Identification of microsatellite loci from Epimedium koreanum and cross‐species amplification in four species of Epimedium (Berberidaceae)

Nineteen polymorphic microsatellite markers were isolated and characterized from a trinucleotide enriched partial genomic library of Epimedium koreanum. The average allele number of these microsatellites was 5.9 per locus, ranging from two to 11. The observed heterozygosity (H_O) and expected heterozygosity (H_E) at the population level were 0.00–0.90 and 0.12–0.90, respectively. In addition, the results of cross‐species amplification of this set of microsatellite markers in four closely related Epimedium species, E. brevicornum, E. sagittatum, E. pubescens and E. wushanense, revealed that these microsatellite markers were useful for population genetic structure evaluation and genotype analysis of major Epimedium species that have been used as traditional Chinese medicines.     

Development of single sequence repeat markers for the ant Aphaenogaster senilis and cross-species amplification in A. iberica, A . gibbosa, A. subterranea and Messor maroccanus

Eight polymorphic microsatellite loci were isolated and characterized for Aphaenogaster senilis, a common ant species distributed in the Western Mediterranean. Characterization of 15 individuals form southern Spain showed moderate to high allelic diversity ranging from 2 to 9 alleles per locus. Cross-species tests on 10 individuals of A. iberica, A. gibbosa, A. subterranea and Messor maroccanus revealed successful amplification for most loci. This set of markers can be useful for population genetic studies and might even prove useful in other phylogenetically close species of the subfamily Myrmicinae.     

Isolation and characterization of microsatellite markers from the sweet passion fruit (Passiflora alata Curtis: Passifloraceae)

Passiflora alata is one of the commercialized species of the passion vines, grown for its edible fruits. It is found in South America, mainly Paraguay, Argentina, Peru and Brazil. The development of a set of microsatellite markers was proposed to provide tools for analysing both genetic structure of wild populations and the reproductive system of the species, which are poorly understood. Seven markers were developed from a P. alata‐genomic library enriched for simple sequence repeats (SSR). Twelve individuals were genotyped, and the levels of the expected heterozygosity (H_E) did show that those markers could be used to develop P. alata genetic studies.     

Development of microsatellite markers for the endangered semi‐shrub Chimaphila umbellata (Ericaceae)

Based on the DNA sequencing reads obtained using 454 pyrosequencing, primers amplifying 16 microsatellite loci were developed for the endangered semi‐shrub Chimaphila umbellata, which occurs sporadically in the Japanese Archipelago. These 16 loci were polymorphic in the populations sampled from the Hokkaido and Tohoku Districts; the mean number of alleles was 3.31 and 3.44, and the mean expected heterozygosity was 0.42 and 0.44, respectively. These loci were not linked to each other and contained no null alleles. Amplification using these primers was also tested in the congeneric species C. japonica, but only three of them successfully amplified DNA of the species. These markers will be used to examine genetic diversity and genetic differentiation in populations of C. umbellata.     

A morphometric analysis and taxonomic study of Panax bipinnatifidus Seem. (Araliaceae) species complex from Sikkim Himalaya, India

Cattleya elongata is a rupicolous orchid species spread throughout and endemic to outcrop islands in campo rupestre vegetation of the Chapada Diamantina, northeastern Brazil. We scored nine natural populations of C. elongata for morphological and genetic variability, covering the whole distribution area of the species, using allozymes and ISSR markers and morphometric multivariate analyses. Genetic variability in allozimes was relatively high (H _e?=?0.12?C0.25), and unexpectedly higher than the values based on ISSR (H _e?=?0.16?C0.19). The populations present moderate structuring (allozymes, ??_PT?=?0.14; ISSR, ??_PT?=?0.18) and low inbreeding (allozymes, F _IS?=?0.06). Genetic similarity among the populations was high in both markers, in spite of the discontinuity of the outcrops of the Chapada Diamantina. We found no particular biogeographical pattern to the distribution of the genetic and morphologic similarity among the populations of C. elongata. We found high morphological variability with moderate differentiation among the populations. We did not find any correlation among genetic, morphological, and geographical distances, and among the variability found in the morphological and genetic markers. The differences observed between the two genetic markers and the various morphological markers examined here indicated that the isolated use of any single parameter of these different populations for conservation planning or management would not consider all of the variability to be found in the species, as found in other Brazilian campos rupestres plants.     

First development and characterization of 27 novel microsatellite markers in the dobsonfly Neoneuromus ignobilis (Megaloptera: Corydalidae) at genome-scale level

The dobsonfly species Neoneuromus ignobilis Navás (Megaloptera: Corydalidae) is endemic to but widely distributed from eastern and southeastern Asia, being an important insect indicator for freshwater biomonitoring. At present, there is no report on the development of microsatellites of Megaloptera. Here, we developed 27 novel microsatellite markers of N. ignobilis from 850,920 candidate microsatellites with the stringent screening criteria considering the amplification success rate, the presence or absence of stutter peaks, the peak intensity, the polymorphism of the loci, the heterozygosity, and the number of alleles. The allele number of 27 microsatellite markers ranges from 3 to 12 with an average value of 6.19 per locus. The observed heterozygosity (H_O) and expected heterozygosity (H_E) revealed a range from 0.000 to 0.947 and 0.000 to 0.842, respectively. We constructed three panels (MP panel, most polymorphic; SS panel, most stringent strategy; ALL panel, total 27 microsatellite markers) and compared the analyses on population genetic diversity and structure. The result showed that the MP panel can significantly improve the analyses of individual assignment and genetic diversity. Accordingly, we advocate selecting the most polymorphic microsatellite marker for analyzing population genetics based on microsatellite data. The present work represents the first study on the microsatellite development of Megaloptera.

     

Isolation and characterization of microsatellite markers for Cycas hainanensis C. J. Chen (Cycadaceae)

Cycas hainanensis is an endangered cycad species endemic to Hainan Island, China. To enrich our scientific conservation for this species, we developed eight microsatellite markers using repetitive DNA enriched libraries. The number of alleles per microsatellite locus varied from 8 to 17. The expected (H _E ) and observed (H _O ) heterozygosities varied from 0.4754 to 0.8846 and from 0.3636 to 0.9600, respectively. These markers will be employed to determine whether the ex situ C. hainanensis individuals in Sourh China Botanical Garden capture a representative portion of genetic diversity of the wild populations.     

Isolation and characterization of eight microsatellite loci from <Emphasis Type="Italic">Lycorma delicatula</Emphasis> (White) (Hemiptera: Fulgoridae) for population genetic analysis in Korea

Lycorma delicatula (White) is native to China but is becoming an important insect pest in Korea. Polymorphic DNA markers like microsatellites are widely used for characterizing dispersal patterns and capacity of invasive insect pests which can contribute to designing effective management of the species. To facilitate such population genetic studies of L. delicatula in Korea, we isolated and characterized eight microsatellite loci for L. delicatula using a hybridization-biotin enrichment method. We further used these novel microsatellite loci to determine population genetic parameters for 33 L. delicatula specimens collected from Cheonan, South Korea where outbreaks of this species were first reported in Korea. The number of alleles per locus ranged from three to ten, with an average of 6.25. The mean expected (H _E) and observed heterozygosities (H _O) were 0.575 and 0.626, respectively. The eight loci showed no deviation from Hardy–Weinberg equilibrium according to the adjusted significance threshold (P = 0.00625), and there was no linkage disequilibrium between each pair of these eight markers. Bayesian cluster analysis using the program structure revealed no evidence of genetic structuring in L. delicatula samples from Cheonan. These new microsatellite markers will be widely applicable to future ecological genetic studies of L. delicatula, including assessment of the level of gene flow and genetic connectivity among populations that are necessary for effective management and monitoring of the species.     

Genetic structure and management of the Neotropical migratory fish Megaleporinus obtusidens on a highly impacted river basin

Habitat fragmentation caused by hydroelectric dams has depleted fish populations worldwide. Restocking actions are usually adopted to recover those populations, but hatchery management protocols rarely guarantee the maintenance of genetic diversity and a balanced contribution among captive breeders each generation. Here, a set of 10 microsatellite markers was used to assess the genetic diversity (average allelic richness A_R?=?10.87 and expected heterozygosity H_E?=?0.742, respectively) and structuring of Megaleporinus obtusidens, a migratory freshwater fish inhabiting over 2500 km of the São Francisco River in Brazil. Three main genetic clusters were identified in this species across the river basin that could be related to the sharply different climatic and hydrologic regimes from the Upper to the Lower course. A significant reduction (> 50%) in genetic diversity was observed in the broodstock when compared to their wild conspecifics, especially in the allelic richness. The information here presented will aid for management of genetic resources of this species in the São Francisco River taking as reference the genetic clusters identified. Furthermore, the results indicated that restocking is not necessary unless signals of population depletion occurs and, if so, hatchery reproductive protocols should rely on artificial fertilization rather than mass spawning.

     

Development and characterization of microsatellite markers from the yellow passion fruit (Passiflora edulis f. flavicarpa)

Here we described the development of the first set of Passiflora microsatellite loci isolated from an enriched genomic library. A sample of 43 individuals from 12 accessions of the yellow passion fruit was used to characterize those loci, which revealed up to 20 alleles per locus. Two loci were monomorphic. The observed (H_O) and expected (H_E) heterozygosities were very similar, as expected for a self‐incompatible species. Allelic diversity (H_T) was 0.444. This set of markers will permit genetic structure analyses of cultivated and wild populations of Passiflora, and contribute for integrating genetic maps based on dominant markers, as they can provide bridge alleles.     

Species identification and comparative population genetics of four coastal houndsharks based on novel NGS‐mined microsatellites

The common smooth‐hound (Mustelus mustelus ) is the topmost bio‐economically and recreationally important shark species in southern Africa, western Africa, and Mediterranean Sea. Here, we used the Illumina HiSeq? 2000 next‐generation sequencing (NGS ) technology to develop novel microsatellite markers for Mustelus mustelus . Two microsatellite multiplex panels were constructed from 11 polymorphic loci and characterized in two populations of Mustelus mustelus representative of its South African distribution. The markers were then tested for cross‐species utility in Galeorhinus galeus , Mustelus palumbes , and Triakis megalopterus , three other demersal coastal sharks also subjected to recreational and/or commercial fishery pressures in South Africa. We assessed genetic diversity (N _A, A _R, H _O, H _E, and PIC) and differentiation (F _ST and D _est) for each species and also examined the potential use of these markers in species assignment. In each of the four species, all 11 microsatellites were variable with up to a mean N _A of 8, A _R up to 7.5, H _E and PIC as high as 0.842. We were able to reject genetic homogeneity for all species investigated here except for T . megalopterus . We found that the panel of the microsatellite markers developed in this study could discriminate between the study species, particularly for those that are morphologically very similar. Our study provides molecular tools to address ecological and evolutionary questions vital to the conservation and management of these locally and globally exploited shark species.     

Case study of microsatellite polymorphism of European perch in selected commercially important lakes of Latvia

Polymorphism of selected microsatellite markers was described for European perch populations of inland Lake Kala and costal Lake Babites in Latvia. The data consisted of ten microsatellite loci analysed for 90 individuals from the Lake Babites (n?=?45) and the Lake Kala (n?=?45). Both lakes differ in water area, connection with the Gulf of Riga (Baltic Sea), lake type, and fish species. In the Lake Kala population, the average number of alleles per locus varied from 4 to 15 and in the population of Lake Babites from 4 to 12. Low but significant genetic differentiation was detected between populations (F_ST?=?0.046). Observed and expected heterozygosity in both lakes was similar (Kala: H_o?=?0.680 and H_e?=?0.816; Babites: H_o?=?0.693 and H_e?=?0.815). The number of unique alleles per locus was 2.3 in Lake Kala and 0.6 in Lake Babites. The present study showed relatively high polymorphism of analysed microsatellite markers.     

Microsatellite markers in Fijian crested iguanas (Brachylophus vitiensis) also amplify Fijian banded iguanas (B. fasciatus)

Five microsatellite markers were isolated from the Fijian crested iguana (Brachylophus vitiensis) using an enrichment technique. These loci also amplified the Fijian banded iguana (B. fasciatus). All five loci were polymorphic with between 2–7 and 4–7 alleles per locus in B. vitiensis and B. fasciatus, respectively. Heterozygosity (H_O) values ranged from 0.069 to 0.875. These markers were useful in resolving kinship relationships and will aid future endeavours by the captive management program to conserve genetic diversity.     

Isolation and characterization of 13 microsatellite loci from Incarvillea mairei (Bignoniaceae), an endemic species to the Himalaya-Hengduan mountains region

Incarvillea mairei (H. Léveillé) Grierson (Bignoniaceae) is an endemic species to the Himalaya-Hengduan Mountains region. Here, we developed 13 microsatellite markers from I. mairei using a modified biotin-streptavidin capture method. Number of alleles per locus (NA) ranged from 3 to 7 with an average of 4.615. The observed (H _O) and expected (H _E) heterozygosities were from 0.050 to 0.800 and from 0.249 to 0.815, respectively. Additionally, among the 13 identified microsatellite markers, 12 of them were successfully amplified in other three congeneric species, and most of them showed polymorphic. Obtained evidences suggest that these markers provide a useful tool for further study of the population genetic structure and the breeding system in this species or/and infra-generic species.     

Isolation and characterization of microsatellite markers from Brown Eared-pheasant (Crossoptilon mantchuricum)

The Brown Eared-pheasant (Crossoptilon mantchuricum) is an endangered species endemic to China. Here we developed eight microsatellite loci using a modified biotin-capture method. In the analyses of 28 individuals sampled, the number of alleles per locus ranged from four to nine. The expected (H _E) and observed (H _O) heterozygosities were 0.466–0.825 and 0.619–0.847, respectively. Results that eight microsatellite loci were highly polymorphic indicated that these markers are sufficiently powerful to address such questions as genetic diversity and population genetic structure of C. mantchuricum.     

Isolation and characterization of microsatellite loci in <Emphasis Type="Italic">Pedicularis verticillata</Emphasis> L. using PCR-based isolation of microsatellite arrays (PIMA)

Pedicularis verticillata L. is a highly valuable herb for traditional Chinese medical treatment. In this report, 11 microsatellite loci from P. verticillata were isolated. The simple sequence repeat (SSR) markers were screened in 23 samples of wild populations of P. verticillata, and eight samples from its sister P. ikomai. The number of alleles ranged from 4 to 13, and value of expected (H _E) and observed (H ₀) heterozygosity was 0.62609–0.89662 and 0–0.95652, respectively. All loci were significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency, indicating a dramatic loss of genetic polymorphisms in the restrictedly distributed species. The markers amplified well in the two species are useful for examining genetic diversity and population genetic structure, which, in turn, can provide information for establishing conservation strategy for these endangered species.     

Isolation and characterization of new microsatellite markers from the Japanese scallop (<Emphasis Type="Italic">Patinopecten yessoensis</Emphasis>)

Twenty polymorphic microsatellite loci were isolated from the Japanese scallop (Patinopecten yessoensis) using a method of hybridizing twice. The genetic diversity was analyzed by these microsatellite markers in field specimens from Zhangzidao Island in Liaoning Province, China. Observed heterozygosity (H _o), expected heterozygosity (H _e), number of effective alleles (N _e), and polymorphism information contents (PIC) were calculated. The heterozygote deficiency or excess was detected by the fixation index (Fis). The Hardy–Weinberg equilibrium was tested by possibilities (P _HWE) using chi-square test. Results indicate that the average of allele numbers was 6.0. The mean values of the parameters H _o, H _e, N _e, and PIC were 0.7921, 0.7076, 4.4347, and 0.6684, respectively. Only 4 of 20 microsatellite markers showed heterozygote deficiency including loci FJ262370, FJ262381, FJ262384, and FJ262400. The P _HWE value indicates the wild population deviated somewhat from the Hardy–Weinberg equilibrium. These newly isolated markers increase the available molecular resources of the Japanese scallop.     

Development of thirteen microsatellite loci for Impatiens lateristachys (Balsaminaceae)

Impatiens lateristachys is a local endemic species of Emei Mountain in southwestern China. In order to study the speciation mode of this species and its closely relatives, it is important to characterize their genetic diversity and differentiation. Here, thirteen polymorphic microsatellite markers have been developed for I. lateristachys. AC/TG microsatellite was enriched by combining biotin capture method. The average allele number of these markers was 3.1, ranging from 2 to 6. The observed (H _O) and expected (H _E) heterozygosities were 0.15−0.55 and 0.21–0.70, respectively. All the 13 markers could be applied in the other four congeneric species.     

Isolation and characterization of eight microsatellite loci from the endangered plant species Hypochaeris salzmanniana (Asteraceae)

We report the isolation and characterization of eight microsatellite loci for Hypochaeris salzmanniana, an endangered species endemic to the southwestern coast of Spain and the Atlantic coast of Morocco. A total of 32 alleles were detected across a sample of 45 individuals, with an average number of 4.0 alleles per locus. The average polymorphic information content (PIC) was 0.533 and the observed (H _O) and expected (H _E) heterozygosity values varied from 0.022 to 0.978 and from 0.434 to 0.759, respectively. Five loci exhibited significant deviation from Hardy–Weinberg equilibrium (P ≤ 0.001) and three pairs of loci showed significant linkage disequilibrium (P ≤ 0.01). The eight loci were tested for transferability in three others species (H. arachnoidea, H. glabra, and H. radicata) belonging to the same section of H. salzmanniana. With the exception of locus Hsalz7, all loci successfully amplified in the three species. These preliminary data confirm the usefulness of microsatellite markers for assessing the ecology and genetic structure of H. salzmanniana and to understand the evolution of species within the section Hypochaeris.