Rate and pattern of mutation at microsatellite loci in maize

Microsatellites are important tools for plant breeding, genetics, and evolution, but few studies have analyzed their mutation pattern in plants. In this study, we estimated the mutation rate for 142 microsatellite loci in maize (Zea mays subsp. mays) in two different experiments of mutation accumulation. The mutation rate per generation was estimated to be 7.7 x 10(-4) for microsatellites with dinucleotide repeat motifs, with a 95% confidence interval from 5.2 x 10(-4) to 1.1 x 10(-3). For microsatellites with repeat motifs of more than 2 bp in length, no mutations were detected; so we could only estimate the upper 95% confidence limit of 5.1 x 10(-5) for the mutation rate. For dinucleotide repeat microsatellites, we also determined that the variance of change in the number of repeats (sigma(m)2) is 3.2. We sequenced 55 of the 73 observed mutations, and all mutations proved to be changes in the number of repeats in the microsatellite or in mononucleotide tracts flanking the microsatellite. There is a higher probability to mutate to an allele of larger size. There is heterogeneity in the mutation rate among dinucleotide microsatellites and a positive correlation between the number of repeats in the progenitor allele and the mutation rate. The microsatellite-based estimate of the effective population size of maize is more than an order of magnitude less than previously reported values based on nucleotide sequence variation.     

MICROSATELLITE EVOLUTION IN VERTEBRATES: INFERENCE FROM AC DINUCLEOTIDE REPEATS

Abstract We analyze published data from 592 AC microsatellite loci from 98 species in five vertebrate classes including fish, reptiles, amphibians, birds, and mammals. We use these data to address nine major questions about microsatellite evolution. First, we find that larger genomes do not have more microsatellite loci and therefore reject the hypothesis that microsatellites function primarily to package DNA into chromosomes. Second, we confirm that microsatellite loci are relatively rare in avian genomes, but reject the hypothesis that this is due to physical constraints imposed by flight. Third, we find that microsatellite variation differs among species within classes, possibly relating to population dynamics. Fourth, we reject the hypothesis that microsatellite structure (length, number of alleles, allele dispersion, range in allele sizes) differs between poikilotherms and homeotherms. The difference is found only in fish, which have longer microsatellites and more alleles than the other classes. Fifth, we find that the range in microsatellite allele size at a locus is largely due to the number of alleles and secondarily to allele dispersion. Sixth, length is a major factor influencing mutation rate. Seventh, there is a directional mutation toward an increase in microsatellite length. Eighth, at the species level, microsatellite and allozyme heterozygosity covary and therefore inferences based on large-scale studies of allozyme variation may also reflect microsatellite genetic diversity. Finally, published microsatellite loci (isolated using conventional hybridization methods) provide a biased estimate of the actual mean repeat length of microsatellites in the genome.     

Analysis of allele frequency of seven microsatellite loci of Y chromosome in three Tuva populations

The allele frequency distribution of seven microsatellite loci of the nonrecombining region of the Y chromosome (Y-STRs) was analyzed in three geographically distant indigenous populations of the Tuva Republic. The populations did not differ in allele frequency distribution of the seven Y-STRs. The Y-chromosome microsatellite loci in Tuvinians showed a high diversity (H = 0.575) that was nearly identical in all three populations. The genetic distance Ddm between the three populations was low, suggesting no subdivision of the modern male population of Tuva. Estimates of the period of linear changes in Ddm showed that Y-chromosome microsatellites can be used to reconstruct evolutionary events dating back no more than 40,000-50,000 years. The problems of human population phylogeny are discussed on the basis of data on Y-chromosome STRs.     

Microsatellites in Zea - variability,patterns of mutations,and use for evolutionary studies

To evaluate the performance of microsatellites or simple sequence repeats (SSRs) for evolutionary studies in Zea, 46 microsatellite loci originally derived from maize were applied to diverse arrays of populations that represent all the diploid species of Zea and 101 maize inbreds. Although null phenotypes and amplification of more than two alleles per plant were observed at modest rates, no practical obstacle was encountered for applying maize microsatellites to other Zea species. Sequencing of microsatellite alleles revealed complex patterns of mutation including frequent indels in the regions flanking microsatellite repeats. In one case, all variation at a microsatellite locus came from indels in the flanking region rather than in the repeat motif. Maize microsatellites show great variability within populations and provide a reliable means to measure intraspecific variation. Phylogeographic relationships of Zea populations were successfully reconstructed with good resolution using a genetic distance based on the infinite allele model, indicating that microsatellite loci are useful in evolutionary studies in Zea. Microsatellite loci show a principal division between tropical and temperate inbred lines, and group inbreds within these two broad germplasm groups in a manner that is largely consistent with their known pedigrees. Received: 10 February 2001 / Accepted: 21 May 2001     

Evolutionary history of microsatellites in the obscura group of Drosophila

The evolutionary origins of microsatellites are not well understood. Some investigators have suggested that point mutations that expand repeat arrays beyond a threshold size trigger microsatellites to become variable. However, little empirical data has been brought forth on this and related issues. In this study, we examine the evolutionary history of microsatellites in six species within the obscura group of Drosophila, tracing changes in microsatellite alleles using both PCR product size and sequence data. We found little evidence supporting a general role of point mutations triggering initial microsatellite expansion, and no consistent threshold size for expansion was observed. Flanking region length variation was extensive when alleles were sequenced in distantly related species, and some species possessed altogether different repeat arrays between the same primer binding sites. Our results suggest extreme caution in using microsatellite allele sizes for phylogenetic analyses or to infer divergences between populations.     

Variability of GATA-microsatellite DNA in populations of the parthenogenetic species of lizard Lacerta unisexualis Darevsky

Multilocus DNA fingerprinting was used to analyze the genome variation of mini- and microsatellite DNA regions in parthenogenetic Caucasian rock lizard Lacerta unisexualis. The DNA fingerprints obtained with probe M13 were nearly identical in all populations examined (the average similarity index S = 0.992). The fingerprints obtained with probe (GATA)4 varied (S = 0.862). Polymorphic fragments were assumed to correspond to allelic variants of genetically unstable GATA loci. Comparison of the fingerprints of animals from four geographically isolated populations revealed several population-specific GATA microsatellite markers. Based on their distribution among the populations, the corresponding alleles were assumed to originate from a common ancestral allele.     

Development and characterization of microsatellite loci in endangered Astrophytum asterias (Cactaceae)

We report the development of polymorphic microsatellite markers for the endangered North American cactus Astrophytum asterias (Cactaceae). Six loci, averaging 8.5 alleles per locus, were found to amplify genomic DNA consistently in 94 individuals from four geographically defined demes in South Texas. These markers will permit the generation of appropriate data for estimating population genetic parameters, population structure and the degree of inbreeding in the small, fragmented populations of A. asterias that currently exist. These are the first microsatellites reported for the genus Astrophytum and for the tribe Cacteae.     

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.     

Genetic variation and differentiation at microsatellite loci in Drosophila simulans. Evidence for founder effects in new world populations.

Drosophila simulans isofemale lines from Africa, South America, and two locations in North America were surveyed for variation at 16 microsatellite loci on the X, second, and third chromosomes, and 18 microsatellites, which are unmapped. D. simulans is thought to have colonized New World habitats only relatively recently (within the last few hundred years). Consistent with a founder effect occurring as colonizers moved into these New World habitats, we find less microsatellite variability in North and South American D. simulans populations than for an African population. Population subdivision as measured at microsatellites is moderate when averaged across all loci (FST = 0.136), but contrasts sharply with previous studies of allozyme variation, which have showed significantly less differentiation in D. simulans than in D. melanogaster. There are substantially fewer private alleles observed in New World populations of D. simulans than seen in a similar survey of D. melanogaster. In addition to possible differences in population size during their evolutionary histories, varying colonization histories or other demographic events may be necessary to explain discrepancies in the patterns of variation observed at various genetic markers between these closely related species.     

Directional evolution of stockiness coevolves with ecology and locomotion in lizards

Although studied in many taxa, directional macroevolution remains difficult to detect and quantify. We present an approach for detecting directional evolution in subclades of species when relatively few species are sampled, and apply it to studying the evolution of stockiness in Phrynosomatine lizards. Our approach is more sensitive to detecting the tempo of directional evolution than other available approaches. We use ancestral reconstruction and phylogenetic mapping of morphology to characterize the direction and magnitude of trait evolution. We demonstrate a directional trend toward stockiness in horned lizards, but not their sister groups, finding that stockier species tend to have relatively short and wide bodies, and relatively short heads, tails, and limbs. Ornstein–Uhlenbeck models show that the directional trend in horned lizards is due to a shift in selective regime and stabilizing selection as opposed to directional selection. Bayesian evolutionary correlation analyses indicate that stockier species run more slowly and eat a larger proportion of ants. Furthermore, species with larger horns tend to be slower and more ant-specialized. Directional evolution toward a stocky body shape has evolved in conjunction with changes in a suite of traits, representing a complex example of directional macroevolution.     

Microsatellite Variation in Two Populations of Free-Ranging Yellow Baboons (Papio hamadryas cynocephalus)

We investigated genetic variation at six microsatellite (simple sequence repeat) loci in yellow baboons (Papio hamadryas cynocephalus) at two localities: the Tana River Primate Reserve in eastern Kenya and Mikumi National Park, central Tanzania. The six loci (D1S158, D2S144, D4S243, D5S1466, D16S508, and D17S804) were all originally cloned from and characterized in the human genome. These microsatellites are polymorphic in both baboon populations, with the average heterozygosity across loci equal to 0.731 in the Tana River sample and 0.787 in the Mikumi sample. The genetic differentiation between the two populations is substantial. Kolmogornov–Smirnov tests indicate that five of the six loci are significantly different in allele frequencies in the two populations. The mean F _ST across loci is 0.069, and Shriver's measure of genetic distance, which was developed for microsatellite loci (Shriver et al., 1995), is 0.255. This genetic distance is larger than corresponding distances among human populations residing in different continents. We conclude that (a) the arrays of alleles present at these six microsatellite loci in two geographically separated populations of yellow baboons are quite similar, but (b) the two populations exhibit significant differences in allele frequencies. This study illustrates the potential value of human microsatellite loci for analyses of population genetic structure in baboons and suggests that this approach will be useful in studies of other Old World monkeys.     

Homoplasy and mutation model at microsatellite loci and their consequences for population genetics analysis

Homoplasy has recently attracted the attention of population geneticists, as a consequence of the popularity of highly variable stepwise mutating markers such as microsatellites. Microsatellite alleles generally refer to DNA fragments of different size (electromorphs). Electromorphs are identical in state (i.e. have identical size), but are not necessarily identical by descent due to convergent mutation(s). Homoplasy occurring at microsatellites is thus referred to as size homoplasy. Using new analytical developments and computer simulations, we first evaluate the effect of the mutation rate, the mutation model, the effective population size and the time of divergence between populations on size homoplasy at the within and between population levels. We then review the few experimental studies that used various molecular techniques to detect size homoplasious events at some microsatellite loci. The relationship between this molecularly accessible size homoplasy size and the actual amount of size homoplasy is not trivial, the former being considerably influenced by the molecular structure of microsatellite core sequences. In a third section, we show that homoplasy at microsatellite electromorphs does not represent a significant problem for many types of population genetics analyses realized by molecular ecologists, the large amount of variability at microsatellite loci often compensating for their homoplasious evolution. The situations where size homoplasy may be more problematic involve high mutation rates and large population sizes together with strong allele size constraints.     

A private allele ubiquitous in the Americas

The three-wave migration hypothesis of Greenberg et al. has permeated the genetic literature on the peopling of the Americas. Greenberg et al. proposed that Na-Dene, Aleut-Eskimo and Amerind are language phyla which represent separate migrations from Asia to the Americas. We show that a unique allele at autosomal microsatellite locus D9S1120 is present in all sampled North and South American populations, including the Na-Dene and Aleut-Eskimo, and in related Western Beringian groups, at an average frequency of 31.7%. This allele was not observed in any sampled putative Asian source populations or in other worldwide populations. Neither selection nor admixture explains the distribution of this regionally specific marker. The simplest explanation for the ubiquity of this allele across the Americas is that the same founding population contributed a large fraction of ancestry to all modern Native American populations.     

Variation of microsatellites BM224 and Bcal7 in populations of green toads (Bufo viridis complex) with various nuclear DNA content and ploidy

We studied variation of microsatellites BM224 and Bcal7 in three species of the Bufo viridis diploid-polyploid complex. We found that locus Bcal7 in all examined samples was monomorphic. Three alleles of microsatellite BM224 were found. Among tetraploid toads, the western species B. oblongus had only one allele variant, whereas the eastern species B. pewzowi had two other alleles. Similar distribution of alleles was observed in triploid specimens, collected in the area borders of tetraploid and diploid species. Among samples of diploid toad B. viridis, we found all three allele variants of microsatellite BM224. Their distribution was geographically determined. A comparison of allele distribution with genome size variation in diploid toads showed very similar patterns.     

Microsatellite loci in eelgrass Zostera marina reveal marked polymorphism within and among populations

Using an enriched genomic library, we developed seven (CT)n/(GA)n microsatellite loci for eelgrass Zostera marina L. Enrichment is described and highly recommended for genomes in which microsatellites are rare, such as in many plants. A test for polymorphism was performed on individuals from three geographically separated populations (N = 15/population) and revealed considerable genetic variation. The number of alleles per locus varied between five and 11 and the observed heterozygosities for single loci ranged from 0.16 to 0.81 within populations. Mean allele lengths were markedly different among populations, indicating that the identified loci will be useful in studying population structure in Z. marina. As the frequency of the most abundant multilocus genotype within populations was always < 1%, these loci have sufficient resolving power to address clone size in predominantly vegetatively reproducing populations.     

Rapid evolution of mammalian X-linked testis microRNAs

Background

Microsatellites in cDNA are useful as molecular markers because they represent transcribed genes and can be used as anchor markers for linkage and comparative mapping, as well as for studying genome evolution. Microsatellites in cDNA can be detected in existing ESTs by data mining. However, in most fish species, no ESTs are available or the number of ESTs is limited, although fishes represent half of the vertebrates on the earth. We developed a simple and efficient method for isolation of microsatellites from cDNA in fish.

Results

The method included normalization of 150 ng cDNA using 0.5 U duplex-specific nuclease (DSN) at 65°C for 30 min, enrichment of microsatellites using biotinylated oligonucleotides and magnetic field, and directional cloning of cDNA into a vector. We tested this method to enrich CA- and GA-microsatellites from cDNA of Asian seabass, and demonstrated that enrichment of microsatellites from normalized cDNA could increased the efficiency of microsatellite isolation over 30 times as compared to direct sequencing of clones from cDNA libraries. One hundred and thirty-nine (36.2%) out of 384 clones from normalized cDNA contained microsatellites. Unique microsatellite sequences accounted for 23.6% (91/384) of sequenced clones. Sixty microsatellites isolated from cDNA were characterized, and 41 were polymorphic. The average allele number of the 41 microsatellites was 4.85 ± 0.54, while the expected heterozygosity was 0.56 ± 0.03. All the isolated microsatellites inherited in a Mendelian pattern.

Conclusion

Normalization of cDNA substantially increased the efficiency of enrichment of microsatellites from cDNA. The described method for isolation of microsatellites from cDNA has the potential to be applied to a wide range of fish species. The microsatellites isolated from cDNA could be useful for linkage and comparative mapping, as well as for studying genome evolution.     

A novel genome‐wide microsatellite resource for species of Eucalyptus with linkage‐to‐physical correspondence on the reference genome sequence

Keystone species in their native ranges, eucalypts, are ecologically and genetically very diverse, growing naturally along extensive latitudinal and altitudinal ranges and variable environments. Besides their ecological importance, eucalypts are also the most widely planted trees for sustainable forestry in the world. We report the development of a novel collection of 535 microsatellites for species of Eucalyptus, 494 designed from ESTs and 41 from genomic libraries. A selected subset of 223 was evaluated for individual identification, parentage testing, and ancestral information content in the two most extensively studied species, Eucalyptus grandis and Eucalyptus globulus. Microsatellites showed high transferability and overlapping allele size range, suggesting they have arisen still in their common ancestor and confirming the extensive genome conservation between these two species. A consensus linkage map with 437 microsatellites, the most comprehensive microsatellite‐only genetic map for Eucalyptus, was built by assembling segregation data from three mapping populations and anchored to the Eucalyptus genome. An overall colinearity between recombination‐based and physical positioning of 84% of the mapped microsatellites was observed, with some ordering discrepancies and sporadic locus duplications, consistent with the recently described whole genome duplication events in Eucalyptus. The linkage map covered 95.2% of the 605.8‐Mbp assembled genome sequence, placing one microsatellite every 1.55 Mbp on average, and an overall estimate of physical to recombination distance of 618 kbp/cM. The genetic parameters estimates together with linkage and physical position data for this large set of microsatellites should assist marker choice for genome‐wide population genetics and comparative mapping in Eucalyptus.     

Strong founder effect in Drosophila pseudoobscura colonizing New Zealand from North America

The North American native species Drosophila pseudoobscura was first identified in New Zealand in the last few decades. Here, we have studied the genetic consequences of its spread across the Pacific Ocean. Using 10 microsatellites that are highly variable in North American populations, we found that the New Zealand population has substantially fewer alleles, a much lower average heterozygosity, and significantly different allele frequencies at these loci. We have discussed the relative sensitivity of these parameters for detecting the founding event. X-linked loci were more strongly differentiated between continents than autosomal loci, as reflected by larger changes in allele frequencies and greater reductions in numbers of alleles and average heterozygosity. The severity of the genetic diversity loss supports a scenario of a few D. pseudoobscura females being introduced to New Zealand from North America.     

Polymorphism in ornamental and common carp strains (Cyprinus carpio L.) as revealed by AFLP analysis and a new set of microsatellite markers

Forty-seven new microsatellite markers were generated and applied, together with the AFLP (Amplified Fragment Length Polymorphism) technique using two different enzyme combinations, to the genetic analysis of two carp species, Cyprinus carpio L. and Ctenopharyngodon idella. The extent of polymorphism and the genetic relationships between nine carp populations were studied. The incidence of microsatellites containing CA and CT motifs was estimated to be one every 17.4 and one every 126.3 kb, respectively, and their average allele numbers were four and five, respectively. Across populations, the average proportion of individuals that were heterozygous for microsatellite markers was 44.2% and the average allele number was 4.02. The EcoRI/TaqI combination generated more analyzable AFLP bands than the EcoRI/MseI pair, making the former preferable for the analysis of carp populations. The proportion of polymorphic AFLP bands within populations ranged from 6.7% in grass carp to 59.9% in Kohaku strain (Koi) of the ornamental carp. The fixation index (FST) for microsatellites in these populations was estimated to be 0.37, and for AFLP markers the value was 0.39. Genetic distance matrices derived from microsatellites and from two AFLP analyses were positively correlated. Grass carp showed fewer AFLP bands than other populations and was genotyped by only half of the microsatellite markers. These findings agree with genetic distance estimates in suggesting that the grass carp is phylogenetically quite remote from all the other populations examined.     

What phylogeny and gene genealogy analyses reveal about homoplasy in citrus microsatellite alleles

Sixty-five microsatellite alleles amplified from ancestral citrus accessions classified in three separate genera were evaluated for sequence polymorphism to establish the basis of inter- and intra-allelic genetic variation, evaluate the extent of size homoplasy, and determine an appropriate model (stepwise or infinite allele) for analysis of citrus microsatellite alleles. Sequences for each locus were aligned and subsequently used to determine relationships between alleles of different taxa via parsimony. Interallelic size variation at each SSR locus examined was due to changes in repeat copy number with one exception. Sequencing these alleles uncovered new distinct point mutations in the microsatellite region and the region flanking the microsatellite. Several of the point mutations were found to be genus, species, or allele specific, and some mutations were informative about the inferred evolutionary relationships among alleles. Overall, homoplasy was observed in alleles from all three loci, where the core microsatellite repeat was changed causing alleles of the same size class to be identical in state but not identical by descent. Because nearly all changes in allele size (with one exception) were due to expansion or contraction of the repeat motif, this suggests that a stepwise mutation model, which assumes homoplasy may occur, would be the most appropriate for analyzing Citrus SSR data. The collected data indicate that microsatellites can be a useful tool for evaluating Citrus species and two related genera since repeat motifs were reasonably well retained. However, this work also demonstrated that the number of microsatellite alleles is clearly an underestimate of the number of sequence variants present.