Hypervariable Microsatellite Markers Research Articles

Genome-wide mining of potentially-hypervariable microsatellites and validation of markers in Momordica charantia L.

Relatively large number of bitter melon microsatellite markers have been reported; however, only few resulted in successful PCR amplification and a small fraction shown polymorphisms. This limited chance of recovering polymorphic markers makes the primer screening a cost-demanding process. To test the hypothesis that microsatellites with longer motifs as well as shorter motifs repeated substantially shall have better prospects to be polymorphic, we performed a genome-wide microsatellite mining. We selected a sample of genome-wide microsatellites with prescribed motif lengths or satisfying a target repeat number, which were considered potentially-hyper variable, for primer designing and validation. Seventy five microsatellites satisfying these criteria were identified, of which 69 were validated through successful PCR amplification. Among them, 40 (53.33% of the markers identified) were polymorphic. This result showed a significantly higher success compared to our initial results of 51 (20.64%) polymorphic markers out of the 188 amplified when 247 previously reported markers were screened. The screening of two cultivars revealed that markers were efficient to identify up to three alleles. The characterization of these 69 new markers with 247 markers previously reported showed that di-nucleotide motifs were most abundant, followed by tri- and tetra-nucleotide motifs. TC motif markers were most polymorphic (12.08%) followed by AG and CT motifs (both 9.89%). Similarly, AGA (6.59%) and TATT (3.29%) were most polymorphic among the tri- and tetra-nucleotide motifs. These 69 hypervariable microsatellite markers along with 188 markers initially validated in this study shall be useful for phylogenetic analyses, studies of linkage, QTL, and association mapping in bitter melon.

Diversity Analysis of Sweet Potato Genetic Resources Using Morphological and Qualitative Traits and Molecular Markers.

The European Union (EU) market for sweet potatoes has increased by 100% over the last five years, and sweet potato cultivation in southern European countries is a new opportunity for the EU to exploit and introduce new genotypes. In view of this demand, the origins of the principal Italian sweet potato clones, compared with a core collection of genotypes from Central and Southern America, were investigated for the first time. This was accomplished by combining a genetic analysis, exploiting 14 hypervariable microsatellite markers, with morphological and chemical measurements based on 16 parameters. From the molecular analyses, Italian accessions were determined to be genetically very similar to the South American germplasm, but they were sub-clustered into two groups. This finding was subsequently confirmed by the morphological and chemical measurements. Moreover, the analysis of the genetic structure of the population suggested that one of the two groups of Italian genotypes may have descended from one of the South American accessions, as predicted on the basis of the shared morphological characteristics and molecular fingerprints. Overall, the combination of two different characterization methods, genetic markers and agronomic traits, was effective in differentiating or clustering the sweet potato genotypes, in agreement with their geographical origin or phenotypic descriptors. This information could be exploited by both breeders and farmers to detect and protect commercial varieties, and hence for traceability purposes.

Characterization of 32 microsatellite loci for the Pacific red snapper, Lutjanus peru, through next generation sequencing.

We developed a set of hypervariable microsatellite markers for the Pacific red snapper (Lutjanus peru), an economically important marine fish for small-scale fisheries in the west coast of Mexico. We performed shotgun genome sequencing with the 454 XL titanium chemistry and used bioinformatic tools to search for perfect microsatellite loci. We selected 66 primer pairs that were synthesized and genotyped in an ABI PRISM 3730XL DNA sequencer in 32 individuals from the Gulf of California. We estimated levels of genetic diversity, deviations from linkage and Hardy-Weinberg equilibrium, estimated the frequency of null alleles and the probability of individual identity for the new markers. We reanalyzed 16 loci in 16 individuals to estimate genotyping error rates. Eighteen loci failed to amplify, 16 loci were discarded due to unspecific amplifications and 32 loci (14 tetranucleotide and 18 dinucleotide) were successfully scored. The average number of alleles per locus was 21 (±6.87, SD) and ranged from 8 to 34. The average observed and expected heterozygosities were 0.787 (±0.144 SD, range 0.250-0.935) and 0.909 (±0.122 SD, range 0.381-0.965), respectively. No significant linkage was detected. Eight loci showed deviations from Hardy-Weinberg equilibrium, and from these, four loci showed moderate null allele frequencies (0.104-0.220). The probability of individual identity for the new loci was 1.46-62. Genotyping error rates averaged 9.58%. The new markers will be useful to investigate patterns of larval dispersal, metapopulation dynamics, fine-scale genetic structure and diversity aimed to inform the implementation of spatially explicit fisheries management strategies in the Gulf of California.

Regional genetic population structure and fine scale genetic cohesion in the Southern blue whiting Micromesistius australis

Southern blue whiting Micromesistus australis support a substantial commercial fishery in South America. There is growing evidence of a high level of demographic independence between stocks associated with the two main spawning grounds, one in the SW Atlantic (SWA) and one in the SE Pacific (SEP), but the potential genetic structuring of these stocks is unknown. In this study adults collected from sites throughout SWA and SEP waters were genotyped at hypervariable microsatellite markers to investigate genetic structuring between and within regions. Allele frequency-based analyses reported highly significant genetic differentiation between regions, indicating low levels of allo-recruitment. Ancillary data on migratory behaviours support natal homing as a prominent stock isolating mechanism. Genetic differentiation was also detected among samples from around the Falkland Islands: kinship analyses indicated that this was due to non-random genetic relatedness within samples. Despite a general pattern of genetic homogeneity among SEP samples, the northernmost sample exhibited significantly high mean relatedness. The data indicate the occurrence of a further level of structuring within both regions that prevents complete mixing, specifically that schools may be hierarchically structured (from putative subpopulations down to kin-containing groups) and exhibit some degree of ontogenetic cohesion, which may also be a component of homing. The importance of homing and group cohesion as factors influencing resilience to, and recovery from, overexploitation is discussed. This study represents an important baseline for future genetic monitoring and assessment of M. australis. The need for such studies is emphasised by the observation of significantly lower levels of genetic variation among SWA samples, which may reflect genetic erosion, and the subsequent collapse of the SWA stock.

Development and characterization of novel microsatellite markers by Next Generation Sequencing for the blue and red shrimp Aristeus antennatus.

The blue and red shrimp, Aristeus antennatus, is a commercially important crustacean, in the Mediterranean Sea, which has been listed as a priority species for fishery management. Hypervariable microsatellite markers could be a useful tool to identify genetic stocks among geographically close fishing grounds. Potential microsatellite markers (97) identified from next-generation sequencing of an individual shrimp using a 454 GS Junior Pyrosequencer were tested on a preliminary panel of 15 individuals representing the four worldwide genetic stocks of the species from which 35 polymorphic loci were identified and used to characterize an additional 20 individuals from the Western Mediterranean Sea. In the Western Mediterranean sample, 32 out of 35 were polymorphic loci and the number of alleles per locus ranged from 2 to 14 and expected heterozygosity ranged from 0.050 to 0.968. No linkage disequilibrium was detected, indicating the independence of the loci. These novel microsatellites provide additional tools to address questions relating to genetic diversity, parentage studies and connectivity patterns of A. antennatus populations and help develop effective strategies to ensure long-term sustainability of this resource.

Genetic diversity in the rice landraces (Oryza sativa L.) of coastal Sundarbans (India) and their adaptation to the local saline condition investigated both at molecular and physiological level

Salinity is one of the major impediments to increase production in rice growing areas worldwide as the crop is sensitive to saline conditions. The key to breed new rice varieties depends on utilization of genetic diversity in landraces that possess many useful genes/alleles. The diversity present among the fifty rice accessions, collected from 4 geographical areas of Sundarbans, India, an area well known for its genetic diversity, was analysed at the genomic level by employing hyper variable microsatellite markers. As these accessions are well adapted to saline conditions, haplotype analysis conducted on Saltol locus, which has four genes that are known to confer salinity in rice, could identify eight different haplotypes. Wide variation was also seen for Na+/K+ ratio among the accessions and an association was detected between markers RM8094, RM3412 and RM493 and Na+/K+ ratio of rice plants under salinity stress. Based on the morphological, physiological and molecular analyses, Nonabokra, Kumrogoor and Kalonunia were identified as potential donors as the tolerance in these genotypes appear to be regulated by alleles other than the ones present in FL478, a well-known donor for salinity in rice.

Molecular Profiling of Major Indian Rice Cultivars Using a Set of Eight Hypervariable Microsatellite Markers

India bred high yielding rice varieties have enriched to a great extent the global rice germplasm since the mid-sixties. Systematic research efforts for development of cultivar-specific DNA fingerprints of major Indian rice cultivars, however, have not received due attention. The present investigation was aimed at development of DNA fingerprints for 90 high yielding rice varieties using hypervariable microsatellite (hvRM) markers. A panel of eight markers, viz. RM11313, RM13584, RM15004, RM5844, RM22250, RM22565, RM24260 and RM8207 was chosen from 52 polymorphic markers based on their highly polymorphic nature, SSR repeat type and number and ability to distinguish genotypes, in order to develop DNA fingerprints of 90 varieties. The remaining high polymorphic hvRM markers could be of immense value in future to distinguish new cultivars, in case they could not be distinguished by the 8 marker panel. Four of the 8 markers, viz. RM22250, RM13584, RM24260 and RM5844 were located in expressed genes and could be of value in DUS (Distinctness, Uniformity and Stability) testing. Thus we suggested, that this set of 8 loci could be used as standard for DNA fingerprinting of Indian rice cultivars.

Impacts of selective logging on inbreeding and gene flow in two Amazonian timber species with contrasting ecological and reproductive characteristics.

Selective logging in Brazil allows for the removal of up to 90% of trees above 50cm diameter of a given timber species, independent of a species' life history characteristics or how quickly it will recover. The genetic and demographic effects of selective logging on two Amazonian timber species (Dipteryx odorata Leguminosae, Jacaranda copaia Bignoniaceae) with contrasting ecological and reproductive characteristics were assessed in the same forest. Genetic diversity and gene flow were characterized by genotyping adults and seed sampled before and after logging, using hypervariable microsatellite markers. Overall, there were no short-term genetic impacts on the J.copaia population, with commercial application of current Brazilian forest management regulations. In contrast, for D.Odorata, selective logging showed a range of genetic impacts, with a 10% loss of alleles, and reductions in siring by pollen from trees within the 546-ha study area (23-11%) and in the number of pollen donors per progeny array (2.8-1.6), illustrating the importance of the surrounding landscape. Asynchrony in flowering between D.odorata trees led to trees with no breeding partners, which could limit the species reproduction and regeneration under current regulations. The results are summarized with other published studies from the same site and the implications for forest management discussed. The different types and levels of impacts associated with each species support the idea that ecological and genetic information by species, ecological guild or reproductive group is essential in helping to derive sustainable logging guidelines for tropical forests.

Molecular Genetic Diversity of Major Indian Rice Cultivars over Decadal Periods

Genetic diversity in representative sets of high yielding varieties of rice released in India between 1970 and 2010 was studied at molecular level employing hypervariable microsatellite markers. Of 64 rice SSR primer pairs studied, 52 showed polymorphism, when screened in 100 rice genotypes. A total of 184 alleles was identified averaging 3.63 alleles per locus. Cluster analysis clearly grouped the 100 genotypes into their respective decadal periods i.e., 1970s, 1980s, 1990s and 2000s. The trend of diversity over the decadal periods estimated based on the number of alleles (Na), allelic richness (Rs), Nei’s genetic diversity index (He), observed heterozygosity (Ho) and polymorphism information content (PIC) revealed increase of diversity over the periods in year of releasewise and longevitywise classification of rice varieties. Analysis of molecular variance (AMOVA) suggested more variation in within the decadal periods than among the decades. Pairwise comparison of population differentiation (Fst) among decadal periods showed significant difference between all the pairs except a few. Analysis of trends of appearing and disappearing alleles over decadal periods showed an increase in the appearance of alleles and decrease in disappearance in both the categories of varieties. It was obvious from the present findings, that genetic diversity was progressively on the rise in the varieties released during the decadal periods, between 1970s and 2000s.

Genetic diversity of microsatellite alleles located at quantitative resistance loci for Ascochyta blight resistance in a global collection of chickpea germplasm

A global collection of 43 chickpea (Cicer arietinum L.) genotypes, resistant and susceptible to Ascochyta blight caused by Ascochyta rabiei was evaluated for the disease under controlled conditions. In this study three known pathotypes (P-I, P-II, and P-III) were used to evaluate the reactions of this collection. the chickpea genotypes were also characterized using 14 microsatellite markers flanking the genomic regions associated with Ascochyta blight resistance quantitative trait loci (QTLs). Phenotyping results indicated that 27 genotypes were re- sistant to P-I, 14 to P-II, and five to P-III, revealing the possible erosion of resistance through the evolution of virulent pathogen pathotypes. the genetic diversity analysis revealed 67 alleles at 14 microsatellite loci with an average of 4.8 alleles per locus among the genotypes tested. Genetic similarity estimates differentiated four subclusters (A, B, C, and D) of the genotypes. However, none of sub-clusters were separated into resistant genotypes for a specific pathotype. The genetic diversity ranged from 0.48 to 0.80 which indicated that there is considerable variation in QTL regions associated with Ascochyta blight resistance among the collections of chickpea genotypes studied, as assessed using the hyper-variable microsatellite markers.

Potentiality of evenly distributed hypervariable microsatellite markers in marker-assisted breeding of rice

With 4 figures and 1 table Abstract To provide ready-to-use markers for background selection in marker-assisted breeding of rice, we have extracted 840 hypervariable microsatellite markers (hvRMs) evenly distributed in the genome from 18 828 Class I simple sequence repeats. These hvRMs were grouped into bins of 5-Mb intervals with 5–8 bins depending on the chromosome length. In each bin, 10 hvRMs were selected based on their microsatellite repeat number and type. Of 840, a representative of 36 hvRMs were selected and its polymorphic information content (PIC) was estimated by amplifying 24 diverse rice cultivars. The findings revealed that hvRMs show high PIC values and more alleles when compared to the 25 randomly chosen microsatellite markers. The informative hvRMs developed for background selection across cultivars in this study along with the already established molecular markers tightly linked to the genes of agronomic value could be of value for the development of a ‘MAS kit’ for speeding up the efforts of plant breeders for targeted crop improvement.

Spawning frequency of brood dams and sires in a marine fish stock‐enhancement hatchery

Parentage analysis, employing five hypervariable microsatellite markers, was used to follow spawning patterns of red drum Sciaenops ocellatus broodfish in two spawning tanks through most of a calendar year in a marine fish hatchery dedicated to stock enhancement. Five of six dams and all four sires spawned at least once during the year. Variation in dam and sire spawning incidence and in number of progeny produced per dam and per sire translated into reduced genetic effective size (N e) per spawn by 40·6% in one tank and 50·8% in the other.

Population structure of Tunisian Leishmania infantum and evidence for the existence of hybrids and gene flow between genetically different populations

Twenty-seven strains of Leishmania infantum from north and central Tunisia belonging to the three main MON zymodemes (the MON-typing system is based on multilocus enzyme electrophoresis (MLEE) of 15 enzymes) found in this country (MON-1, MON-24 and MON-80) and representing different pathologies (visceral, cutaneous and canine leishmaniasis) have been studied to understand the genetic polymorphism within this species. Intraspecific variation could be detected in L. infantum by the use of 14 hypervariable microsatellite markers. In addition to microsatellite repeat length variation, a high degree of allelic heterozygosity has been observed among the strains investigated, suggestive of sexual recombination within L. infantum groups. The two major clusters found by using Bayesian statistics as well as distance analysis are consistent with the classification based on isoenzymes, dividing Tunisian L. infantum into MON-1 and MON-24/MON-80. Moreover, the existence of hybrid strains between the MON-1 and the non-MON-1 populations has been shown and verified by analysis of clones of one of these strains. Substructure analysis discriminated four groups of L. infantum. The major MON-1 cluster split into two groups, one comprising only Tunisian strains and the second both Tunisian and European strains. The major MON-24 cluster was subdivided into two groups with geographical and clinical feature correlations: a dermotropic group of strains mainly from the north, and a viscerotropic group of strains from the centre of Tunisia. The four viscerotropic hybrid strains all originated from central Tunisia and were typed by MLEE as MON-24 or MON-80. To our knowledge, this is the first report describing relationships between clinical picture and population substructure of L. infantum MON-24 based on genotype data, as well as the existence of hybrids between zymodemes MON-1 and MON-24/MON-80, and proving one of these hybrid strains by molecular analysis of the parent strain and its clones.

Vicariance patterns in the Mediterranean Sea: east–west cleavage and low dispersal in the endemic seagrass Posidonia oceanica

AbstractAim The seagrass, Posidonia oceanica is a clonal angiosperm endemic to the Mediterranean Sea. Previous studies have suggested that clonal growth is far greater than sexual recruitment and thus leads to low clonal diversity within meadows. However, recently developed microsatellite markers indicate that there are many different genotypes, and therefore many distinct clones present. The low resolution of markers used in the past limited our ability to estimate clonality and assess the individual level. New high‐resolution dinucleotide microsatellites now allow genetically distinct individuals to be identified, enabling more reliable estimation of population genetic parameters across the Mediterranean Basin. We investigated the biogeography and dispersal of P. oceanica at various spatial scales in order to assess the influence of different evolutionary factors shaping the distribution of genetic diversity in this species.Location The Mediterranean.Methods We used seven hypervariable microsatellite markers, in addition to the five previously existing markers, to describe the spatial distribution of genetic variability in 34 meadows spread throughout the Mediterranean, on the basis of an average of 35.6 (± 6.3) ramets sampled.Results At the scale of the Mediterranean Sea as a whole, a strong east–west cleavage was detected (amova). These results are in line with those obtained using previous markers. The new results showed the presence of a putative secondary contact zone at the Siculo‐Tunisian Strait, which exhibited high allelic richness and shared alleles absent from the eastern and western basins. F statistics (pairwise θ ranges between 0.09 and 0.71) revealed high genetic structure between meadows, both at a small scale (about 2 to 200 km) and at a medium scale within the eastern and western basins, independent of geographical distance. At the intrameadow scale, significant spatial autocorrelation in six out of 15 locations revealed that dispersal can be restricted to the scale of a few metres.Main conclusions A stochastic pattern of effective migration due to low population size, turnover and seed survival is the most likely explanation for this pattern of highly restricted gene flow, despite the importance of an a priori seed dispersal potential. The east–west cleavage probably represents the outline of vicariance caused by the last Pleistocene ice age and maintained to this day by low gene flow. These results emphasize the diversity of evolutionary processes shaping the genetic structure at different spatial scales.

Population structure and evolutionary origins of Microsporum canis, M. ferrugineum and M. audouinii

The recurrent evolutionary emergence of asexual lineages within sexual zoo- and anthropophilic dermatophyte species living in animal-frequented soil is likely to be triggered by changes in ecological niche, i.e., shifts of host animal. Subsequent adaptation to the new host species is noted. Sometimes geographic isolation or intrinsic host factors like human race may also play a role in speciation. In the present study, we elaborate concepts of speciation in dermatophytes using the Microsporum canis complex as an example. The group consists of a cluster of phylogenetically closely related anamorphs: the anthropophilic taxa Microsporum audouinii and M. ferrugineum, and the zoophilic taxon M. canis. The sexually reproducing species underlying this complex is Arthroderma otae. The study is done by an analysis of the population structure of about 200 isolates and using intergenic spacers, non-translated regions of genes as well as hypervariable microsatellite markers that are known to evolve at high mutation rates. The results suggest that sympatric speciation took place already during the period where mating ability was maintained and thus that strictly clonal fungal species emerged in Africa and led to genetically isolated clonal species elsewhere.

Genetic epidemiology of Sarcoptes scabiei (Acari: Sarcoptidae) in northern Australia

Utilising three hypervariable microsatellite markers we have previously shown that scabies mites on people are genetically distinct from those on dogs in sympatric populations in northern Australia. This had important ramifications on the formulation of public health control policies. In contrast phylogenetic analyses using mitochondrial markers on scabies mites infecting multiple animal hosts elsewhere in the world could not differentiate any genetic variation between mite haplotype and host species. Here we further analyse the intra-specific relationship of Sarcoptes scabiei var. hominis with S. scabiei var. canis by using both mitochondrial DNA and an expanded nuclear microsatellite marker system. Phylogenetic studies using sequences from the mitochondrial genes coding for 16S rRNA and Cytochrome Oxidase subunit I demonstrated significant relationships between S. scabiei MtDNA haplotypes, host species and geographical location. Multi-locus genotyping using 15 microsatellite markers substantiated previous data that gene flow between scabies mite populations on human and dog hosts is extremely rare in northern Australia. These data clearly support our previous contention that control programs for human scabies in endemic areas with sympatric S. scabiei var. hominis and var. canis populations must focus on human-to-human transmission. The genetic division of dog and human derived scabies mites also has important implications in vaccine and diagnostic test development as well as the emergence and monitoring of drug resistance in S. scabiei in northern Australia.

Molecular population genetic analysis of the spectacled bear (Tremarctos ornatus) in the northern Andean area.

Eighty-two Andean bear samples obtained in three South American Andean countries were analyzed using five hypervariable microsatellite markers. Neither the overall sample nor the samples coming from each of the countries analyzed were in Hardy-Weinberg equilibrium. This is attributable to the Wahlund effect caused by a fragmentation of the bear populations. The genetic variability levels found were in general low for this kind of molecular markers (H = 0.38 for the global population). The situation of the Ecuadorian bear population seems to be endangered (H = 0.24). This is the lowest level recorded for any bear population. The genetic heterogeneity among the three populations was large and highly significant (F(ST) = 0.39; R(ST) = 0.32-0.49). Likewise, the gene flow estimates were remarkably low among these populations (Nm = 0.2-0.3). This means that the populations are strongly isolated. Different methods were applied to determine the effective numbers in these populations. A significant spatial structure of the genotypes was analyzed with different procedures. In all cases, an isolation-by-distance structure was detected. This could be a consequence of the original South America colonization. There was no evidence for a recent genetic bottleneck. This could mean that the low heterozygosity and the population fragmentation could be explained by ancient events related to the bear colonization, with the arrival of humans beings in the Americas, and/or with the glacial maximum, 16.000-30.000 years ago.

Haplotyping and mapping a large cluster of downy mildew resistance gene candidates in sunflower using multilocus intron fragment length polymorphisms

Downy mildew (Plasmopara halstedii (Farl.) Berlese et de Toni) is a serious foliar pathogen of cultivated sunflower (Helianthus annuus L.). Genetic resistance is conditioned by several linked downy mildew resistance gene specificities in the HaRGC1 cluster of TIR-NBS-LRR resistance gene candidates (RGCs) on linkage group 8. The complexity and diversity of the HaRGC1 cluster was assessed by multilocus intron fragment length polymorphism (IFLP) genotyping using a single pair of primers flanking a hypervariable intron located between the TIR and NBS domains. Two to 23 bands were amplified per germplasm accession. The size of the included intron ranged from 89 to 858 nucleotides. Forty-eight unique markers were distinguished among 24 elite inbred lines, six partially isogenic inbred lines, nine open-pollinated populations, four Native American land races, and 20 wild H. annuus populations. Nine haplotypes (based on 24 RGCs) were identified among elite inbred lines and were correlated with known downy mildew resistance specificities. Sixteen out of 39 RGCs identified in wild H. annuus populations were not observed in elite germplasm. Five partially isogenic downy mildew resistant lines developed from wild H. annuus and H. praecox donors carried eight RGCs not found in other elite inbred lines. Twenty-four HaRGC1 loci were mapped to a 2-4 cM segment of linkage group 8. The multilocus IFLP marker and duplicated, hypervariable microsatellite markers tightly linked to the HaRGC1 cluster are powerful tools for distinguishing downy mildew resistance gene specificities and identifying and introgressing new downy mildew resistance gene specificities from wild sunflowers.

Size, distribution, and persistence of genets in local populations of the late-stage ectomycorrhizal basidiomycete, Russula brevipes.

• The spatial mapping of genets from sporocarps allows determination of the primary modes of reproduction of ectomycorrhizal fungi. The goals of this research were to determine the relative size, density, and persistence of genets of Russula brevipes , a late successional basidiomycete, in mature stands of lodgepole pine ( Pinus contorta ) and Sitka spruce ( Picea sitchensis ). • Sporocarps were mapped and genets were identified using species-specific hypervariable microsatellite markers. • Most of the sporocarps sampled in clumped aggregations were the same genet and occupied areas less than 3m. The largest distance measured between two sporocarps of the same genet was 18m. In local populations of R. brevipes associated with Sitka spruce, genets were comprised of largely related individuals, whereas in lodgepole pine, most genets appeared unrelated. • High genotypic diversity in Sitka spruce stands suggests frequent mating and recombination between local inhabitants from primary establishment of basidiospores. In lodgepole pine, fewer, unrelated genotypes of R. brevipes indicates that multiple sources of colonization arose from infrequent, long distance spore dispersal events.

Heterogeneity in the rate and pattern of germline mutation at individual microsatellite loci.

There is a lack of information on how individual microsatellite loci differ with respect to their mutation properties. Such variation will have an important bearing on our understanding of the ubiquitous occurrence of simple repeat sequences in eukaryotic genomes and on deriving proper mutation models that can be incorporated into genetic distance estimates. We genotyped approximately 100 families of the bird barn swallow (Hirundo rustica) for two hypervariable (heterozygosity >95%) microsatellite markers: HrU6, an (AAAG)n tetranucleotide repeat, and HrU10, an (AAGAG)n pentanucleotide repeat. A total of 27 germline mutation events were documented, corresponding to mutation rates of 0.57% (HrU6) and 1.56% (HrU10). The mutation rate increased with allele size, at approximately 0.1% per repeat unit over the observed range of allele sizes (approximately 10-100 repeat units). Single repeat unit changes dominated, with 21/27 mutations representing the gain or loss of one repeat unit. There was no clear difference in the number of gains versus losses nor was there an effect of allele size on the magnitude or direction of mutation. Unexpectedly, the mutation rate of females (maternally transmitted mutations) was 2.5-5 times higher than that of males. Contrasting these observations with mutation data from other microsatellite loci reveals differences not only in the mutation rate, but also in the magnitude, direction and effect of sex on mutation. Thus, microsatellite mutation and evolution may be viewed as a dynamic and variable process.