Isolation and characterization of microsatellites in Drosophila montana and their cross‐species amplification in D. virilis

We report the isolation of 20 microsatellite loci from Drosophila montana and their cross amplification in the relative D. virilis. All microsatellite loci were polymorphic in the focal species D. montana, with gene diversities ranging from 0.23 to 0.93. In D. virilis only eight loci (40%) amplified and two loci were polymorphic (10%). These markers represent the first report of microsatellites isolated in D. montana. They could be applied for studying population structure and phylogeography. The largest benefit, however, will be their use in studies of quantitative trait loci, such as the mapping of behavioural quantitative trait loci.     

Isolation and characterization of microsatellites in Drosophila virilis and their cross species amplification in members of the D. virilis group

We report the isolation and cross species amplification of 42 Drosophila virilis microsatellite loci. Nine loci were isolated from mapped P1 bacteriophage clones and 33 were obtained from genomic DNA or GenBank searches. Cross species amplification was tested for all members of the D. virilis group. The amplification success was high (varying from 45% to 100%) and most of the loci were polymorphic. This set of loci can be applied for several genetic studies such as mapping behavioural quantitative trait loci (QTL) and for studying population structure in a phylogeographical framework in D. virilis group species.     

Rapid divergence of microsatellite abundance among species of Drosophila

Among major taxonomic groups, microsatellites exhibit considerable variation in composition and allele length, but they also show considerable conservation within many major groups. This variation may be explained by slow microsatellite evolution so that all species within a group have similar patterns of variation, or by taxon-specific mutational or selective constraints. Unfortunately, comparing microsatellites across species and studies can be problematic because of biases that may exist among different isolation and analysis protocols. We present microsatellite data from five Drosophila species in the Drosophila subgenus: D. arizonae, D. mojavensis, and D. pachea (three cactophilic species), and D. neotestacea and D. recens (two mycophagous species), all isolated at the same time using identical protocols. For each species, we compared the relative abundance of motifs, the distribution of repeat size, and the average number of repeats. Dimers were the most abundant microsatellites for each species. However, we found considerable variation in the relative abundance of motif size classes among species, even between sister taxa. Frequency differences among motifs within size classes for the three cactophilic species, but not the two mycophagous species, are consistent with other studied Drosophila. Frequency distributions of repeat number, as well as mean size, show significant differences among motif size classes but not across species. Sizes of microsatellites in these five species are consistent with D. virilis, another species in the subgenus Drosophila, but they have consistently higher means than in D. melanogaster, in the subgenus Sophophora. These results confirm that many aspects of microsatellite variation evolve quickly but also are subject to taxon-specific constraints. In addition, the nature of microsatellite evolution is dependent on temporal and taxonomic scales, and some variation is conserved across broad taxonomic levels despite relatively high rates of mutation for these loci.     

Evidence for complex mutations at microsatellite loci in Drosophila.

Fifteen lines each of Drosophila melanogaster, D. simulans, and D. sechellia were scored for 19 microsatellite loci. One to four alleles of each locus in each species were sequenced, and microsatellite variability was compared with sequence structure. Only 7 loci had their size variation among species consistent with the occurrence of strictly stepwise mutations in the repeat array, the others showing extensive variability in the flanking region compared to that within the microsatellite itself. Polymorphisms apparently resulting from complex nonstepwise mutations involving the microsatellite were also observed, both within and between species. Maximum number of perfect repeats and variance of repeat count were found to be strongly correlated in microsatellites showing an apparently stepwise mutation pattern. These data indicate that many microsatellite mutation events are more complex than represented even by generalized stepwise mutation models. Care should therefore be taken in inferring population or phylogenetic relationships from microsatellite size data alone. The analysis also indicates, however, that evaluation of sequence structure may allow selection of microsatellites that more closely match the assumptions of stepwise models.     

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.     

Mitochondrial microsatellite variability in common wheat and its ancestral species

On the basis of the entire mitochondrial DNA sequence of common wheat, Triticum aestivum, 21 mitochondrial microsatellite loci having more than ten mononucleotide repeats were identified. The mitochondrial microsatellite variability at all loci was examined with 43 accessions from 11 Triticum and Aegilops species involved in wheat polyploidy evolution. Polymorphic banding patterns were obtained at 15 out of 21 mitochondrial microsatellite loci. The number of alleles per polymorphic microsatellite ranged from 2 to 5 with an average of 3.07, and the diversity values (H) ranged from 0.09 to 0.50 with an average of 0.29. These values are almost two third of wheat chloroplast microsatellite values, indicating that variability of mitochondrial microsatellite is much less than that of chloroplast microsatellite. Based on the allele variation at all loci, a total of seven mitochondrial haplotypes were identified among common wheat and its ancestral species. Three diploid species showed their own specific haplotypes and timopheevi group (11 accessions) had three types, whereas 29 accessions of emmer and common wheat groups shared the same haplotype. These results indicate that a single mitochondrial haplotype determined by microsatellite analysis has conservatively been maintained in the evolutionary lineage from wild tetraploid to cultivated hexaploid species.     

Characterization and isolation of novel microsatellites from the Drosophila dunni subgroup

We have isolated and characterized 77 novel microsatellites from two species, Drosophila dunni and Drosophila nigrodunni, which are closely related Caribbean-island endemics from the Drosophila cardini species group. These species are very distantly related to all other Drosophila from which microsatellites have previously been characterized. We find that the average length of microsatellites isolated in these species is quite small, with an overall mean length of 9.8 repeat units for dinucleotide microsatellites in the two study species. The nucleotide composition of dinucleotides differs between the two species: D. nigrodunni has a predominance of (AC/GT)n repeats, whereas D. dunni has equal numbers of (AC/GT)n and (AG/CT)n repeats. Tri- and tetranucleotide repeats are not abundant in either species. We assayed the variability of eight microsatellites in a closely related third species, Drosophila arawakana, using wild-caught individuals from the island of Guadeloupe. We found the microsatellites to be extremely variable in this population, with observed heterozygosities ranging from 0.541 to 0.889. DNA amplification trials suggest that these eight microsatellites are widely conserved across the D. cardini group, with five of the eight producing amplification products in every species tested. However, the loci are very poorly conserved over greater phylogenetic distances. DNA amplification of the microsatellite loci was unreliable in members of the closely related Drosophila quinaria, Drosophila calloptera, Drosophila guarani and Drosophila tripunctata species groups. Furthermore, these microsatellites could not be detected in the genome of Drosophila melanogaster, despite the conservation of microsatellite flanking regions at some loci. These data indicate that Drosophila microsatellite loci are quite short lived over evolutionary timescales relative to many other taxa.     

High density of long dinucleotide microsatellites in Drosophila subobscura

We isolated 96 dinucleotide repeats with five or more tandemly repeated units from a subgenomic Drosophila subobscura library. The mean repeat unit length of microsatellite clones in D. subobscura is 15, higher than that observed in other Drosophila species. Population variation was assayed in 32-40 chromosomes from Barcelona, Spain, using 18 randomly chosen microsatellite loci. Positive correlation between measures of variation and perfect repeat length measures (mean size, most common, and longest allele) is consistent with a higher mutation rate in loci with longer repeat units. Levels of microsatellite variation measured as variance in repeat number and heterozygosity in D. subobscura were similar to those of Drosophila pseudoobscura and higher than those of Drosophila melanogaster and Drosophila simulans. Our data suggest that higher levels of microsatellite variation, and possibly density, in D. subobscura compared with D. melanogaster are due to both a higher average effective population and a higher intrinsic slippage rate in the former species.     

Drosophila simulans' response to laboratory selection for tolerance to a toxic food source used by its sister species D. sechellia

Drosophila sechellia is a specialist species which feeds and breeds on a toxic plant, Morinda citrifolia. All other Drosophila species are killed by ripe fruits of Morinda. D. simulans was subjected to laboratory selection for survival in presence of octanoic acid, the toxic compound of M. citrifolia. After 20 generations of selection, selected lines showed an increased tolerance to octanoic acid, although the response was small compared to the interspecific differences. The genetic response to selection was assessed by studying the changes in allele frequencies at 28 microsatellite loci. Three loci, located in three distinct genomic regions, showed changes in allele frequencies significantly different from what is expected under drift alone in the selected lines. An oligogenic determination for tolerance to octanoic acid is in agreement with published results based on interspecific crosses.     

Development of microsatellite markers for the red tide‐forming harmful species Chattonella antiqua,C. marina,and C. ovata (Raphidophyceae)

We have developed 11 microsatellite markers that are specific to Chattonella antiqua, C. marina, and C. ovata, the red tide‐forming harmful phytoplanktons. The 11 loci were amplified in the three species. The number of alleles per locus ranged from 5 to 16. The three species shared most microsatellite regions, although the genetic differences in specific loci were detected among them. These markers of the Chattonella species will be beneficial for biogeographical, detailed taxonomic, studies.     

Use of microsatellites to evaluate genetic diversity and species relationships in the genus Lycopersicon

In order to determine how informative a set of microsatellites from tomato is across the genus Lycopersicon, 17 microsatellite loci, derived from regions in and around genes, were tested on 31 accessions comprising the nine species of the genus. The microsatellite polymorphisms were used to estimate the distribution of diversity throughout the genus and to evaluate the efficacy of microsatellites for establishing species relationships in comparison with existing phylogeny reconstructions. Gene diversity and genetic distances were calculated. A high level of polymorphism was found, as well as a large number of alleles unique for species. The level of polymorphism detected with the microsatellite loci within and among species was highly correlated with the respective mating systems, cross-pollinating species having a significantly higher gene diversity compared to self-pollinating species. In general, microsatellite-based trees were consistent with a published RFLP-based dendrogram as well as with a published classification based on morphology and the mating system. A tree constructed with low-polymorphic loci (gene diversity <0.245) was shown to represent a more-reliable topology than a tree constructed with more-highly polymorphic loci. Received: 19 February 2001 / Accepted: 26 March 2001     

Isolation and characterization of microsatellite loci in western North American Anodonta species

We have developed and characterized 13 microsatellite loci from a group of Anodonta species in western North America, and demonstrated their utility in populations representing two major clades in this genus. Allelic diversity and polymorphic information content were high for all loci, although these characteristics varied across populations. Deviations from Hardy-Weinberg genotypic ratios were not detected, although the estimated frequency of null alleles was high in one population for one locus. This is the first set of microsatellite loci to be developed for freshwater mussels in western North America, and will be useful for describing gene flow patterns among populations.     

Microsatellite markers for <Emphasis Type="Italic">Juglans cinerea</Emphasis> L. and their utility in other Juglandaceae species

Ten polymorphic microsatellite markers were found to amplify in butternut (Juglans cinerea; Juglandaceae). These microsatellite loci were found to amplify across most of nine other species and five hybrids examined. Loci were highly polymorphic, with 18 to 32 alleles per locus across species. These nuclear microsatellite markers will be useful in examining genetic diversity within and among populations of butternut, and in distinguishing butternut from interspecific hybrids.     

Isolation and characterization of microsatellites in Sparganium emersum and cross‐species amplification in the related species S. erectum

We developed seven novel polymorphic microsatellite loci for the aquatic macrophyte Sparganium emersum (Sparganiaceae). These were characterized on 62 individuals collected from nine different populations. In this set of individuals, seven to 20 alleles per locus were detected and observed heterozygosity ranged between 0.16 and 0.95. Cross‐species amplification was tested in the related species Sparganium erectum, and was successful for five of the seven microsatellite loci.     

Isolation, characterization, and localization of beta-tubulin genomic clones of three Drosophila montium subgroup species.

Genomic libraries were constructed from three Drosophila species, namely Drosophila auraria, Drosophila serrata, and Drosophila kikkawai, belonging to the Drosophila montium subgroup of the Drosophila melanogaster species group. Clones containing beta-tubulin specific sequences were isolated, characterized by restriction endonuclease digestions and Southern hybridizations, and mapped by in situ hybridization on the polytene chromosomes of the species studied. The distribution of the beta-tubulin loci was found to be similar in D. montium species and D. melanogaster.     

Characterisation and analysis of microsatellite loci in a mangrove species, Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

An enriched microsatellite library of the mangrove species Avicennia marina was constructed, in which 85.8% of the clones contained microsatellite sequences. Of the microsatellite repeat sequences isolated, 55.0% were di-nucleotides, 34.2% were tri-nucleotides, 50.0% were perfect, 24.2% were imperfect, and 15.0% were compound. Four different di-nucleotide repeats were isolated with repeat lengths ranging from 5 to 33; ten different tri-nucleotide repeats were isolated with repeat lengths ranging from 3 to 25. The most common di-nucleotide was the AC/TG repeat; the most common tri-nucleotide was the CCG/GGC repeat. Sixteen microsatellite sequences were selected for primer design, and 6 primers were selected to investigate the polymorphism detected among 15 individuals of A. marina from three natural populations in Australia. A total of 40 alleles were detected at 6 microsatellite loci. The number of alleles per microsatellite locus ranged from 5 to 13. On average, 7 alleles were detected per locus. All microsatellite loci showed high levels of gene diversity (heterozygosity), with values ranging from 0.53 to 0.88; the mean value of gene diversity was 0.70. Microsatellite loci were also tested for conservation across Avicennia species. There was a decline in amplification success with increasing divergence between Avicennia species. The results indicate that microsatellites are abundant in the Avicennia genome and can be valuable genetic markers for assessing the effects of deforestation and forest fragmentation in mangrove communities, which is an important issue for mangrove conservation and afforestation schemes. Received: 8 June 1999 / Accepted: 21 September 1999     

Microsatellite variation and evolution in the Mimulus guttatus species complex with contrasting mating systems

Mutational variability at microsatellite loci is shaped by both population history and the mating system. In turn, alternate mating systems in flowering plants can resolve aspects of microsatellite loci evolution. Five species of yellow monkeyflowers (Mimulus sect. Simiolis) differing for historical rates of inbreeding were surveyed for variation at six microsatellite loci. High levels of diversity at these loci were found in both outcrossing and selfing taxa. In line with allozyme studies, inbreeders showed more partitioning of diversity among populations, and diversity in selfing taxa was lower than expected from reductions in effective population size due to selfing alone, suggesting the presence of either population bottlenecks or background selection in selfers. Evaluation of the stepwise mutation model (a model of DNA replication slippage) suggests that these loci evolve in a stepwise fashion. Inferred coalescent times of microsatellite alleles indicate that past bottlenecks of population size or colonization events were important in reducing diversity in the inbreeding taxon.      

Microsatellite loci in the cactophilic species Drosophila antonietae (Diptera; Drosophilidae)

Morphometric and isoenzymatic data have showed little variation among Drosophila antonietae populations. This suggests historic gene flow, due to the distribution of these populations, which are associated with cacti along rivers, and natural selection on these markers. Microsatellite loci have high variability and are ideal for detecting gene flow and analysing population structure. Here we describe the isolation of seven polymorphic microsatellite loci in D. antonietae.     

Microsatellite variation in Drosophila melanogaster and Drosophila simulans: a reciprocal test of the ascertainment bias hypothesis

Interspecific comparisons of microsatellite loci have repeatedly shown that the loci are longer and more variable in the species from which they are derived (the focal species) than are homologous loci in other (nonfocal) species. There is debate as to whether this is due to directional evolution or to an ascertainment bias during the cloning and locus selection processes. This study tests these hypotheses by performing a reciprocal study. Eighteen perfect dinucleotide microsatellite loci identified from a Drosophila simulans library screen and 18 previously identified in an identical Drosophila melanogaster library screen were used to survey natural populations of each species. No difference between focal and nonfocal species was observed for mean PCR fragment length. However, heterozygosity and number of alleles were significantly higher in the focal species than in the nonfocal species. The most common allele in the Zimbabwe population of both species was sequenced for 31 of the 36 loci. The length of the longest stretch of perfect repeat units is, on average, longer in the focal species than in the non-focal species. There is a positive correlation between the length of the longest stretch of perfect repeats and heterozygosity. The difference in heterozygosity can thus be explained by a reduction in the length of the longest stretch of perfect repeats in the nonfocal species. Furthermore, flanking-sequence length difference was noted between the two species at 58% of the loci sequenced. These data do not support the predictions of the directional-evolution hypothesis; however, consistent with the ascertainment bias hypothesis, the lower variability in nonfocal species is an artifact of the microsatellite cloning and isolation process. Our results also suggest that the magnitude of ascertainment bias for repeat unit length is a function of the microsatellite size distribution in the genomes of different species.