size ranged from 167 to 445 base-pairs (bp) and the number of alleles per locus varied between 5 and 16 (mean = 9.7). Only five of the loci deviated significantly from Hardy–Weinberg expectations after Bonferroni corrections. The development of these new microsatellite loci will greatly facilitate future genetic studies on L. nasuta.     

Mapping of rabbit microsatellite markers using chromosome-specific libraries

Recently, rabbit microsatellite markers were developed from a chromosome 1-specific library, and seven new markers were incorporated into the genetic map of the rabbit. We have now developed microsatellite markers from chromosomes 3-, 5-, 6-, 7-, 12-, and 19-specific libraries. Linkage analysis was performed with use of these new markers, five recently physically mapped markers (PMP2, TCRB, ALOX15, MT1, and Sol33), microsatellite markers located in the HBA gene cluster, the MHC region and FABP6 gene, and seven biochemical markers (Es-1, Es-3, Est-2, Est-4, Est-6, Est-X, and HP). This analysis enabled us to verify the specificity of the libraries and to determine the position and orientation of the linkage groups on the chromosomes.     

32 species validation of a new Illumina paired-end approach for the development of microsatellites

Development and optimization of novel species-specific microsatellites, or simple sequence repeats (SSRs) remains an important step for studies in ecology, evolution, and behavior. Numerous approaches exist for identifying new SSRs that vary widely in terms of both time and cost investments. A recent approach of using paired-end Illumina sequence data in conjunction with the bioinformatics pipeline, PAL_FINDER, has the potential to substantially reduce the cost and labor investment while also improving efficiency. However, it does not appear that the approach has been widely adopted, perhaps due to concerns over its broad applicability across taxa. Therefore, to validate the utility of the approach we developed SSRs for 32 species representing 30 families, 25 orders, 11 classes, and six phyla and optimized SSRs for 13 of the species. Overall the IPE method worked extremely well and we identified 1000s of SSRs for all species (mean = 128,485), with 17% of loci being potentially amplifiable loci, and 25% of these met our most stringent criteria designed to that avoid SSRs associated with repetitive elements. Approximately 61% of screened primers yielded strong amplification of a single locus.     

Cosmix-plexing: a novel recombinatorial approach for evolutionary selection from combinatorial libraries

The efficiency of existing combinatorial biological library methods has been moderate in terms of the success rates, the affinities of the ligands selected and the time and effort involved in trying to optimize the initial leads. Although mimicking natural evolution, existing strategies take little notice of the importance of recombination within a selected population to generate increased diversity. We present an overview of our recent progress which has resulted in the successful development of such a strategy, which we designate cosmix-plexing. We incorporate recombination as a central feature in obtaining high success rates and high affinities, even for short monomer peptides, in a very short time. The method uses type II restriction enzymes to re-assort small hypervariable DNA cassettes from an intermediate pre-selected population (e.g. from a phagemid display library), while maintaining the original open-reading frame. Since, in the naive library, each cassette contains all possible combinations of the polypeptide sequences it encodes, much longer regions can be optimized than was possible with methods which depend on a simple selection from the naive library. Short peptides can now be rapidly selected, which exhibit the same, or higher, specificity and affinity for a defined target molecule, than (say) an antibody or even the natural ligand.     

Characterization of new Schistosoma mansoni microsatellite loci in sequences obtained from public DNA databases and microsatellite enriched genomic libraries

In the last decade microsatellites have become one of the most useful genetic markers used in a large number of organisms due to their abundance and high level of polymorphism. Microsatellites have been used for individual identification, paternity tests, forensic studies and population genetics. Data on microsatellite abundance comes preferentially from microsatellite enriched libraries and DNA sequence databases. We have conducted a search in GenBank of more than 16,000 Schistosoma mansoni ESTs and 42,000 BAC sequences. In addition, we obtained 300 sequences from CA and AT microsatellite enriched genomic libraries. The sequences were searched for simple repeats using the RepeatMasker software. Of 16,022 ESTs, we detected 481 (3%) sequences that contained 622 microsatellites (434 perfect, 164 imperfect and 24 compounds). Of the 481 ESTs, 194 were grouped in 63 clusters containing 2 to 15 ESTs per cluster. Polymorphisms were observed in 16 clusters. The 287 remaining ESTs were orphan sequences. Of the 42,017 BAC end sequences, 1,598 (3.8%) contained microsatellites (2,335 perfect, 287 imperfect and 79 compounds). The 1,598 BAC end sequences 80 were grouped into 17 clusters containing 3 to 17 BAC end sequences per cluster. Microsatellites were present in 67 out of 300 sequences from microsatellite enriched libraries (55 perfect, 38 imperfect and 15 compounds). From all of the observed loci 55 were selected for having the longest perfect repeats and flanking regions that allowed the design of primers for PCR amplification. Additionally we describe two new polymorphic microsatellite loci.     

Genome scan for loci predisposing to anxiety disorders using a novel multivariate approach: strong evidence for a chromosome 4 risk locus

We conducted a 10-centimorgan linkage autosomal genome scan in a set of 19 extended American pedigrees (219 subjects) ascertained through probands with panic disorder. Several anxiety disorders--including social phobia, agoraphobia, and simple phobia--in addition to panic disorder segregate in these families. In previous studies of this sample, linkage analyses were based separately on each of the individual categorical affection diagnoses. Given the substantial comorbidity between anxiety disorders and their probable shared genetic liability, it is clear that this method discards a considerable amount of information. In this article, we propose a new approach that considers panic disorder, simple phobia, social phobia, and agoraphobia as expressions of the same multivariate, putatively genetically influenced trait. We applied the most powerful multipoint Haseman-Elston method, using the grade of membership score generated from a fuzzy clustering of these phenotypes as the dependent variable in Haseman-Elston regression. One region on chromosome 4q31-q34, at marker D4S413 (with multipoint and single-point nominal P values < .00001), showed strong evidence of linkage (genomewide significance at P<.05). The same region is known to be the site of a neuropeptide Y receptor gene, NPY1R (4q31-q32), that was recently connected to anxiolytic-like effects in rats. Several other regions on four chromosomes (4q21.21-22.3, 5q14.2-14.3, 8p23.1, and 14q22.3-23.3) met criteria for suggestive linkage (multipoint nominal P values < .01). Family-by-family analysis did not show any strong evidence of heterogeneity. Our findings support the notion that the major anxiety disorders, including phobias and panic disorder, are complex traits that share at least one susceptibility locus. This method could be applied to other complex traits for which shared genetic-liability factors are thought to be important, such as substance dependencies.     

New polymorphic microsatellite loci for Theobroma cacao: isolation and characterization of microsatellites from enriched genomic libraries

Seventeen polymorphic microsatellite markers were isolated from enriched genomic libraries for Theobroma cacao, providing additional tools for studying the genetic diversity and map saturation of this species. These markers were characterized in 32 accessions of the T. cacao germplasm collection from the Centro de Pesquisas do Cacau. The number of alleles at each locus varied from 2 to 8, with an average of 4.41 alleles per locus. The polymorphism information content varied from 0.060 to 0.695, with an average of 0.333. The markers characterized in this study will be employed in map saturation studies and diversity assessments of cacao genotypes.     

High-resolution typing of Toxoplasma gondii using microsatellite loci.

High-resolution typing of Toxoplasma gondii is essential to understand the effect of genetic differences among strains on the variation in disease manifestation and transmission patterns. Current typing methods discern 3 lineages with minimal within-lineage variation. Described here are 6 new variable loci. These loci, including a minisatellite and 5 microsatellites, were more polymorphic than allozymes, restriction fragment length polymorphisms, and sequence variation in introns. Most importantly, these loci revealed, for the first time, substantial within-lineage variation that was over 6-fold higher than that detected by other markers. Genotyping at these loci facilitates classification of isolates beyond the lineage level.     

The effects of read length,quality and quantity on microsatellite discovery and primer development: from Illumina to PacBio

The advent of next‐generation sequencing (NGS) technologies has transformed the way microsatellites are isolated for ecological and evolutionary investigations. Recent attempts to employ NGS for microsatellite discovery have used the 454, Illumina, and Ion Torrent platforms, but other methods including single‐molecule real‐time DNA sequencing (Pacific Biosciences or PacBio) remain viable alternatives. We outline a workflow from sequence quality control to microsatellite marker validation in three plant species using PacBio circular consensus sequencing (CCS). We then evaluate the performance of PacBio CCS in comparison with other NGS platforms for microsatellite isolation, through simulations that focus on variations in read length, read quantity and sequencing error rate. Although quality control of CCS reads reduced microsatellite yield by around 50%, hundreds of microsatellite loci that are expected to have improved conversion efficiency to functional markers were retrieved for each species. The simulations quantitatively validate the advantages of long reads and emphasize the detrimental effects of sequencing errors on NGS‐enabled microsatellite development. In view of the continuing improvement in read length on NGS platforms, sequence quality and the corresponding strategies of quality control will become the primary factors to consider for effective microsatellite isolation. Among current options, PacBio CCS may be optimal for rapid, small‐scale microsatellite development due to its flexibility in scaling sequencing effort, while platforms such as Illumina MiSeq will provide cost‐efficient solutions for multispecies microsatellite projects.     

Isolation and characterisation of di and tri nucleotide microsatellite loci in Rosmarinus officinalis (Lamiaceae), using enriched genomic libraries

An enrichment protocol was used to isolate and characterise microsatellite loci in Rosmarinus officinalis, a Mediterranean chamephyte. Twelve microsatellite loci were characterised and amplified a total of 117 alleles in a sample of 30 individuals from one population, with an average of 9.75 alleles per locus. Observed heterozygosities ranged from 0.333 to 0.900. Cross-species transferability was also assayed in the two other species of the genus. The cumulated probabilities of exclusion for paternity and parentage of the 12 loci were of 0.999971 and 1, respectively, supporting the usefulness of these microsatellite loci for parentage analyses. Nine out of 12 microsatellite loci amplified in the two species and were polymorphic detecting a total of 49 and 45 in R. eriocalyx and R. tomentosus, respectively. Twenty-two alleles were exclusive of R. eriocalyx and 12 of R. tomentosus, additionally, three alleles were shared between these two species but were otherwise absent in the analysed individuals of R. officinalis. In total, this set of markers amplified 154 different microsatellite alleles, supporting their usefulness to conduct population genetic, reproductive biology and hybridisation studies in Rosmarinus.     

Revealing environmentally driven population dynamics of an Arctic diatom using a novel microsatellite PoolSeq barcoding approach

Ecological stability under environmental change is determined by both interspecific and intraspecific processes. Particularly for planktonic microorganisms, it is challenging to follow intraspecific dynamics over space and time. We propose a new method, microsatellite PoolSeq barcoding (MPB), for tracing allele frequency changes in protist populations. We successfully applied this method to experimental community incubations and field samples of the diatom Thalassiosira hyalina from the Arctic, a rapidly changing ecosystem. Validation of the method found compelling accuracy in comparison with established genotyping approaches within different diversity contexts. In experimental and environmental samples, we show that MPB can detect meaningful patterns of population dynamics, resolving allelic stability and shifts within a key diatom species in response to experimental treatments as well as different bloom phases and years. Through our novel MPB approach, we produced a large dataset of populations at different time-points and locations with comparably little effort. Results like this can add insights into the roles of selection and plasticity in natural protist populations under stable experimental but also variable field conditions. Especially for organisms where genotype sampling remains challenging, MPB holds great potential to efficiently resolve eco-evolutionary dynamics and to assess the mechanisms and limits of resilience to environmental stressors.     

Ten novel microsatellite loci characterized for a remarkably widespread fish: Galaxias maculatus (Galaxiidae)

Ten polymorphic microsatellite markers (five tetra‐, one compound tetra‐, one octa‐ and three dinucleotides) were isolated and characterized for Galaxias maculatus, a fish species widely distributed in the Southern Hemisphere. Markers were tested in 89 individual samples from a single location and the number of alleles ranged between 2 and 28. Observed and expected heterozygosities ranged from 0.103 to 0.910 and 0.098 to 0.935 respectively. No evidence was detected for either linkage disequilibrium (P‐values > 0.05 for each locus pair) or deviations from HWE (P‐values > 0.05 for every loci).