Development and characterization of polymorphic microsatellite markers for Neolitsea sericea using Illumina paired‐end draft sequencing data

Simple sequence repeat (SSR) markers were developed and characterized for Neolitsea sericea (Bl.) Koidz. (Lauraceae). Out of 196 designed primer pairs, a total of 144 pairs showed amplification, of which 44 had clear and stable chromatograms. Polymorphism of these 44 loci was tested using 32 individuals sampled from a single population of N. sericea. The number of alleles and the polymorphism information content varied from 3 to 12 and 0.271 to 0.853, respectively. A significant departure from the Hardy‐Weinberg equilibrium was observed in one of the 44 loci. These SSR markers are useful for population genetic studies and parentage analysis in N. sericea, which is one of the most common evergreen species in coastal Pinus thunbergii forests in central‐western Japan.     

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 tri‐ and tetra‐repeat microsatellite loci in the white‐spotted charr Salvelinus leucomaenis (Salmonidae)

Tri‐ and tetra‐motif repeat microsatellite marker loci were developed for the white‐spotted charr Salvelinus leucomaenis. The 454 pyrosequencing was used to discover repeat arrays, and eight microsatellite‐primer sets, available for the estimation of polymorphisms, were identified. The number of alleles in a wild population ranged from two to four and the observed and expected heterozygosities were 0·180–0·600 and 0·188–0·599, respectively.     

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.     

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.     

Analysis of genetic relatedness among Indian cattle (Bos indicus) using genotyping‐by‐sequencing markers

Genetic relatedness of 24 animals belonging to seven Indian cattle breeds was studied using high throughput genotyping‐by‐sequencing (GBS) markers. GBS produced 93.6 million reads with an average of about 3.9 million reads per animal. A total of 107 488 SNPs were identified in these individuals. When only one SNP per read was considered, a total of 60 261 SNPs representing independent reads were identified with an average SNP‐to‐SNP distance of 45 kb across the bovine reference genome. About 24% of the GBS‐SNP markers were more than 100 kb apart. Of these, 58 322 SNPs mapped to autosomes, 1645 to the X chromosome and 28 to the Y chromosome. The average SNP‐to‐SNP distance on the X chromosome was 91.3 kb, whereas on the Y chromosome it was 1546.4 kb. The minor allele frequency within the Indian cattle varied from 0.103 (Ongole) to 0.177 (Siri), whereas Holstein cattle had the lowest value of 0.089. This is the first application of GBS in cattle of South Asia. The baseline information generated in this study might prompt implementation of GBS in breeding of cattle belonging to this region.     

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.     

Development and characterization of 10 microsatellite markers for the semi-evergreen tree species, Ligustrum ovalifolium (Oleaceae)

Ligustrum ovalifolium is a semi-evergreen tree distributed in the western part of Japan and southern Korea. This species contains an insular endemic variety, L. ovalifolium var. pacificum; this variety occurs only on the Izu Islands located south of the Japanese mainland Honshu. We isolated 10 polymorphic microsatellite loci from the species, and characterized them for 21 individuals from a population of L. ovalifolium. The primers developed in this study yielded an average 12.2 alleles per locus and an average expected heterozygosity of 0.78. These markers will be powerful tools for studying the genetic differentiation within the species.     

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 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.     

Isolation and characterization of 10 microsatellite loci in Callicarpa subpubescens (Verbenaceae), an endemic species of the Bonin Islands

Ten microsatellite loci were isolated and characterized for Callicarpa subpubescens (Verbenaceae), an endemic tree species of the Bonin Islands. The observed number of alleles at each locus ranged from two to eight with an average of 4.9, and the expected heterozygosity ranged from 0.238 to 0.690 with an average of 0.483. All 10 loci were screened in cross-amplification tests for two other endemic Callicarpa species that also inhabit the Bonin Islands. All loci were successfully amplified in these species.     

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 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.     

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.     

Isolation and characterization of 16 polymorphic microsatellite loci for Euchiloglanis kishinouyei

A total of 16 novel polymorphic microsatellite marker loci were isolated from a genomic library of Euchiloglanis kishinouyei and further characterized using a sample from a wild population consisting of 40 individuals. The number of alleles among loci ranged from three to 17 and the observed and expected heterozygosities ranged from 0·030 to 0·950 and 0·163 to 0·980, respectively. The average polymorphic information content (PIC) of all loci was 0·450.     

Isolation and characterization of microsatellite loci in Hexagrammos otakii based on 2b-RAD method

In this study, we isolated 20 novel microsatellites loci associated with the growth of Hexagrammos otakii (Fat greenling) by using 2b-RAD sequencing method. The characteristics of 20 microsatellite loci were amplified in 105 H. otakii individuals which came from four different groups and tested by Capillary electrophoresis. The number of allele of the 20 microsatellite loci ranged from 8 to 26 with an average of 19.95. PIC value ranged from 0.1659 to 0.9227 with an average of 0.7555. Observed and expected heterozygosity varied from 0.0667 to 0.8571 and from 0.1710 to 0.9314, respectively. Highly polymorphic characteristics were observed in each microsatellite loci of the study except HO6. Further research showed that 10 microsatellite loci had transferability in Hexagrammos agrammus. In this study, high polymorphism and genetic diversity using the 15 polymorphic microsatellite loci suggest that they are suitable for investigating the fine-scale population structure, genetic relationship and further evaluating the artificial reproduction strategy of H. otakii.     

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.