Isolation and characterization of microsatellite markers for Viola mirabilis (Violaceae) using a next‐generation sequencing platform

We isolated and characterized microsatellite loci in Viola mirabilis (Violaceae), an endangered species from South Korea. Twenty‐three polymorphic microsatellite loci were developed and tested in Korean, Chinese and Japanese populations. The number of alleles per locus varied from two to eight. The observed and expected heterozygosities within the three populations were 0.000–0.625 and 0.469–0.695, respectively. A total of six loci in the Korean population, one locus in the Chinese population and seven loci in the Japanese population deviated from Hardy–Weinberg equilibrium. We expect that these newly developed microsatellite markers will contribute to understanding the phylogeography and population genetics of V. mirabilis, which will aid in developing conservation strategies for this species.     

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.     

Isolation and characterization of microsatellite loci in Greater Sage‐Grouse (Centrocercus urophasianus)

Primers for five polymorphic microsatellite loci were developed for Greater Sage‐Grouse (Centrocercus urophasianus) using an enrichment/detection protocol. The high level of polymorphism (nine to 33 alleles) suggests that these loci will be applicable for investigating mating systems and paternity analysis as well as population genetics. Cross‐species amplification was successful for each locus in at least two other galliform species.     

Isolation and characterization of microsatellite loci in Cylindrocladium parasiticum

Twelve polymorphic microsatellite markers were developed for Cylindrocladium parasiticum, a plant pathogen with a wide host range and the causal agent of the serious disease of peanuts (Arachis hypogaea) known as cylindrocladium black rot (CBR). Polymorphism was evaluated on 17 isolates from different hosts and regions. Each locus had between two and six alleles. Cross‐species transferability tested for 20 other Cylindrocladium species found amplification only in Cylindrocladium pacificum, which is phylogenetically closely related to C. parasiticum.     

Isolation of microsatellite loci from spotted gum (Corymbia variegata), and cross‐species amplification in Corymbia and Eucalyptus

Corymbia variegata (spotted gum) is an important commercial hardwood timber species in Australia. Fourteen polymorphic microsatellite loci were isolated from C. variegata, with 3–5 alleles amplified in three individuals examined. Cross‐species amplification in Corymbia was successful for all primer pairs, while 10 loci (71%) were successfully transferred to at least one species in the closely related genus Eucalyptus.     

Isolation and characterization of microsatellite loci in the Bang's leaf‐nosed bat Hipposideros turpis

The Bang's leaf‐nose bat, Hipposideros turpis, is an endangered cave‐dwelling species inhabiting the southwesternmost islands of Japan. We isolated six dinucleotide microsatellite markers from the partial genomic library of the bat, and examined their allelic variation using a sample (N = 33) from the largest colony in Japan. All the loci showed a moderate allelic variation ranging from two to eight alleles, with the observed heterozygosities from 0.33 to 0.73, and conformed to Hardy–Weinberg expectations. The present microsatellite markers will be useful in assessing population genetic structure and gene flow among populations of this species.     

Polymorphic tetranucleotide microsatellite DNA loci from the southern dusky salamander (Desmognathus auriculatus)

We describe polymerase chain reaction (PCR) primers and amplification conditions for seven tetranucleotide microsatellite DNA loci isolated from the southern dusky salamander (Desmognathus auriculatus). Primers were tested on 16 individuals from one population in Aiken County, South Carolina. We detected an average of 6.57 alleles per locus, an observed heterozygosity range of 0.44–0.94, and high polymorphic information contents (mean of 0.68).     

Isolation and characterization of microsatellite loci in Cylindrocladium pauciramosum

Ten polymorphic microsatellite markers were developed for Cylindrocladium pauciramosum, a plant pathogen with a wide host range, which poses a serious problem in South African Eucalyptus nurseries. Polymorphism was evaluated on 43 isolates collected from Colombia and South Africa. Each locus had between three and six alleles. Testing for random mating showed multilocus equilibrium for a population of 40 isolates from a South African forestry nursery. Cross‐species transferability tested for 19 other Cylindrocladium species found amplification only in C. spathulatum, which is phylogenetically closely related to C. pauciramosum.     

Rapid,economical single‐nucleotide polymorphism and microsatellite discovery based on de novo assembly of a reduced representation genome in a non‐model organism: a case study of Atlantic cod Gadus morhua

By combining next‐generation sequencing technology (454) and reduced representation library (RRL) construction, the rapid and economical isolation of over 25 000 potential single‐nucleotide polymorphisms (SNP) and >6000 putative microsatellite loci from c. 2% of the genome of the non‐model teleost, Atlantic cod Gadus morhua from the Celtic Sea, south of Ireland, was demonstrated. A small‐scale validation of markers indicated that 80% (11 of 14) of SNP loci and 40% (6 of 15) of the microsatellite loci could be amplified and showed variability. The results clearly show that small‐scale next‐generation sequencing of RRL genomes is an economical and rapid approach for simultaneous SNP and microsatellite discovery that is applicable to any species. The low cost and relatively small investment in time allows for positive exploitation of ascertainment bias to design markers applicable to specific populations and study questions.     

Isolation and characterization of microsatellite loci in Fragaria

This study reports the development and characterization of 20 microsatellite primer pairs in wild strawberry Fragaria vesca. One hundred primers were obtained from an AC‐enriched library developed in the cultivar ‘Ilaria’. A set of eight F. vesca genotypes was used to detect the polymorphism resulting in an average of 7.0 alleles, an average observed heterozygosity of 0.32 and an average expected heterozygosity of 0.73. Nineteen (95%) of the primers also amplified the cultivated octoploid strawberry Fragaria×ananassa.     

Development and characterization of 24 microsatellite markers in Primula tosaensis,an endangered primrose,using MiSeq

Primula tosaensis (Primulaceae) is an endangered primrose endemic to Japan. In this study, 24 novel microsatellite markers were developed using Illumina MiSeq sequencing to facilitate conservation of this endangered species. The genetic diversity and polymorphisms of these novel markers were measured in 32 individuals from a wild P. tosaensis population. The number of alleles and expected heterozygosities ranged from 2 to 5 (mean = 2.8) and from 0.119 to 0.724 (mean = 0.395), respectively. All loci were in Hardy–Weinberg equilibrium. The markers developed in this study will provide a powerful and practical tool for investigating the population structure and genetic diversity of P. tosaensis.     

Fifty‐three polymorphic microsatellite loci in the chestnut blight fungus,Cryphonectria parasitica

We report on 53 microsatellite loci for use in population genetic or linkage mapping studies in Cryphonectria parasitica. In 40 isolates collected from throughout the Northern Hemisphere, the number of alleles per locus ranged from two to 14 (mean 5.17) with gene diversity values ranging from 0.049 to 0.859 (mean 0.437). Samples from Asia were more diverse than those from Europe and North America. Most of the markers (48 of 53) were developed from an expressed sequence tag library, and hence, offer the opportunity to examine population structure or provide genome location information for specific expressed genes vs. anonymous genomic regions.     

Displacement of native white‐spotted charr Salvelinus leucomaenis by non‐native brown trout Salmo trutta after resolution of habitat fragmentation by a migration barrier

After resolution of habitat fragmentation by an erosion‐control dam, non‐native brown trout Salmo trutta invaded the upstream side of the dam and displaced native white‐spotted charr Salvelinus leucomaenis in Monbetsu stream, Hokkaido, northern Japan.     

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.     

Identification and characterization of (GA/CT) n - microsatellite loci from Quercus petraea

In this study a size selected genomic library from Quercus petraea was screened for (GA/CT)_n-microsatellite sequences. The resulting loci were analysed by PCR for their usefulness as molecular markers in Q. petraea and Q. robur. 17 out of 52 tested primer pairs resulted in the amplification of a polymorphic single-locus pattern. The number of alleles found per locus varied from 6 to 16. Combining the genetic variation observed for the characterized loci provides a unique genotype for all the individuals tested. Using intraspecific controlled crosses of Q. robur trees Mendelian inheritance could be shown for five loci.     

Comparing High‐throughput Platforms for Sequencing the V4 Region of SSU‐rDNA in Environmental Microbial Eukaryotic Diversity Surveys

High‐throughput sequencing platforms are continuing to increase resulting read lengths, which is allowing for a deeper and more accurate depiction of environmental microbial diversity. With the nascent Reagent Kit v3, Illumina MiSeq now has the ability to sequence the eukaryotic hyper‐variable V4 region of the SSU‐rDNA locus with paired‐end reads. Using DNA collected from soils with analyses of strictly‐ and nearly identical amplicons, here we ask how the new Illumina MiSeq data compares with what we can obtain with Roche/454 GS FLX with regard to quantity and quality, presence and absence, and abundance perspectives. We show that there is an easy qualitative transition from the Roche/454 to the Illumina MiSeq platforms. The ease of this transition is more nuanced quantitatively for low‐abundant amplicons, although estimates of abundances are known to also vary within platforms.     

Isolation and characterization of microsatellite markers for Achyranthes bidentata (Amaranthaceae) using next-generation sequencing platform

Achyranthes bidentata Blume (Amaranthaceae) is a perennial herb that is widely distributed in India, Java, China, and Japan. The natural resources of A. bidentata in its geo-authentic product area have rapidly declined in recent years because of the over-collection of its roots. To devise adequate conservation and management strategies for this species, its genetic diversity and population structure should be characterized. Roche 454 pyrosequencing combined with magnetic bead enrichment was used to develop microsatellite markers for A. bidentata. A total of 903 microsatellite loci were identified from 42,004 individual sequence reads. One hundred microsatellite loci were selected to test the primer amplification efficiency across 16 individuals from two A. bidentata populations. Of these tested markers, 8 yielded polymorphic amplification products, 29 yielded single alleles. For polymorphic primer pairs, the number of alleles per locus ranged from 2 to 4, with an average of 2.75. The observed and expected heterozygosities ranged from 0.353 to 0.671 and 0.250 to 0.938, respectively. The inbreeding coefficient varied from −0.692 to 0.627. This set of markers will provide useful tools for examining genetic diversity and population structure, and aid in better understanding of the conservation of A. bidentata.     

Isolation and characterization of 12 tetranucleotide repeat microsatellite loci from the green‐backed tit (Parus monticolus)

From a partial genomic library enriched for GATA short tandem repeats, we developed 12 polymorphic microsatellite loci from the green‐backed tit (Parus monticolus). We characterized these loci by genotyping 30 adult individuals with unknown relationship. The number of alleles ranged from four to 17 per locus (mean = 9.3 alleles) and the observed heterozygosity for each locus ranged from 0.633 to 0.933 (mean = 0.789). All loci conformed to Hardy–Weinberg expectations. Four of 66 possible pairwise comparisons between loci showed significant gametic disequilibrium.