Isolation and characterization of polymorphic microsatellite loci from the Dungeness crab Cancer magister

The Dungeness crab, Cancer magister, is the focus of one of the most intensely harvested fisheries in North America. Given its economic importance, there is considerable interest in assessing the degree and spatial pattern of genetic structure in C. magister. To that end, we developed a series of 17 hypervariable microsatellite loci. Six of these 17 loci could be amplified in a single multiplex PCR reaction. Using dye‐labelled primers all six loci can be coamplified and scored simultaneously on an automated sequencer. The ability to multiplex multiple loci greatly increases the ease and speed of genotyping for this species.     

Pattern of Inheritance of Microsatellite Loci in the Squid Loligo pealeii (Mollusca: Cephalopoda)

Six microsatellite loci are described for the squid Loligo pealeii. All loci exhibit some degree of allelic diversity. The pattern of inheritance was tested for 3 loci through an analysis of the filial genotypes from a female-male mating. At all 3 loci, the ratios of the filial genotypes conformed to the ratios expected by Mendelian inheritance. The hypervariable loci will be useful in studies on sexual selection in this species, whereas the relatively less variable loci will be useful to address questions of population structure. Received March 13, 2000; accepted May 22, 2000.     

Microsatellite and major histocompatibility complex variation in an endangered rattlesnake,the Eastern Massasauga (Sistrurus catenatus)

Genetic diversity is fundamental to maintaining the long‐term viability of populations, yet reduced genetic variation is often associated with small, isolated populations. To examine the relationship between demography and genetic variation, variation at hypervariable loci (e.g., microsatellite DNA loci) is often measured. However, these loci are selectively neutral (or near neutral) and may not accurately reflect genomewide variation. Variation at functional trait loci, such as the major histocompatibility complex (MHC), can provide a better assessment of adaptive genetic variation in fragmented populations. We compared patterns of microsatellite and MHC variation across three Eastern Massasauga (Sistrurus catenatus) populations representing a gradient of demographic histories to assess the relative roles of natural selection and genetic drift. Using 454 deep amplicon sequencing, we identified 24 putatively functional MHC IIB exon 2 alleles belonging to a minimum of six loci. Analysis of synonymous and nonsynonymous substitution rates provided evidence of historical positive selection at the nucleotide level, and Tajima's D provided support for balancing selection in each population. As predicted, estimates of microsatellite allelic richness, observed, heterozygosity, and expected heterozygosity varied among populations in a pattern qualitatively consistent with demographic history and abundance. While MHC allelic richness at the population and individual levels revealed similar trends, MHC nucleotide diversity was unexpectedly high in the smallest population. Overall, these results suggest that genetic variation in the Eastern Massasauga populations in Illinois has been shaped by multiple evolutionary mechanisms. Thus, conservation efforts should consider both neutral and functional genetic variation when managing captive and wild Eastern Massasauga populations.     

Microsatellite multiplexes for high‐throughput genotyping of French grunts (Haemulon flavolineatum,Pisces: Haemulidae) and their utility in other grunt species

Grunts of the genus Haemulon are abundant in the western Atlantic and are an important component of regional fisheries. Management efforts for these and other Caribbean reef fish require an understanding of population structure and the extent of dispersal among populations. We characterized 11 polymorphic microsatellite loci for French grunts (Haemulon flavolineatum) and grouped them into three PCR multiplexes to facilitate high‐throughput genotyping. These loci will be useful for population genetic studies of French grunts. Cross‐species amplifications suggest these loci are also suitable for population genetic studies of H. aurolineatum, H. plumieri, and H. sciurus.     

Polymorphic microsatellite DNA markers from the Oriental Fruit Fly Bactrocera dorsalis (Hendel)

Six polymorphic microsatellite loci are isolated from the Oriental fruit fly Bactrocera dorsalis (Hendel), an agricultural pest in Asia, including Taiwan. To assess their potential utility as high‐resolution genetic markers, polymerase chain reaction (PCR) primers, amplification conditions, and an automated fluorescence detection protocol were developed. In analyses of 71 individual flies from six different areas of Taiwan, allele numbers ranged from five to 25 were detected for each locus. The observed heterozygosity ranged between 0.268 and 0.737 among these loci. No linkage disequilibrium was found. These microsatellite markers have potential utility to population structure and gene flow studies of B. dorsalis (Hendel).     

Characterization and PCR multiplexing of polymorphic microsatellite loci for the locust Locusta migratoria

Because of the scarcity of polymorphic genetic markers available in locust species, only a few population genetics studies have been carried out on this taxon. We isolated and characterized 11 polymorphic microsatellite loci in the pest locust Locusta migratoria capito, and described experimental conditions for polymerase chain reaction (PCR) multiplexing and simultaneously genotyping these loci. The number of alleles per locus ranged from six to 25, and the expected heterozygosity ranged from 0.431 to 0.957. Results of cross‐taxon amplification tests are reported in six other Locusta migratoria subspecies, six species of the Oedipodinae subfamily and two other pest locust species.     

Development of new microsatellite loci for the genus <Emphasis Type="Italic">Polistes</Emphasis> from publicly available expressed sequence tag sequences

Over the last 20 years, microsatellites have revolutionized the study of cooperation in the social insects. The Polistes paper wasps have been an important model system for investigations of cooperative behavior. Recently, an expressed sequence tag (EST) library has been developed for P. metricus, allowing researchers to investigate the genetic basis of cooperative behavior in primitive social insect societies for the first time. We searched these freely available EST sequences for microsatellite motifs. This represents a relatively new approach to the development of microsatellite loci that allows for the development of a greater number of loci at less expense. We designed 32 PCR primer pairs, of which 23 amplified PCR products and 18 were polymorphic. These loci exhibited high levels of polymorphism, comparable to anonymous loci isolated via screens of partial genomic libraries. Thus, they are appropriate for population genetic studies as well as the reconstruction of colony genetic structure. A screen of the entire EST database found a total of 708 di-, tri-, tetra- and penta-nucleotide repeats with large repeat units typical of polymorphic loci and at least 30 bp of flanking sequence for primer design. This pool of potential loci represents a new genetic tool for P. metricus, as well as Polistes more generally, as there is great promise for cross amplification in other species.     

Identification of Microsatellite Repeats in Turbot (Scophthalmus maximus) and Dover Sole (Solea solea) using a RAPD-Based Technique: Characterization of Microsatellite Markers in Dover Sole

We have used a RAPD-based technique to identify several microsatellite repeats in turbot (Scophthalmus maximus) and Dover sole (Solea solea) and report the characterization of six novel polymorphic microsatellite markers for Dover sole. These are the first such markers to be developed for this flatfish species. They exhibit much higher levels of heterozygosity than those previously observed with allozyme loci and should prove useful in addressing population genetic questions as well as more fundamental aquaculture-related questions. Received ; accepted July 13, 1999.     

Microsatellite loci for Myrmica kotokui and their application in some congeneric ant species distributed in northern Japan

We developed seven polymorphic microsatellite loci for Myrmica kotokui from RAPD (rapid analysis of polymorphic DNA) fragments. These loci showed two to six alleles with expected and observed heterozygosity of 0.13–0.73 and 0.14–0.78, respectively. These loci are transferable to the congeneric species as Myrmica rubra, Myrmica luteola and Myrmica taediosa from northern Japan. These loci will allow analyses of genetic structure of Myrmica species at both the colony level and population levels.     

Characterization of polymorphic microsatellite markers and genetic diversity in wild bronze featherback, Notopterus notopterus (Pallas, 1769)

Six polymorphic microsatellite DNA loci were identified in the primitive fish, bronze featherback, Notopterus notopterus for the first time and demonstrated significant population genetic structure. Out of the six primers, one primer (NN90) was specific to N. notopterus (microsatellite sequence within the RAG1 gene) and five primers were product of successful cross-species amplification. Sixty-four primers available from 3 fish species of order Osteoglossiformes and families Notopteridae and Osteoglossidae were tested to amplify homologous microsatellite loci in N. notopterus. Fifteen primer pairs exhibited successful cross-priming PCR product. However, polymorphism was detected only at five loci. To assess the significance of these six loci (including NN90) in population genetic study, 215 samples of N. notopterus from five rivers, viz Satluj, Gomti, Yamuna, Brahmaputra and Mahanadi were analyzed. The five sample sets displayed different diversity levels and observed heterozygosity ranged from 0.6036 to 0.7373. Significant genotype heterogeneity (P < 0.0001) and high F_ST (0.2205) over all loci indicated that the samples are not drawn from the same genepool. The identified microsatellite loci are promising for use in fine-scale population structure analysis of N. notopterus.     

Extensive allozyme monomorphism in a threatened species of freshwater mussel, Margaritifera hembeli Conrad (Bivalvia: Margaritiferidae)

A threatened but under-studied component offreshwater biodiversity in North America is thenative freshwater mussels (Bivalvia:Unionoidea: Margaritiferidae and Unionidae). Genetic data suggest that these mussel speciesgenerally exhibit levels of variability similarto other invertebrates. We surveyed allozymevariation in the Louisiana Pearlshell, Margaritifera hembeli (Margaritiferidae), athreatened freshwater mussel. Five examinedpopulations are monomorphic for 25 allozymeloci, the first report of a native freshwatermussel species with extensive allozymemonomorphism. Low genetic diversity appears tobe characteristic of margaritiferids, as anANOVA indicated that mussels of the familyMargaritiferidae have significantly lowerlevels of heterozygosity than the mussels ofthe family Unionidae. Margaritiferids havestrong habitat preferences and modification ofhabitat leads to rapid loss of populations.Although bottlenecks are known to cause lowgenetic variability, margaritiferids mayexhibit meta-population structure withextinction/re-colonization dynamics leading tolow genetic variability. Margaritiferidsgenerally exhibit a patchy distribution with acolonization rate that is approximately twicethat of extinction. Tests of themetapopulation hypothesis will requireadditional allozyme population genetic data aswell as hypervariable microsatellite loci.     

Population genetic structure of Sorex unguiculatus and Sorex caecutiens (Soricidae, Mammalia) in Hokkaido, based on microsatellite DNA polymorphism

We investigated the genetic structure of Sorex unguiculatus and Sorex caecutiens populations in Hokkaido, Japan, using hypervariable microsatellite DNA markers. We used five microsatellite loci to type 475 S. unguiculatus individuals from 20 localities on the Hokkaido mainland and four localities from each of four offshore islands (and 11 shrews from one locality in southern Sakhalin for a particular analysis). We used six microsatellite loci to type 240 S. caecutiens individuals from 13 localities on the Hokkaido mainland. Genetic variation was high in mainland populations of both species and low in the island populations of S. unguiculatus. Allelic richness and island size were positively correlated for S. unguiculatus, suggesting that genetic drift occurred on those islands due to small population size. In addition, four insular populations of S. unguiculatus were genetically differentiated from the mainland populations, although clear phylogeographic clustering was not confirmed among populations on the Hokkaido mainland for either S. unguiculatus or S. caecutiens. Heterozygosity excess was observed in more than half of the populations including the mainland populations of the two species, suggesting recent bottleneck events in these populations. Population dynamics of the shrews might be explained by a metapopulation scheme. According to autocorrelation analysis, the extent of non-random spatial genetic structure was approximately 100 km. Isolation by distance was observed in S. unguiculatus, but not in S. caecutiens although there is a positive trend. The lack of correlation for S. caecutiens might have been due to small sample size. Thus, no obvious differences in population genetic structure were found between the two species on the Hokkaido mainland in the present study, while previous investigations using mitochondrial DNA sequences inferred that these two species might have rather different biogeographic histories.     

Characterization of microsatellite markers for the North African gundi,Ctenodactylus gundi (Rodentia: Ctenodactylidae)

Eleven highly polymorphic microsatellite loci were isolated from the North African gundi (Ctenodactylus gundi), a gregarious rock‐dwelling rodent found in northern Africa. These loci exhibited high genetic diversity (eight to 19 alleles per locus) and high levels of heterozygosity (62–98%). Collectively, these microsatellite markers are sufficiently variable to estimate levels of relatedness and population genetic structure for C. gundi.     

Microsatellite DNA markers for population-genetic studies of Labeo dyocheilus (McClelland, 1839)

Fifty‐four primers published for six cyprinid fishes were tested to amplify homologous microsatellite loci in Labeo dyocheilus. Fifteen primers yielded successful amplification and seven were polymorphic with 3–9 alleles. To evaluate utility of the identified loci in population genetic study, 84 samples were analysed. The samples were collected from four rivers viz. Beas, Satluj, Yamuna and Jiabharali. The four sample sets displayed different diversity levels, with observed heterozygosity from 0.34 to 0.53. Significant genotype heterogeneity (P < 0.001) over all loci indicated that the samples are not drawn from the same genepool. The identified microsatellite loci are promising for use in fine‐scale population structure analysis of L. dyocheilus.     

Nineteen new microsatellite DNA polymorphisms in pigtailed macaques (Macaca nemestrina)

Microsatellite loci known to be polymorphic in baboons (Papio hamadryas) and/or humans were tested in pigtailed macaques (Macaca nemestrina) from the Washington Regional Primate Research Center. Nineteen polymorphisms were identified in the macaques, with an average of 9.2 alleles per locus and an average heterozygosity of 0.76. Seven loci were analyzed using radiolabelled PCR primers and standard gel electrophoresis. Twelve loci were studied using fluorescently labelled primers and the Perkin-Elmer ABI 377 genotyping system. Of these 19 pigtailed macaque polymorphisms, 12 were used to perform paternity testing among captive animals. In a set of 15 infants, this panel of 12 genetic polymorphisms was sufficient to establish paternity in all cases. The number of alleles per locus in pigtailed macaques was compared with the number of alleles in a sample of baboons, and no significant correlation was observed. This indicates that population genetic processes such as genetic drift and recurrent mutation act rapidly enough on these loci to eliminate any relationship in levels of polymorphism across those two species. These 19 loci will be valuable for a range of genetic studies in pigtailed macaques, including paternity testing, analysis of population structure and differentiation among wild populations, and genetic linkage mapping.     

Isolation and characterization of Ligustrum micranthum (Oleaceae) microsatellite loci using paired‐end Illumina reads

Twenty‐six microsatellite loci were developed and characterized for Ligustrum micranthum, a species endemic to the Ogasawara Islands, Japan. The genetic structure of this species must be clarified in order to restore the island's ecosystem. A total of 8511 primer pairs were designed from de novo sequencing. Of the 48 primer pairs selected, amplification and polymorphisms were tested using one population each from the Chichijima and Hahajima Islands of the Ogasawara Islands. Twenty‐six microsatellite loci were successfully amplified and the number of alleles for these loci ranged from five to 31 per locus, and the mean expected heterozygosities were 0.858 and 0.849, respectively. No significant deviation from the Hardy–Weinberg equilibrium was observed in either population, and no significant linkage disequilibrium was detected between any locus pair. The microsatellite loci reported in this study can be used in future studies to evaluate the genetic structure and mating system of L. micranthum.     

Isolation and characterization of highly polymorphic microsatellite markers in Hypochaeris radicata (Asteraceae)

We developed five highly polymorphic dinucleotide microsatellite loci for the grassland species Hypochaeris radicata (Asteraceae). Polymorphism of these markers was examined in six populations in the Netherlands. All loci were polymorphic in all populations. The number of alleles per locus varied between 18 and 43. Expected heterozygosity was between 0.86 and 0.91. Cross‐species amplification was tested in six Hypochaeris species and was successful for three different loci in four species. These microsatellites are a useful tool in population genetic, dispersal and metapopulation studies or in testing levels of inbreeding.     

Polymorphic microsatellite loci for paternity analysis in the Madagascar paradise flycatcher (Terpsiphone mutata: Aves)

Nine polymorphic microsatellite loci from the Madagascar paradise flycatcher Terpsiphone mutata were isolated using nonradioactive polymerase chain reaction (PCR)‐based techniques to screen an enriched genomic library. Seven polymorphic loci showed no evidence of null alleles and exhibited high levels of variation in 18 unrelated individuals (mean diversity = 0.80, mean number of alleles = 13.6). These loci are therefore suitable for analysis of population structure and paternity (exclusion probability for six unlinked loci = 0.9998).     

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.     

Polymorphic CA/GT and GA/CT microsatellite loci for Ostrinia nubilalis (Lepidoptera: Crambidae)

Ten polymorphic dinucleotide (CA/GT and GA/CT) microsatellite loci suitable for population genetic screening were characterized from enriched partial Ostrinia nubilalis genomic libraries. Sequence from 126 enriched small insert genomic library clones identified 25 CA/GT and 58 GA/CT loci that were unique. Perfect repeats tended to be short (n = 10–12). Ten microsatellites, PCR amplified from a Crawfordsville Iowa population showed a mean of 10 alleles per locus (range six to 20), and six of 10 loci showed heterozygote deficiency. Amplification of eight loci was observed in the sister species O. furnicalis.