hw‐quickcheck: an easy‐to‐use computer program for checking genotypes for agreement with Hardy–Weinberg expectations

Deviations from Hardy–Weinberg expectations are frequently a sign of genotyping error. hw‐quickcheck is an easy‐to‐use computer program for detecting departures from Hardy–Weinberg equilibrium. hw‐quickcheck uses exact tests for all of its calculations. These tests include a global test for heterozygote excess/deficiency and genotype‐specific tests.     

Significant deviations from Hardy-Weinberg equilibrium caused by low levels of microsatellite genotyping errors

Microsatellite genotyping from samples with varying quality can result in an uneven distribution of errors. Previous studies reporting error rates have focused on estimating the effects of both randomly distributed and locus‐specific errors. Sample‐specific errors, however, can also significantly affect results in population studies despite a large sample size. From two studies including six microsatellite markers genotyped from 272 sperm whale DNA samples, and 33 microsatellites genotyped from 213 bowhead whales, we investigated the effects of sample‐ and locus‐specific errors on calculations of Hardy–Weinberg equilibrium. The results of a jackknife analysis in these two studies identified seven individuals that were highly influential on estimates of Hardy–Weinberg equilibrium for six different markers. In each case, the influential individual was homozygous for a rare allele. Our results demonstrate that Hardy–Weinberg P values are very sensitive to homozygosity in rare alleles for single individuals, and that > 50% of these cases involved genotype errors likely due to low sample quality. This raises the possibility that even small, normal levels of laboratory errors can result in an overestimate of the degree to which markers are out of Hardy–Weinberg equilibrium and hence overestimate population structure. To avoid such bias, we recommend routine identification of influential individuals and multiple replications of those samples.     

Characterization of 14 microsatellite marker loci in the grey bamboo lemur (Hapalemur griseus)

Fourteen nuclear microsatellite loci were isolated from a genomic DNA library derived from a free‐ranging bamboo lemur (Hapalemur griseus griseus) from Ranomafana National Park, Madagascar. Expected and observed heterozygosity levels were determined for populations of bamboo lemurs from Ranomafana National Park and a nonprotected area near Tsinjoarivo, Madagascar. Although genotype frequencies for all but one marker were within Hardy–Weinberg expectations for the Tsinjoarivo population, this was not the case for the Ranomafana samples. It is probable that the deviation of genotype frequencies from Hardy–Weinberg expectations in Ranomafana was precipitated by the presence of two taxa within the sample population.     

Isolation and characterization of 11 polymorphic trinucleotide microsatellite markers in the stonefly Arcynopteryx compacta (Plecoptera: Perlodidae)

We describe the isolation of 11 polymorphic trinucleotide microsatellite loci from the stonefly Arcynopteryx compacta. Loci were highly variable with 3 to 14 alleles (mean = 6.45). Observed heterozygosity ranged from 0 to 0.867. Seven loci showed significant deviation from Hardy–Weinberg equilibrium across both populations. There was no evidence for null alleles, and thus, Hardy–Weinberg departures could have resulted from genetic structure between populations or subpopulations. No linkage between loci was found. The 11 loci should prove highly informative for population genetic studies.     

Characterization of 24 microsatellite loci in delta smelt, Hypomesus transpacificus, and their cross-species amplification in two other smelt species of the Osmeridae family

We characterized 24 polymorphic tetranucleotide microsatellite loci for delta smelt (Hypomesus transpacificus) endemic to the San Francisco Bay Estuary, CA, USA. Screening of samples (n = 30) yielded two to 26 alleles per locus with observed levels of heterozygosity ranging from 0.17 to 1.0. Only one locus deviated from Hardy–Weinberg equilibrium, suggesting these individuals originate from a single panmictic population. Linkage disequilibrium was found in two pairs of loci after excluding the locus out of Hardy–Weinberg equilibrium. Twenty‐two primer pairs cross‐amplified in wakasagi smelt (Hypomesus nipponensis), and 15 primer pairs cross‐amplified in longfin smelt (Spirinchus thaleichthys).     

Interpreting Whole-Genome Marker Data

The challenges of whole-genome data, when genotypes are available from hundreds of thousands of genetic markers, are explored for four topics in statistical genetics: Hardy–Weinberg testing, estimating linkage disequilibrium from unphased genotypic data, association mapping and characterizing population structure.     

Perl and Python codes to estimate Hardy–Weinberg‐ and linkage‐disequilibrium‐related parameters

We have developed two pieces of Perl and Python codes to calculate the natural log of lambda, a quantity that expresses deviations from Hardy–Weinberg equilibrium, and R, a weighted correlation coefficient between alleles of locus pairs as a quantity that expresses linkage disequilibrium. These quantities can be used in an estimation approach as tools to facilitate the characterization and the comparison of populations.     

Development and characterisation of microsatellite markers for the medicinal plant Scutellaria baicalensis (Lamiaceae)

Scutellaria baicalensis is a popular medicinal plant that is on the verge of extinction due to uncontrolled harvesting, habitat destruction and deterioration of its ecosystem. We isolated and characterised 21 microsatellite loci in this species. Ninety-four individuals from six populations were used to test the polymorphism of the microsatellite loci. The number of alleles per locus ranged from 1 to 13, with a mean of 7.2. Observed and expected heterozygosities varied from 0.000 to 1.000 and 0.000 to 0.938, respectively. Among these new microsatellite markers, only two loci showed significant deviation from Hardy–Weinberg equilibrium. No locus pairs showed significant linkage disequilibrium. The 21 primer pairs were tested in other Scutellaria species. Most of these primer pairs worked successfully, except for Scut18. These new microsatellite markers could be applied to investigate the genetic diversity and population genetic structure of S. baicalensis and its closely related species.     

Isolation and characterization of microsatellite DNA markers in the Pacific abalone,Haliotis discus hannai

We developed 17 new microsatellite markers in Haliotis discus hannai. All loci were found to be polymorphic with an average of 13.1 alleles per locus (range 3–28). The mean observed and expected heterozygosities were 0.77 (range 0.17–1.00) and 0.79 (range 0.42–0.96), respectively. Six loci deviated significantly from Hardy–Weinberg proportions, and thus should be used with caution. The high variabilities revealed in this study suggest that these microsatellite loci should provide useful markers for studies of trait mapping, kinship and population genetics.     

Polymorphic tetranucleotide microsatellite markers in the Caribbean spiny lobster,Panulirus argus

Ten tetranucleotide microsatellite loci are described for the Caribbean spiny lobster Panulirus argus. Loci were polymorphic (4–15 alleles per locus) and exhibited high levels of expected (0.553–0.921) and observed heterozygosity (0.469–0.906) from samples caught off Belize and Puerto Rico coasts. No significant departure from Hardy–Weinberg equilibrium conditions were observed for any locus. All microsatellite loci should be useful for assessing population discrimination for this valuable marine animal currently subjected to excessive fishing efforts.     

Isolation and characterization of microsatellite loci from the Japanese conger eel Conger myriaster

Five polymorphic microsatellite markers were developed in the Japanese conger eel (Conger myriaster) to understand the recruitment mechanisms and population structure of this species, the spawning sites and migration routes of which are unknown. Three of the loci were highly polymorphic and will serve as powerful tools for detailed population studies of the Japanese conger eel. One of the other two loci showed significant departure from Hardy–Weinberg equilibrium and the other showed low allelic diversity.     

Characterization of nine microsatellite loci in impala (Aepyceros melampus)

Nine microsatellite loci were isolated from a genomic DNA library created from impala (Aepyceros melampus). Observed and expected levels of heterozygosity were computed utilizing 25 individuals from a population in central Kenya. Tests for Hardy–Weinberg equilibria were conducted and found that three of the nine loci deviated from equilibrium in this population. These markers were developed to analyse the genetic effects of culling and isolation on a game preserve in Kenya.     

Microsatellite loci for studies of wild and hatchery Australian Murray cod Maccullochella peelii peelii (Percichthyidae)

Murray cod is a species of conservation concern that is subject both to wild harvest and to aquaculture production. Six polymorphic microsatellite loci have been developed for this species, which will facilitate studies of wild‐stock structure, will help the management of hatchery diversity, and will aid genetic improvement programmes. The markers exhibited nonindependence of genotypes between loci and deviations from Hardy–Weinberg expectations, although these most probably reflect practices at the hatchery from which the genotyped individuals were sourced.     

Isolation of polymorphic microsatellite markers in the great tit (Parus major minor)

Six microsatellite loci were developed for a passerine bird, the great tit (Parus major), using two methods. These loci were polymorphic (3–8 alleles per locus) and exhibited expected heterozygosities from 0.45 to 0.77. At one locus the genotypic frequencies deviated significantly from Hardy–Weinberg expectations.     

Genetic structure in a montane mayfly Baetis bicaudatus (Ephemeroptera: Baetidae), from the Rocky Mountains, Colorado

1. Populations of a number of sub‐tropical stream insect species have been found to show unexpected patterns of genetic variation, with more differences between samples from the same stream than between whole streams or between subcatchments. Many samples also showed deviations from Hardy–Weinberg proportions. It has been proposed that these patterns result from limited numbers of matings contributing to a given stream reach, because adults emerge throughout the year, and low levels of larval drift between reaches. These patterns may be less likely in a northern hemisphere montane species with synchronous emergence of adults and high levels of drift. We tested the hypothesis that patterns of genetic variation in a montane mayfly from the Rocky Mountains, Colorado, would reflect a pattern of ‘isolation by distance’ with samples from the same creek being more similar than samples from different creeks and that deviations from Hardy–Weinberg proportions would be minimal. 2. Based on allozyme variation, the hypothesis of minimal deviations from Hardy–Weinberg proportions was not supported and there was no evidence of isolation by distance. Nevertheless the levels of differentiation among samples from within the same stream were less than those reported for most subtropical species. 3. Results from analysis of a fragment of the cytochrome oxidase gene (subunit 1) revealed contrasting patterns. The levels of genetic differentiation were an order of magnitude higher between streams than among samples within streams. In addition, although there was no significant isolation by distance effect overall, a nested clade analysis provided evidence for restricted gene flow with isolation by distance for some clades. 4. We suggest that these contrasting results may reflect the differences in male and female dispersal patterns. While differentiation at nuclear gene markers (allozymes) give information about both male and female dispersal, mitochondrial DNA markers reflect only female dispersal. We suggest that in this species, female dispersal may be more restricted, perhaps mostly along stream channels, whereas male dispersal is more widespread. An alternative explanation for the different results is the different evolutionary rates of the mitochondrial and nuclear markers.     

Isolation and characterization of highly polymorphic microsatellite loci in a pelagic‐spawning reef fish Hypodytes rubripinnis

Group spawning (1 female and more than 2 males) and streaking behaviour of subordinate males are common in pelagic‐spawning reef fishes such as the temperate tetrarogidid fish Hypodytes rubripinnis. Five microsatellite loci were isolated and characterized for paternity analysis of this species. Four of the five loci were highly polymorphic, having 11–34 alleles with observed heterozygosities between 0.89 and 1.00 in 37 unrelated individuals. These four loci conformed to Hardy–Weinberg equilibrium. These loci should be useful in future studies that assess how fertilization is partitioned among male H. rubripinnis in multimale spawning events.     

Polymorphic microsatellite markers for studies of the conservation and reproductive genetics of imperilled sand tiger sharks (Carcharias taurus)

We report on the isolation of eight microsatellites from the sand tiger shark, Carcharias taurus, using an enrichment protocol. All loci, with the exception of Cta45–183, were in Hardy–Weinberg equilibrium. Loci exhibited three to 15 alleles, and observed and expected heterozygosities of 0.095–1.000 and 0.284–0.924, respectively. An additional marker (Iox‐12) developed from a shortfin mako library was variable in sand tigers. These markers will be used to examine population genetics and mating patterns of this imperilled species.     

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

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

Development of microsatellite loci in Pacific herring (Clupea pallasi)

Fifteen polymorphic di‐, tri‐ and tetranucleotide microsatellite markers were developed in Pacific herring (Clupea pallasi) to aid in the delineation of population structure of herring in British Columbia. Twelve of the microsatellite loci were analysed in over 4000 wild herring. Expected heterozygosities ranged 0.73–0.95, and only two loci significantly deviated from Hardy–Weinberg equilibrium.     

Isolation and characterization of microsatellite loci from Galápagos lava lizards (Microlophus spp.)

We describe the isolation of microsatellite loci from Galápagos lava lizards (Microlophus spp.) using an enriched genomic library. Twelve loci that are polymorphic among six populations from two species are described. Characterization of these loci in 20 individuals within one population (Isla Plaza Sur) showed seven to be polymorphic with 3–11 alleles. Heterozygosities within this population were high (0.32–0.90) and did not deviate from Hardy–Weinberg expectations. We suggest that these markers will be useful in studies of population differentiation within and among islands across the Galápagos archipelago.