Isolation and characterization of polymorphic microsatellites in a Yangtze River fish,brass gudgeon (Coreius heterodon Bleeker)

The brass gudgeon (Coreius heterodon) is a fish of economic importance in the Yangtze River. From a (GATA)_n‐enriched genomic library, 25 microsatellites were developed by employing the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Nine loci exhibited polymorphism with two to 12 alleles (mean 3.9 alleles/locus) in a test population, and observed heterozygosity ranged from 0.1111 to 0.9630 (mean 0.4426). Three of the nine loci showed polymorphism in a congeneric species, the largemouth bronze gudgeon Coreius guichenoti. These loci should provide sufficient level of genetic diversity to evaluate the fine‐scale population structure of C. heterodon.     

Microsatellite loci for paternity assessment in the black swan (Cygnus atratus: Aves)

Seven polymorphic tetranucleotide microsatellite loci were isolated and characterized from the black swan Cygnus atratus Latham, using nonradioactive PCR‐based techniques to screen an enriched genomic library. These loci were highly variable (mean H _E  = 0.75, mean number of alleles = 9.1), and showed no evidence of linkage disequilibrium or null alleles. This locus set is suitable for detecting extra‐pair copulations (combined exclusion probability = 0.999) and for other population genetic applications.     

Isolation and characterization of microsatellites in Chinese soft‐shelled turtle,Pelodiscus sinensis

Fifteen polymorphic microsatellite loci were developed for the Chinese soft‐shelled turtle (Pelodiscus sinensis) from the (GT)_n microsatellite‐enriched genomic library, using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol. The polymorphism of all 15 loci ranged from two to seven alleles with observed heterozygosities ranging from 0.03 to 0.98 (mean 0.43) in one population of 40 individuals. These novel loci will be helpful for understanding the population structure at genetic level and marker‐assisted breeding of this vulnerable species.     

Accounting for missing data in the estimation of contemporary genetic effective population size (Ne)

Theoretical models are often applied to population genetic data sets without fully considering the effect of missing data. Researchers can deal with missing data by removing individuals that have failed to yield genotypes and/or by removing loci that have failed to yield allelic determinations, but despite their best efforts, most data sets still contain some missing data. As a consequence, realized sample size differs among loci, and this poses a problem for unbiased methods that must explicitly account for random sampling error. One commonly used solution for the calculation of contemporary effective population size (N_e) is to calculate the effective sample size as an unweighted mean or harmonic mean across loci. This is not ideal because it fails to account for the fact that loci with different numbers of alleles have different information content. Here we consider this problem for genetic estimators of contemporary effective population size (N_e). To evaluate bias and precision of several statistical approaches for dealing with missing data, we simulated populations with known N_e and various degrees of missing data. Across all scenarios, one method of correcting for missing data (fixed‐inverse variance‐weighted harmonic mean) consistently performed the best for both single‐sample and two‐sample (temporal) methods of estimating N_e and outperformed some methods currently in widespread use. The approach adopted here may be a starting point to adjust other population genetics methods that include per‐locus sample size components.     

Novel polymorphic microsatellite markers for paternity analysis in the red‐capped robin (Petroica goodenovii: Aves)

Seven microsatellite loci were isolated and characterized from the red‐capped robin Petroica goodenovii, using nonradioactive polymerase chain reaction (PCR)‐based techniques to screen an enriched genomic library. Five loci showed no evidence of null alleles and were variable [mean heterozygosity (H_E) = 0.440, mean number of alleles = 8]. Cross‐amplification using primers for microsatellites in Phylloscopus occipitalis and Emberiza schoeniclus yielded another two polymorphic loci. The combined set of five red‐capped robin and two cross‐amplified loci are suitable for paternity assignment (exclusion probability for seven unlinked loci = 0.9760).     

Development of EST‐derived microsatellite markers for Arabidopsis lyrata subspecies petraea (L.)

A set of expressed sequence tag–simple sequence repeat (EST‐SSR) loci has been developed for Arabidopsis lyrata ssp. petraea. From 768 root cDNA clones, 126 microsatellites, including di‐, tri‐, tetra‐ and pentanucleotide repeat motifs were identified and primers were designed to 24 EST‐SSRs. Eleven loci were subsequently screened on 150 individuals sampled from five natural populations, which revealed three to nine alleles per locus (mean 5.36) and expected heterozygosity (H_E) estimates ranging from 0.046 to 0.698. Significant deviations from random mating were observed at 10 EST‐SSR loci, likely due to inbreeding (global F_IS = 0.151) and population structure (global F_ST = 0.246).     

Development of polymorphic microsatellite loci in the hermaphroditic freshwater snails Drepanotrema surinamense and Drepanotrema depressissimum

We characterized new variable microsatellites in two congeneric species of hermaphroditic freshwater snails (Drepanotrema depressissimum and D. surinamense), as well as conditions for multiplexing and simultaneously genotyping sets of loci. D. depressissimum had high mean gene diversity (> 0.8) and large number of alleles (= 10.9) per population. Most loci and populations were at Hardy–Weinberg equilibrium. The F_ST estimates were low among lesser Antilles populations and larger with a Venezuelan population. Far less diversity was found in D. surinamense with mean number of alleles and gene diversity per population of 2.8 and 0.34, respectively. Very few heterozygous individuals were observed. The most likely explanation is a high selfing rate (> 0.825) in this species. Unsurprisingly, the F_ST estimates among populations were much higher than in D. depressissimum. Cross‐species amplification in three congeneric species was on the whole unsuccessful.     

Intra‐ and interpopulation genetic differentiation and gene flow in a group of isolated populations of Bradybaena fruticum (O. F. Müller, 1774) in South Poland

Intra‐ and interpopulation variation was studied, by means of cellulose acetate allozyme electrophoresis, on 16 populations of helicoid snail Bradybaena fruticum (O. F. Müller, 1774) in South Poland. Four enzyme systems, coded by seven loci, were analysed. Calculated with Fisher's technique and Ohta's D‐statistics, four cases of linkage disequilibrium were detected, reflecting population subdivision. The mean number of alleles per locus equalled 2.16 and the mean expected heterozygosity was 0.287. Exact multipopulation and multilocus tests for Hardy–Weinberg equilibrium indicated a statistically significant homozygote excess in all the loci and all populations but three. Each population, however, was at Hardy–Weinberg equilibrium for most loci, though the values of f (F_IS) were usually high. Homozygote excess was ascribed partly to inbreeding and partly to Wahlund's effect (spatial subdivision of population; at least two cohorts of adult, reproducing snails), disrupting selection in this polymorphic species not excluded. F‐statistics showed relatively low values of θ (F_ST ; mean for all loci = 0.224) and those of Nm usually below 1 (mean 0.866). Pairwise values of either θ or Cavalli‐Sforza and Edwards arc distance were statistically significantly associated with geographic distances. Contrary to this, no geographic pattern of interpopulation differences was detected by correspondence analysis on allele frequencies, non‐linear multidimensional scaling, UPGMA clustering or neighbour‐joining trees constructed on θ and Cavalli‐Sforza and Edwards arc distance. Accordingly, some most distant populations were more similar to one another than the close ones.     

Isolation and characterization of microsatellite markers in the southern emu‐wren (Stipiturus malachurus: Aves)

We isolated and characterized eight novel microsatellite loci in the southern emu‐wren (Stipiturus malachurus). We used nonradioactive polymerase chain reaction (PCR)‐based techniques to screen an enriched genomic DNA library. Based on genotypes from a single population, six loci showed no evidence of null alleles and were polymorphic (allele range = 2–9, mean heterozygosity = 0.57), and one locus was sex‐linked (N_A = 4). These loci were variable and had different allele size ranges in three other populations of southern emu‐wrens, and are therefore useful for determining levels of genetic diversity within and between populations of the species.     

RAPID GENETIC DIFFERENTIATION AND FOUNDER EFFECT IN COLONIZING POPULATIONS OF COMMON MYNAS (ACRIDOTHERES TRISTIS)

Populations of common mynas introduced to Australia, New Zealand, Fiji, Hawaii, and South Africa from India during the last century were compared genetically with the extant native population using isozyme electrophoresis of 39 presumptive loci. Average heterozygosity, mean number of alleles/locus, and the percentage of polymorphic loci are lower in the introduced populations, and the 18% loss of alleles involves only alleles that are rare in the native population. The native population is only weakly subdivided genetically (F_ST = 0.032) whereas the introduced populations are much more differentiated (F_ST = 0.123), and the mean genetic distance among them is significantly greater than among native samples. The reduction in mean number of alleles/locus and average heterozygosity is greatest in the South African population, consistent with a very small effective size in the founder population. In the introduced populations, random drift is implicated by the different subsets of polymorphic loci they possess, by their greater variance in allele frequencies, and by shifts either side of the native means. It is concluded that in the evolutionarily short period of 100–120 years, bottlenecks and random drift have promoted genetic shifts equal to those between different subspecies of birds.     

Isolation and characterization of polymorphic microsatellite markers in the gray,short‐tailed opossum (Monodelphis domestica)

Twenty‐six polymorphic microsatellite markers were isolated from (AC)_n and (AG)_n microsatellite‐enhanced genomic libraries of the gray, short‐tailed opossum Monodelphis domestica. All 26 loci showed high allelic diversity, with allele numbers ranging from five to 11 in a subset of 35 animals. Normal Mendelian inheritance was confirmed for 24 loci by analysing allelic segregation in 10, two‐generation, families. Non‐amplifying (null) alleles were detected at two loci, which we recommend be used only if pedigree data are available. We conclude that all of these microsatellite markers would be useful for quantitative trait locus mapping and population genetic studies.     

Microsatellite loci for behavioural studies of rainbow bee‐eaters (Merops ornatus: Aves)

A set of polymorphic microsatellite loci was identified for population and behavioural studies of the rainbow bee‐eater (Merops ornatus). Eight loci were newly isolated from a rainbow bee‐eater genomic library enriched for GA‐containing repeats, and six loci from those previously reported for the European bee‐eater (Merops apiaster). From these two sources, the seven most variable loci that amplified reliably were unlinked and in Hardy–Weinberg equilibrium, had five to 13 alleles (mean 7.7) and an expected heterozygosity of 0.60–0.86 (mean 0.73).     

Genetic diversity and differentiation in Portuguese cattle breeds using microsatellites

Genotype data from 30 microsatellites were used to assess genetic diversity and relationships among 10 native Portuguese cattle breeds, American Charolais and the Brazilian Caracú. Hardy–Weinberg equilibrium was observed for all loci/population combinations except for five loci in Brava de Lide and one locus in Alentejana that exhibited heterozygote deficiency. Estimates of average observed and expected heterozygosities, total number of alleles (TNA) per breed and mean number of alleles (MNA) per locus/population were obtained. A total of 390 alleles were detected. TNA among Iberian cattle ranged from 170 to 237 and MNA ranged from 5.67 to 8.07. The highest observed heterozygosities were found in the Caracú, Maronesa, Garvonesa and Arouquesa and the lowest in Brava de Lide and Mirandesa. Estimation of population subdivision using Wright's F_ST index showed that the average proportion of genetic variation explained by breed differences was 9%. Neighbour‐joining phylogenetic trees based on D_A distances showed that the genetic relationships of present‐day Portuguese native breeds are consistent with historical origins in the Brown Concave (Arouquesa, Mirandesa, Marinhoa) and Red Convex (Mertolenga, Alentejana, Garvonesa, Minhota) evolutionary groups. The Iberian Black Orthoide group, represented by Brava de Lide and Maronesa, and the Barrosã breed appeared to be more closely related to the Brown Concave group but may represent a separate lineage. The Caracú breed was not found to be closely associated with any of the native Portuguese breeds.     

Microsatellite loci for population and behavioural studies of Horsfield's bronze‐cuckoo (Chalcites basalis: Aves)

Eight polymorphic microsatellite loci were identified for Horsfield's bronze‐cuckoo (Chalcites basalis). These include seven newly isolated loci from cuckoo genomic libraries enriched for GA and GAAA repeat‐containing clones. These loci have a mean expected heterozygosity of 0.71, a mean number of alleles of 13.8 and a combined exclusion probability (one parent known) of 0.9999. Two loci (Cba01 and Cba07) showed a significant deficiency of heterozygotes and may therefore have null alleles, although this effect could be the result of nonrandom population sampling.     

Isolation of microsatellite loci from the common buzzard,Buteo buteo (Aves: Accipitridae)

We isolated 56 common buzzard (Buteo buteo) microsatellite loci from (AC)_n‐ and (AAAG)_n‐enriched genomic libraries. Eleven loci were tested on 90 individuals from Eastern Westphalia in Germany, yielding two to 17 alleles per locus (average 5.7) and expected heterozygosities ranging from 0.11 to 0.93 (average 0.53). These highly variable microsatellite markers provide a powerful means for investigating population genetic diversity in the common buzzard, a little‐understood aspect of this widespread species.     

Characterization of microsatellite loci in the Blue‐and‐gold Macaw,Ara ararauna (Psittaciformes: Aves)

Microsatellite loci were isolated from the Blue‐and‐gold Macaw (Ara ararauna), a Neotropical parrot, from a GT_n and CT_n enriched genomic library. Six loci were characterized varying from one to 11 alleles per locus. Five loci exhibited greater than 50% heterozygosity within the 49 individuals genotyped. Furthermore, the primers also amplified the DNA from two additional genera of Neotropical parrots, indicating the potential utility of these markers for population‐level studies and conservation efforts of Neotropical parrots.     

Microsatellite loci for studies on population differentiation and connectivity of the red‐bellied yellow tail fusilier,Caesio cuning (Caesionidae)

Ten polymorphic microsatellites were isolated from the red‐bellied yellow tail fusilier Caesio cuning, a reef‐associated fish which occurs widely in the Indo‐Pacific region. The species is exploited by both small‐scale and commercial fisheries. Fifty individuals from six populations were genotyped using primers that reliably amplified 10 polymorphic loci. The number of alleles per locus ranged from three to 11. Observed heterozygosity (H_O) and expected heterozygosity (H_E) ranged between 0.400 to 0.880 and 0.337 to 0.843, respectively. These microsatellite loci may be used to study population structure, genetic diversity and connectivity of C. cuning in the range of its distribution.     

Isolation and characterization of polymorphic tetranucleotide microsatellite loci in the chestnut-crowned babbler (Pomatostomus ruficeps)

We describe 18 microsatellite markers isolated in the cooperatively breeding chestnut‐crowned babbler (Pomatostomus ruficeps). The number of alleles ranged from seven to 16 per locus (mean N_a = 10.4 ± 0.54 SE) and the expected heterozygosity ranged from 0.732 to 0.889 (mean H_E = 0.836 ± 0.01 SE). Three of the 18 loci exhibited significant heterozygote deficiency, but the remaining 15 will be used to analyse population genetic structure and the mating system of this highly social species.     

Polymorphic dinucleotide microsatellites in tongue sole (Cynoglossus semilaevis)

The tongue sole, Cynoglossus semilaevis, is a rare marine flatfish distributed in Chinese coastal waters. From a (GT)_n‐enriched genomic library, 57 microsatellites were isolated and characterized. Seventeen of these loci were polymorphic in a test population with alleles ranging from three to 13, and observed and expected heterozygosities from 0.1613 to 1.0000 and from 0.2126 to 0.8983, respectively. Five loci deviated from the Hardy–Weinberg equilibrium in the sampled population, and linkage disequilibrium between two loci was significant after applying Bonferroni correction. Three additional fish species assessed for cross‐species amplification revealed that only one locus was also polymorphic in one species. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the breeding strategy and investigate the fine‐scale population structure in C. semilaevis.     

Characterization of 10 trinucleotide microsatellite loci in the Critically Endangered Pyrenean yam Borderea chouardii (Dioscoreaceae)

The low levels of allozymic variability found in the Critically Endangered Borderea chouardii prompted us to develop microsatellite markers to assess the genetic variability and population structure for the adequate conservation management of this species. A (CTT)_n‐enriched partial genomic library was constructed. Ten polymorphic microsatellite loci were isolated from it, rendering 51 alleles in 47 individuals analysed. The allelic pattern observed for all of the loci with more than two alleles suggests that B. chouardii is tetraploid.