Isolation and characterization of polymorphic microsatellites in Iris ensata (Iridaceae)

• Premise of the study: Microsatellite primers were developed in Iris ensata (Iridaceae) to provide polymorphic markers for further studies into population genetics. • Methods and Results: Thirteen polymorphic microsatellite loci were isolated from I. ensata. These loci were successfully amplified in two natural populations of I. ensata from eastern China (Longwangshan, Zhejiang Province) and northeastern China (Jinchuan, Jilin Province). There was no significant linkage disequilibrium found for any pair of loci. These loci contained between two and 12 alleles per locus across all 48 individuals of I. ensata. The number of alleles per locus varied from two to 10 at the population level and the observed and expected heterozygosities ranged from 0.167 to 0.958 and from 0.284 to 0.853, respectively. • Conclusions: These loci showed high levels of polymorphism and could be used to study the population genetic structure, genetic relationships, and phylogeography of I. ensata.     

Isolation and characterization of 11 microsatellite loci in Scotophilus kuhlii (Lesser Asiatic Yellow House Bat)

We used enriched genomic library method to isolate and characterize 11 di-nucleotide microsatellite loci in the Lesser Asiatic Yellow House Bat, Scotophilus kuhlii. The polymorphism of these loci was tested on a natural population of 36 individuals from Hainan Province, South China. All loci revealed the polymorphism ranging from 5 to 18 alleles. The observed heterozygosity values were from 0.694 to 1.000 and expected heterozygosity values were from 0.558 to 0.929. No significant linkage disequilibrium was detected between any pairs of loci. These polymorphic markers will be used to assess population structure in S. kuhlii.     

Microsatellite markers for the common vole (<Emphasis Type="Italic">Microtus arvalis</Emphasis>) and their cross-species utility

Fragmentation of natural habitats of the common vole (Microtus arvalis) provides an excellent model system to study the consequences of restricted gene flow and small population sizes for isolated populations. Here we describe the isolation and characteristics of 10 autosomal and one X-linked microsatellite marker. These new markers were tested in 24 voles from a natural population in eastern Germany. Loci were highly polymorphic with numbers of alleles per locus ranging from three to 26 and expected heterozygosities from 0.51 to 0.97. All loci except for the X-linked locus Mar105 followed Hardy–Weinberg expectations. Cross-species amplifications revealed that most loci were polymorphic as well in M. agrestis, M. thomasi, and M. pennsylvanicus.     

Isolation and characterization of 10 microsatellite loci from Iris hexagona (Iridaceae)

Ten microsatellite loci isolated from Iris hexagona are described. All these loci are polymorphic, with three to 13 alleles across 24 individuals from a single natural population. Heterozygosity ranged from 0.125 to 0.870. Three loci depart significantly from Hardy–Weinberg equilibrium in our test population. The test population shows significant heterozygote deficiency in these and two other loci. Three loci exhibit significant linkage disequilibrium. These loci will be utilized to investigate patterns of genetic variation in the species throughout the Florida peninsula.     

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.     

Eight microsatellite loci in Phaedranassa schizantha Baker (Amaryllidaceae) and cross-amplification in other Phaedranassa species

Phaedranassa schizantha is a species endemic to Ecuador from which eight polymorphic microsatellite loci were isolated from an enriched genomic library. A total of 31 alleles with an average of four alleles per locus were detected across 29 individuals from a single natural population of P. schizantha. Observed heterozygosity ranged from 0.05 to 0.68. Most of the eight loci were successfully amplified in six Phaedranassa species. Five of those species are either “Vulnerable” or “Endangered” under IUCN criteria. These loci will be used to investigate patterns of inter- and intraspecific variation of Phaedranassa species, which will contribute data relevant to their conservation status.     

Isolation and characterization of microsatellite loci from the endangered highlands scrub hypericum (Hypericum cumulicola)

We report the isolation of 19 primer pairs for amplification of polymorphic microsatellite loci for Hypericum cumulicola. These markers were evaluated in 24 individuals from one population; two to four alleles were detected per locus, and observed heterozygosity ranged from 0 to 0.5. Two loci demonstrated significant heterozygote deficiencies, possibly due to null alleles, and significant linkage disequilibrium was found between six pairs of loci. The remaining microsatellite loci will help determine if genetic differentiation is responsible for life‐history differences between natural and anthropogenically disturbed populations of H. cumulicola.     

A population genetic evaluation of ecological restoration with the case study on <Emphasis Type="Italic">Cyclobalanopsis myrsinaefolia</Emphasis> (Fagaceae)

The ultimate goal of ecological restoration is to create a self-sustaining ecosystem that is resilient to perturbation without further assistance. Genetic variation is a prerequisite for evolutionary response to environmental changes. However, few studies have evaluated the genetic structure of restored populations of dominant plants. In this study, we compared genetic variation of the restored populations with the natural ones in Cyclobalanopsis myrsinaefolia, a dominant species of evergreen broadleaved forest. Using eight polymorphic microsatellite loci, we analyzed samples collected from restored populations and the donor population as well as two other natural populations. We compared the genetic diversity of restored and natural populations. Differences in genetic composition were evaluated using measurements of genetic differentiation and assignment tests. The mean number of alleles per locus was 4.65. Three parameters (A, A _R, and expected heterozygosity) of genetic variation were found to be lower, but not significantly, in the restored populations than they were in the natural populations, indicating a founder effect during the restoration. Significant but low F _ST (0.061) was observed over all loci, indicating high gene flow among populations, as expected from its wind-pollination. Differentiation between the two restored populations was smallest. However, differences between the donor population and the restored populations were higher than those between other natural populations and the restored populations. Only 13.5% and 25.7% individuals in the two restored populations were assigned to the donor population, but 54.1 and 40% were assigned to another natural population. The genetic variation of the donor population was lowest, and geographic distances from the restoration sites to the donor site were much higher than the other natural populations, indicating that the present donor likely was not the best donor for these ecological restoration efforts. However, no deleterious consequences might be observed in restored populations due to high observed heterozygosity and high gene flow. This study demonstrates that during the restoration process, genetic structures of the restored populations may be biased from the donor population. The results also highlight population genetic knowledge, especially of gene flow-limited species, in ecological restoration.     

Characterization of 15 polymorphic microsatellite loci in an endangered tropical tree Hopea bilitonensis (Dipterocarpaceae) in Peninsular Malaysia

This study reports the isolation and characterization of 15 polymorphic microsatellite loci in an endangered tropical tree Hopea bilitonensis (Dipterocarpaceae). The primers were designed from a genomic library enriched for dinucleotide (CT) repeats and screened on 24 adult trees from a natural population. The number of alleles ranged from two to eight, and the observed heterozygosity ranged from 0.042 to 1.000. These loci will allow mating system and population analyses to be carried out in this species.     

Isolation and characterization of polymorphic microsatellite markers for the endangered tree <Emphasis Type="Italic">Handeliodendron bodinieri</Emphasis> (Sapindaceae)

Nine polymorphic microsatellite loci were developed and characterized for Handeliodendron bodinieri, an endangered tree endemic to karst forest in southwest China. Each locus displayed between 4 and 11 alleles. The observed heterozygosity ranged from 0.345 to 0.931 and expected heterozygosity varied from 0.414 to 0.824. The combined probabilities of paternity exclusion over all loci were 0.931 for P _r(Ex₁) and 0.993 for P _r(Ex₂), respectively. These loci represent a powerful tool in investigating gene flow and population structure in natural populations of H. bodinieri.     

Development and characterization of microsatellite markers for Psychotria tenuinervis (Rubiaceae), a shrub species from the Atlantic forest, and primers transferability from Coffea

The shrub species Psychotria tenuinervis (Rubiaceae) is native to the Brazilian Atlantic forest and is largely found within natural and disturbed forest fragments. Aiming to develop studies on population genetic structure of forest fragment species, eigth microsatellite markers were developed for P. tenuinervis. Also, 15 loci already developed for Coffea (Rubiaceae) were tested for transferability to this species. We utilized 45 individuals from natural populations of three different fragments-anthropic edge, interior fragment and natural edge, within the Brazilian Atlantic forest. The average number of alleles per locus was 2.5 (two–four alleles/locus). These loci will be useful for future population genetic studies aiming to the conservation and management of this species.     

Identification of polymorphic microsatellite loci in the mud crab Scylla serrata (Brachyura: Portunidae)

Five microsatellite loci are identified and characterized from the genome of Scylla serrata, a widespread and commercially important species of coastal marine crab. The loci were detected by randomly screening for di‐ and tri‐nucleotide repeat units within a partial genomic library developed for the species. The five loci consist of dinucleotide repeats and are both co‐dominant and polymorphic within the species. A sample (N = 36) of S. serrata from one Australian population has an average observed heterozygosity of 0.875 and provides no evidence of either linkage among loci or significant deviation from random mating expectations across loci. PCR products for each of the five loci were also observed from a small sample of three other species within the Scylla genus. These markers may provide genetic information that will be useful for both aquaculture and studies of natural populations of the genus.     

Analysis of microsatellite DNA of rainbow trout (<Emphasis Type="Italic">Parasalmo</Emphasis> (<Emphasis Type="Italic">Oncorhynchus</Emphasis>) <Emphasis Type="Italic">mykiss</Emphasis>) of Kamchatka: Selection of loci and optimization of the method

The variation of a sample of rainbow trout (Parasalmo (Oncorhynchus) mykiss) from natural populations of several rivers of the Kamchatka Peninsula with respect to 43 microsatellite DNA loci has been studied. These loci were earlier used for analysis of Asian populations of closely related salmonids. Ten of them may be regarded as markers and seen promising for further studies on intraspecific relationships of rainbow trout of Kamchatka. Their use in studies on more numerous samples from different localities and populations of Parasalmo (O.) mykiss in the Asian part of the species range will ensure efficient population genetic analysis of the Kamchatka population group of this species.     

Isolation and characterization of eight microsatellite loci from Phaedranassa tunguraguae (Amaryllidaceae)

Phaedranassa tunguraguae is an endangered species endemic to Ecuador. Eight highly polymorphic microsatellite loci were isolated from an enriched genomic library for this species. Levels of polymorphism were evaluated using a total of 31 individuals from a single natural population. An average of 14.1 alleles per locus was detected, and observed heterozygosity ranged from 0.387 to 0.903. All but one locus depart significantly from Hardy–Weinberg equilibrium. These loci are the first microsatellite primers isolated for Amaryllidaceae and will be utilized to investigate patterns of genetic variation of P. tunguraguae, which will contribute data relevant to the conservation of the species.     

Microsatellite loci for tucumã of Amazonas (Astrocaryum aculeatum) and amplification in other Arecaceae

• Premise of the study: Microsatellite loci were developed for tucumã of Amazonas (Astrocaryum aculeatum), and cross-species amplification was performed in six other Arecaceae, to investigate genetic diversity and population structure and to provide support for natural populations management. • Methods and Results: Fourteen microsatellite loci were isolated from a microsatellite-enriched genomic library and used to characterize two wild populations of tucumã of Amazonas (Manaus and Manicoré cities). The investigated loci displayed high polymorphism for both A. aculeatum populations, with a mean observed heterozygosity of 0.498. Amplification rates ranging from 50% to 93% were found for four Astrocaryum species and two additional species of Arecaceae. • Conclusions: The information derived from the microsatellite markers developed here provides significant gains in conserved allelic richness and supports the implementation of several molecular breeding strategies for the Amazonian tucumã.     

Development of microsatellite markers for Carapa guianensis (Aublet), a tree species from the Amazon forest

Carapa guianensis is a timber species found in Central America and the north of South America. We have developed microsatellite primers which will allow analysis of gene flow and population genetic structure in natural populations of this tree species. Polymorphism of five microsatellite loci was evaluated using a total of 12 adult trees from a natural population. An average of 4.2 alleles per locus was detected, and expected heterozygosity ranging from 0.397 to 0.806. These loci are being used for genetic population analysis in a managed forest in the state of Pará, in Amazonian Brazil, as part of the Dendrogene project.     

Microsatellite allele sequencing in population analyses of the South American cactophilic species Drosophila antonietae (Diptera: Drosophilidae)

Drosophila antonietae belongs to the Drosophila buzzatii cluster, a cactophilic group of species naturally endemic to South America. Morphological and genetic analyses indicate that its populations are the most homogenous in the cluster and that the diversity observed is mainly a result of variation within populations. Seven polymorphic microsatellite loci were described for this species and used in the present study to investigate the genetic diversity of natural populations of D. antonietae by both length and sequence variation. The study aimed to understand how homoplasy and null alleles affect inferences about the population history of this species and to obtain an accurate interpretation of population inferences where these loci could be applied. The results provide useful information on the interpretation of genetic data derived from the microsatellite loci described for D. antonietae and on evolutionary aspects of cactophilic Drosophila. Importantly, the results indicate that size homoplasy and null alleles do not represent significant problems for the population genetics analyses because the large amount of variability at microsatellite loci compensate the low frequency of these problems in the populations. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 573–584.     

Isolation and characterization of polymorphic microsatellite loci in the Australian smelt,Retropinna semoni

Five polymorphic microsatellite loci were isolated from the Australian smelt, Retropinna semoni, in order to study levels of connectivity among populations at various spatial scales. Screening of one natural population (n = 30) from central Victoria, southeastern Australia, yielded nine to 14 alleles per locus with observed levels of heterozygosity ranging from 0.40 to 0.80. All loci were in Hardy–Weinberg equilibrium after Bonferroni correction for multiple tests. These loci should provide a useful tool in further understanding the population genetics of this species.     

Identification and characterisation of thirteen new microsatellites for Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis> L.) from a repeat-enriched library

A total of 13 polymorphic microsatellite markers were developed for Atlantic cod (Gadus morhua L.) from a repeat-enriched library. Polymorphism of each locus was assessed in 96 unrelated individuals from a natural population. The number of alleles per locus varied from 8 to 45. The ranges of observed and expected heterozygosity were 0.122–0.907 and 0.673–0.965, respectively. Four of the loci (Gmo-G24, Gmo-G40, Gmo-G46 and Gmo-G49) followed Hardy–Weinberg expectation. No evidence for linkage disequilibrium between pairs of loci was found in any combination of loci pairs, except between Gmo-G40 and Gmo-G43. These microsatellite markers provide useful tools for studies of population genetics, reproductive ecology and for constructing linkage maps of Atlantic cod. Jon-Ivar Westgaard and Tekle Tafese have contributed equally to the work.     

Molecular genetic variation following a population crash in the endangered Mauna Kea silversword, Argyroxiphium sandwicense ssp. sandwicense (Asteraceae)

The endangered Mauna Kea silversword, Argyroxiphium sandwicense ssp. sandwicense (Asteraceae), has experienced a severe decline in distribution and abundance because of predation by alien ungulates. The small remnant natural population on the Mauna Kea volcano contains only 46 individuals. By contrast, the Haleakala silversword, A. sandwicense ssp. macrocephalum, consists of a large, vigorous population exceeding 60 000 individuals. Molecular genetic variation in the two populations was assessed using random amplified polymorphic DNA (RAPD) loci. Despite its severe crash in size, the Mauna Kea population did not differ significantly from the Haleakala population in the number of detectably polymorphic loci or in heterozygosity. The lack of substantial reduction in genetic variation, at least as measured with RAPD loci, suggests that the Mauna Kea population may not yet have gone through multiple generations at very small size.