Characterization of 10 trinucleotide microsatellite loci in the Critically Endangered Pyrenean yam Borderea chouardii (Dioscoreaceae)

The low levels of allozymic variability found in the Critically Endangered Borderea chouardii prompted us to develop microsatellite markers to assess the genetic variability and population structure for the adequate conservation management of this species. A (CTT)_n‐enriched partial genomic library was constructed. Ten polymorphic microsatellite loci were isolated from it, rendering 51 alleles in 47 individuals analysed. The allelic pattern observed for all of the loci with more than two alleles suggests that B. chouardii is tetraploid.     

A Bayesian approach for discriminating among alternative inheritance hypotheses in plant polyploids: the allotetraploid origin of genus Borderea (Dioscoreaceae)

Polyploidy is a common phenomenon occurring in a vast number of land plants. Investigations of patterns of inheritance and the origins of plants (i.e., autopolyploidy vs. allopolyploidy) usually involve cytogenetic and molecular studies of chromosome pairing, chromosome mapping, and marker segregation analysis through experimental crosses and progeny tests. Such studies are missing for most wild species, for which artificial crosses are difficult, not feasible, or unaffordable. We report here a Bayesian method to discriminate between alternative inheritance patterns in the two extant, tetraploid species of the monocot genus Borderea (Dioscoreaceae), which does not involve progeny array tests. Our approach is based on the screening of a large number of SSR genotypes, which were obtained from successful amplifications of 17 microsatellite regions in individuals of both B. chouardii and B. pyrenaica. We tested for tetrasomic vs. disomic modes of inheritance, using the Bayes factor test. Assignment of genotypes under both alternatives could be unequivocally done for 14 and 11 of the 17 studied microsatellite regions in B. chouardii and B. pyrenaica, respectively, totaling 9502 analyzed genotypes. The comparison of posterior probabilities for the two competing hypotheses across the surveyed loci clearly favored a disomic inheritance pattern. Linkage tests indicated that none of the studied SSR loci were in linkage disequilibrium, thus representing independent samples of the Borderea genome. These results, along with previous allozyme data, support the allotetraploid origin of this paleoendemic genus and reveal the lowest reported chromosome base number for the family of the yams.     

Nunatak survival vs. tabula rasa in the Central Pyrenees: a study on the endemic plant species Borderea pyrenaica (Dioscoreaceae)

Aim  Borderea pyrenaica (Dioscoreaceae) is a Tertiary relict plant endemic to the Central Pyrenees. Because of its narrow distribution in a small geographical area and the fact that it is restricted to high alpine habitats, it constitutes an ideal model species for inferring the historical dynamics of population survival and migration during and after Quaternary glaciations in the Pyrenees.
Location  Central Pyrenees and pre-Pyrenees, Spain–France.
Methods  Eleven primer pairs were used to amplify 18 microsatellite loci in this allotetraploid species in a sample of 804 individuals from 15 populations, revealing a total of 77 alleles. Genotypic data of individuals and populations were analysed using clustering and Bayesian methods of analysis of population structure.
Results  A higher number of private alleles and a significantly higher allelic richness ( A *) were found in the southern area (21, A * = 2.295) than in the northern area (5, A * = 1.791). Furthermore, the allelic composition of the northern area represented a subset of that from the southern area.
Main conclusions  The hypothesis of in situ survival in northern Pyrenean nunataks was rejected, while peripheral refugia were considered to be restricted to the southern Pyrenees and pre-Pyrenees, where historical geographical fragmentation probably caused the divergence among southern Pyrenean populations. Molecular evidence indicates that these refugial populations probably colonized the northern area after sheet-ice retreat. Borderea pyrenaica lineages followed two migratory pathways in their northward colonization, suggesting several founder events for the populations that eventually reached the territory of the Gavarnie cirque.     

Population structure and conservation genetics of wild rice Oryza rufipogon (Poaceae): a region-wide perspective from microsatellite variation

Oryza rufipogon Griff. is the most agriculturally important but seriously endangered wild rice species. To better estimate how genetic structure can be used to obtained a conservation perspective of the species, genetic variability at six polymorphic microsatellite DNA loci was examined. High levels of genetic variability were detected at six loci in 1245 individuals of 47 natural populations covering most of the species' range in China (overall RS = 3.0740, HO = 0.2290, HS = 0.6700). Partitioning of genetic variability (FST = 0.246) showed that most microsatellite variation was distributed within populations. Significant departures from Hardy-Weinberg expectations and very strong linkage disequilibrium indicate a high degree of inbreeding in the species and severe subdivision within populations. A mean Nm value of 0.7662 suggested a limited gene flow among the assayed populations. Our study suggests that conservation and restoration genetics should focus in particular on the maintenance of historically significant processes such as high levels of outbreeding and gene flow and large effective population size in the species.     

Characterization of microsatellite loci isolated in trumpeter swan (Cygnus buccinator)

Primers for 16 microsatellite loci were developed for the trumpeter swan (Cygnus buccinator), a species recovering from a recent population bottleneck. In a screen of 158 individuals, the 16 loci were found to have levels of variability ranging from two to seven alleles. No loci were found to be linked, although two loci repeatedly revealed significant departures from Hardy–Weinberg equilibrium. Amplification in the closely related tundra swan (Cygnus columbianus) was successful for all except one locus. These microsatellite loci will be applicable for population genetic analyses and ultimately aid in management efforts.     

A panel of polymorphic microsatellites in the threatened Apennine chamois (Rupicapra pyrenaica ornata)

The Apennine chamois (Rupicapra pyrenaica ornata) is a highly endangered mountain ungulate. About 800 individuals still survive in central Apennines, Italy. Current conservation projects involve genetic identification of single animals, paternity tests, and assessment of relatedness. A suite of 60 microsatellite primer pairs from domestic bovids and cervids revealed polymorphisms for 10 loci, providing evidence for a low level of genetic variability, which might be a consequence of severe population bottlenecks and prolonged permanence at low numbers.     

Isolation of polymorphic microsatellite loci in Crossosoma californicum (Crossosomataceae)

Crossosoma californicum (Crossosomataceae) is a rare shrub species endemic to the California Channel Islands. Previous studies based on allozymes revealed little genetic variability in this plant species. We have isolated 13 polymorphic microsatellite loci from C. californicum. These loci show intermediate levels of variability, averaging 4.2 alleles per locus and expected heterozygosity of 0.376. Two loci did not fit Hardy–Weinberg expectations with significant deficits of heterozygous genotypes consistent with the presence of null alleles or population subdivision.     

Genetic structure of Korean populations of bumblebees Bombus ignitus (Hymenoptera: Apidae) as revealed by microsatellite markers

Bumblebee Bombus ignitus, which is indigenous to Korea, Japan, and China, has been recognized as a valuable pollinator for both crops and wild plants. Bombus ignitus has now become commercially important as a pollinator because of its use in the agricultural industry, particularly for greenhouse pollination. For long‐term management and effective conservation of B. ignitus, an understanding of the genetic structure of its naturally occurring populations is practically important. In this study, the genetic structure among the five populations of B. ignitus in South Korea was assessed using nine microsatellite loci. These markers showed high levels of genetic variability, with the number of alleles ranging from 6 to 22 (mean = 13.4) and the frequency of the most common allele ranging from 0.11 to 0.66. Only the Sabuk (SB) population showed a genetic signature of a recent bottleneck, which was further supported by the lowest level of allelic richness (AR) (mean AR = 3.944). Genetic differentiation was highly significant among all pairs of populations (P < 0.001) across the nine microsatellite markers, suggesting a lack of gene flow among those populations. Interestingly, F_ST (and R_ST and D_est) values were always greater for the Taebaek population than for the four remaining populations. The phylogenetic analysis showed evidence supporting our hypothesis that the Taebaek population is genetically more divergent than the other populations. Overall, our results suggest that the Korean populations of B. ignitus might have undergone geographic isolation and have become highly separated spatially from one another, thereby having a limited range of migration among their local habitats.     

Ten polymorphic microsatellite loci in the giant scallop (Mizuhopecten yessoensis)

Ten novel microsatellite loci were isolated from giant scallop Mizuhopecten yessoensis, and the polymorphisms were examined to estimate genetic variability. The genetic variabilities varied depending on the locus. The number of alleles ranged from two to 17, and the observed and expected heterozygosities ranged from 0.17 to 0.99 and 0.33 to 0.90, respectively. Six loci showed significant Hardy–Weinberg disequilibrium. The high variabilities revealed in this study suggest that microsatellites should prove useful for various genetic investigations.     

Development of 18 microsatellite loci for the freshwater snail Bellamya aeruginosa (Mollusca, Gastropoda)

Although it is a major freshwater gastropod species, genetic diversity of Bellamya aeruginosa was completely unknown. Eighteen microsatellite loci were isolated and characterized from (AC)(15)-enriched genomic libraries of the freshwater snail B. aeruginosa. Most of the 18 loci were successfully amplified and high polymorphic information content values were found, ranging from 0.244 to 0.792 (mean 0.541). The number of alleles per locus ranged from 5 to 13 (mean 8.8), the expected heterozygosity varied from 0.347 to 0.950 (mean 0.815) and the observed heterozygosity varied from 0.087 to 0.782 (mean 0.431). Eight loci showed significant deviation from Hardy-Weinberg equilibrium after Bonferroni's correction and no significant genotypic linkage disequilibrium was detected between most locus pairs, except for TXH79-TXH97 and TXH113-TXH121. These 18 polymorphic microsatellite loci should be useful for population genetics analysis and species identification of Bellamya.     

Genetic diversity and bottleneck analysis of Nagpuri buffalo breed of India based on microsatellite data

In this study, 25 heterologous bovine microsatellite markers have been used for the assessment of genetic diversity in Nagpuri buffalo, an important breed of Central India. For this, 48 DNA samples of unrelated individuals of Nagpuri buffalo were PCR amplified and microsatellite alleles were resolved in 6% denaturing, silver stained Urea-PAGE gel. Genotypic status of individuals at each locus was identified manually and data analysis carried out using POPGENE software. Observed number of alleles varied from 2 (ILSTS073 locus) to 8 (HEL13 & ILSTS058 loci) with a mean of 5.24 alleles per locus. Moderate level of heterozygosity (0.45) indicated sufficient genetic diversity existing in this buffalo population. PIC values for the microsatellite loci analysed, ranged from 0.10 (ILSTS0I9 locus) to 0.81 (ILSTS058 locus) with a mean of 0.53. No shift in the frequency distribution of alleles and a normal L-shaped curve indicated non-existence of any bottleneck in Nagpuri. The study thus highlights the usefulness of heterologous bovine microsatellite markers to assess the genetic variability in buffalo breds as well. Also various diversity indices suggest sufficient genetic variability within Nagpuri buffalo that can be utilized as initial guidelines for future breeding strategies and conservation. The article is published in the original.     

Isolation and characterization of eight polymorphic microsatellite loci for the coconut pest, Brontispa longissima (Coleoptera: Hispidae)

Brontispa longissima is one of the most serious insect pests of coconut in Southeast Asia; it was first discovered on Hainan Island in June 2002. Despite the economic risk associated with this pest, genetic aspects of the invasion process have remained relatively unexplored. Using microsatellite markers, we investigated the population structure, genetic variability and pattern of invasion in various geographic populations. The methodology was based on a modified biotin-capture method. Eight polymorphic microsatellite loci were isolated and characterized for the pest. The allele number per locus varied from 2 to 3 (N = 30). The expected and observed heterozygosities of the eight loci ranged from 0.042 to 0.509 and from 0.042 to 0.963, respectively. Although the frequency of polymorphisms was not very high in this population, the microsatellite loci that were isolated will be useful for investigating the genetic diversity and migration routes of B. longissima populations.     

PERMANENT GENETIC RESOURCES: Isolation of microsatellite loci from the endangered plant Sibara filifolia (Brassicaceae)

Sibara filifolia (Brassicaceae) is a federally endangered annual herb found on two of the California Channel Islands. Previous studies based on allozymes revealed little genetic variability on San Clemente Island. Nine polymorphic microsatellite loci were isolated from individuals on San Clemente Island. We found low levels of allelic variation (mean N_A = 2.3), with seven loci exhibiting significant deviations from Hardy–Weinberg equilibrium (P < 0.01) and 10 pairs of loci exhibiting significant linkage disequilibrium (P < 0.01). Most of the observed variability (mean H_O = 0.003) occurred among populations or in rare homozygous individuals.     

New microsatellite markers for the snow crab Chionoecetes opilio (Brachyura: Majidae)

Nine novel microsatellite loci were isolated from Chionoecetes opilio by screening an enriched genomic library using nonradioactive polymerase chain reaction techniques, and the polymorphisms were examined to estimate genetic variability. The genetic variabilities varied depending on the locus. All loci were found to be polymorphic with an average of 9.7 alleles per locus (range 3–25). The observed and expected heterozygosities ranged from 0.80 to 0.98 and from 0.56 to 0.95, respectively. Five loci showed significant Hardy–Weinberg disequilibrium at the P < 0.05 level. The high variabilities revealed in this study suggest that these microsatellite loci should provide useful markers for genetic variation monitoring of C. opilio.     

Development of microsatellite markers in Byrsonima crassifolia (Malpighiaceae)

? Premise of the study: We developed and characterized microsatellite markers for Byrsonima crassifolia (Malpighiaceae), a widely distributed neotropical fruit tree. ? Methods and Results: Eight polymorphic and two monomorphic microsatellite loci were identified and screened in 60 samples from four geographically disparate populations (Bolivia, Brazil, Mexico, and Panama). Each locus exhibited between two and 11 alleles. Expected heterozygosity ranged from 0 to 0.839. All loci amplify in the congeners B. variabilis and B. basiloba, four amplify in B. bucidaefolia, and seven amplify in B. variabilis, although levels of polymorphism have not been assessed. ? Conclusions: These loci will provide novel tools for comparing genetic diversity present in cultivated and noncultivated populations of B. crassifolia throughout its range, and may prove valuable in related species.     

New microsatellite loci in the dwarf yams Dioscorea group Epipetrum (Dioscoreaceae)

? Premise of the study: Microsatellite loci were isolated and characterized from enriched genomic libraries of two taxa of the Chilean Epipetrum group of Dioscorea to assess their levels of genetic diversity and population differentiation. ? Methods and Results: Eleven microsatellite loci were identified. Six out of nine microsatellites from D. biloba amplified in D. humilis, and the two microsatellites from D. humilis amplified in both taxa. Two different sets of eight loci amplified in each of the two tested taxa, D. biloba and D. humilis. The average number of alleles was 5.75 and 5 for D. biloba and D. humilis, respectively. Higher levels of mean genetic diversity were found in D. biloba (H(E) = 0.639) than in D. humilis (H(E) = 0.414). ? Conclusions: These microsatellite primers will be useful in population genetic studies and to establish conservation strategies in the endangered taxa of the Epipetrum group of Dioscorea.     

Isolation and characterization of 16 novel microsatellite loci in two inbred strains of the Chinese hamster (Cricetulus griseus)

The Shanyi inbred A and E strains of the Chinese hamster are widely used in biomedical research, but detailed genetic characterization has been lacking. We developed microsatellite markers that could be used for genetic diversity analysis and linkage map construction. We isolated and characterized 16 novel microsatellite loci from a microsatellite-enriched genomic DNA library. These loci were genotyped in 48 animals from the two strains, and the polymorphic information content was determined. In the Shanyi A and E populations, 14 and 15 loci were found to be polymorphic, respectively, with polymorphic information content ranging from 0.1393 to 0.8082 and from 0.1109 to 0.7397, respectively. A total of 115 alleles were found for the 16 microsatellite loci in the two populations; the mean observed heterozygosity (H(O)) was 0.5191 and 0.4333 for the A and E populations, respectively, indicating marked genetic variation within the two populations. Correspondingly, the F(ST) values ranged from 0.002 to 0.9253, with an overall mean of 0.1935, indicating significant genetic difference between the two strains. The population differentiation levels were substantiated by Nei's genetic distance and full Bayesian analyses computed with STRUCTURE. Despite the genetic diversity and differentiation within and between the two inbred populations, the 48 individuals were correctly allocated into their original populations with high statistical confidence based on these 16 microsatellite loci. These novel microsatellite loci should be useful genetic markers for these two Chinese hamster inbred strains.     

Contrasting patterns of mitochondrial and microsatellite population structure in fragmented populations of greater prairie-chickens

Greater prairie-chickens (Tympanuchus cupido pinnatus) were once found throughout the tallgrass prairie of midwestern North America but over the last century these prairies have been lost or fragmented by human land use. As a consequence, many current populations of prairie-chickens have become isolated and small. This fragmentation of populations is expected to lead to reductions in genetic variation as a result of random genetic drift and a decrease in gene flow. As expected, we found that genetic variation at both microsatellite DNA and mitochondrial DNA (mtDNA) markers was reduced in smaller populations, particularly in Wisconsin. There was relatively little range-wide geographical structure (FST) when we examined mtDNA haplotypes but there was a significant positive relationship between genetic (FST) and geographical distance (isolation by distance). In contrast, microsatellite DNA loci revealed significant geographical structure (FST) and a weak effect of isolation by distance throughout the range. These patterns were much stronger when populations with reduced levels of genetic variability (Wisconsin) were removed from the analyses. This suggests that the effects of genetic drift were stronger than gene flow at microsatellite loci, whereas these forces were in range-wide equilibrium at mtDNA markers. These differences between the two molecular markers may be explained by a larger effective population size (Ne) for mtDNA, which is expected in species such as prairie-chickens that have female-biased dispersal and high levels of polygyny. Our results suggest that historic populations of prairie-chickens were once interconnected by gene flow but current populations are now isolated. Thus, maintaining gene flow may be important for the long-term persistence of prairie-chicken populations.     

Assessment of population genetic structure in common wild rice Oryza rufipogon Griff. using microsatellite and allozyme markers

The genetic structure of five natural populations of common wild rice Oryza rufipogon Griff. from China, was investigated with 21 microsatellite loci and compared to estimates of genetic diversity and genetic differentiation detected by 22 allozyme loci. Microsatellite loci, as expected, have much higher levels of genetic diversity (mean values of A = 3.1, P = 73.3%, Ho = 0.358 and He = 0.345) than allozyme loci (mean values of A = 1.2, P = 12.7%, Ho = 0.020 and He = 0.030). Genetic differentiation detected by microsatellite loci ( FST = 0.468, mean I = 0.472) was higher than that for allozyme loci ( FST =0.388, mean I = 0.976). However, microsatellite markers showed less deviation from Hardy-Weinberg expectation (Wright's inbreeding coefficient FIS = -0.069) than do allozymes ( FIS = 0.337). These results suggest that microsatellite markers are powerful high-resolution tools for the accurate assessment of important parameters in population biology and conservation genetics of O. rufipogon, and offer advantages over allozyme markers.     

Isolation and characterization of polymorphic microsatellite loci in Candidia barbata (Cyprinidae) using PCR-based isolation of microsatellite arrays (PIMA)

Candidia barbata is an endemic cyprinid fish in Taiwan and suffers habitat destruction caused by human overexploitation. In this study, a total of 14 polymorphic microsatellite loci from C. barbata were isolated and characterized using an optimized protocol to construct a microsatellite-enriched genomic library. The analysis of variability was performed in 30 specimens of Taiwan. The mean number of alleles across loci was 4.92 ± 1.44. The levels of expected and observed heterozygosities varied from 0.1266 to 0.5079, and from 0.0667 to 0.9667, respectively. Frequencies of null alleles of the 14 loci are not significantly greater than zero. No linkage disequilibrium was detected between loci in either population.