Remotely plucked hair genotyping: a reliable and non-invasive method for censusing pine marten (Martes martes,L. 1758) populations

We investigated the feasibility of using genetic techniques to census pine marten (Martes martes) populations by genotyping non-invasively collected samples (plucked hair and scats), with particular reference to the genetically depauperate Irish population. Novel real-time polymerase chain reaction methods were developed for species and sex identification, targeting short DNA sequences. Background genetic variation at 17 microsatellite loci was very low in the Irish population, with an average of 2.29 alleles per locus and expected heterozygosity of 0.35. Despite such low polymorphism, a panel of eight loci with a sibling probability of identity of 0.011 reliably identified individual pine marten and their gender, as determined by reference to genotypes of live trapped individuals. With high nuclear DNA amplification success rates (93.8%) and low genotyping error rates (1.8%), plucked hairs may represent a more reliable and cost-effective DNA source than scats for monitoring populations of this elusive carnivore, and similar taxa such as the sympatric stone marten Martes foina.

     

Polymorphic tetranucleotide microsatellite loci in the Hwamei (Garrulax canorus canorus) (Timaliidae)

Eight polymorphic tetrarepeat (GATA)_n, microsatellite loci were isolated from a babbler, Hwamei (Garrulax canorus canorus). We evaluated the polymorphism of these microsatellite loci by genotyping 36–48 individuals from the Asian mainland. The number of alleles for each locus ranged from eight to 29. The heterozygosity was between 0.587 and 0.978. Except for one locus, genotype frequencies of these microsatellites did not significantly deviate from the Hardy–Weinberg expectation. These markers should be useful for monitoring potential hybridization between different Hwamei subspecies and provide new insights into the mating system and geographical differentiation of these birds.     

Buccal swabs allow efficient and reliable microsatellite genotyping in amphibians

Buccal swabs have recently been used as a minimally invasive sampling method in genetic studies of wild populations, including amphibian species. Yet it is not known to date what is the level of reliability for microsatellite genotypes obtained using such samples. Allelic dropout and false alleles may affect the genotyping derived from buccal samples. Here we quantified the success of microsatellite amplification and the rates of genotyping errors using buccal swabs in two amphibian species, the Alpine newt Triturus alpestris and the Green tree frog Hyla arborea, and we estimated two important parameters for downstream analyses, namely the number of repetitions required to achieve typing reliability and the probability of identity among genotypes. Amplification success was high, and only one locus tested required two to three repetitions to achieve reliable genotypes, showing that buccal swabbing is a very efficient approach allowing good quality DNA retrieval. This sampling method which allows avoiding the controversial toe-clipping will likely prove very useful in the context of amphibian conservation.     

Microsatellites in Cassava (Manihot esculenta Crantz): discovery, inheritance and variability

 Fourteen microsatellites containing GA-repeats were isolated and characterized in cassava (Manihot esculenta Crantz, Euphorbiaceae). Microsatellite heterozygosity (h) was estimated in 48 accessions using (³²P)-end-labeled primers and in more than 500 accessions using fluorescence-based genotyping. Heterozygosity values ranged from 0.00 to 0.88 and the number of alleles detected varied from 1 to 15. The reproducibility of allele sizing was also assessed using fluorescence-based genotyping. The average inter-gel size difference was 1.03 nucleotides. Chi-square tests (χ²) were performed to analyse segregation distortion and the linkage between alleles segregating from either or both parents in an F₁ mapping population. Most microsatellite loci segregated in the expected 1 : 1, 1 : 2 : 1 or 1 : 1 : 1 : 1 ratio. Linkage was detected between loci segregating from either parent, and segregation distortion from the male parent was detected for locus GA-131. Approximately 80% of the microsatellites detected one or two alleles per accession, suggesting a low degree of microsatellite locus duplication, an unexpected finding for a putative allopolyploid, highly heterozygous species. The high h values of most microsatellites, their amplification in other Manihot taxa and their suitability for high-throughput, fluorescence-based genotyping, make microsatellites the marker of choice for germplasm characterization and saturation of the cassava map. Received: 4 September 1997 / Accepted 16 March 1998     

Transferability and characterization of microsatellite markers from Byrsonima cydoniifolia A. Juss. (MALPIGHIACEAE) in seven related taxa from Cerrado biome reveal genetic relationships

Byrsonima Rich. is one of the largest genera of the Malpighiaceae family with 97 species occurrence in Brazil and multiple potentialities, including pharmaceutical and food industries. In this study, 17 microsatellite markers characterized in Byrsonima cydoniifolia were tested for seven related taxa, all species are native to Brazil and four are endemic. Genomic DNA was extracted from leaves tissues and 17 microsatellite markers were used to cross-amplification of microsatellite regions. Polymorphism and genetic diversity were evaluated for B. intermedia, B. verbascifolia, B. laxiflora, B. subterranea, B. umbellata, B. linearifolia. from 16 individuals and for B. viminifolia from 14 individuals. Transferred microsatellite markers panels ranged from 11 (64.8%) in B. viminifolia to 6 (35.2%) in B. umbellata. The total number of alleles per locus ranged from 5 (B. linearifolia) to 8 (B. subterranea) alleles. B. umbellata showed lower values of observed and expected heterozygosity (H_O?=?0.312; H_E?=?0.436) and B. subterranea presented the highest values (H_O?=?0.687; H_E?=?0.778). A greater number of microsatellite markers should be developed for B. umbellata. The microsatellite marker panels transferred to the species B. intermedia, B. verbascifolia, B. laxiflora, B. subterranea, B. viminifolia and B. linearifolia are very informative, with a high combined probability of exclusion of paternity (Q?≥?0.976) and the low combined probability of identity (I?≤?9.91?×?10^–6), potentially suitable for future genetic-population studies, supporting strategies for maintaining the genetic diversity and for exploration of Byrsonima species as genetic resources.

     

megasat: automated inference of microsatellite genotypes from sequence data

megasat is software that enables genotyping of microsatellite loci using next‐generation sequencing data. Microsatellites are amplified in large multiplexes, and then sequenced in pooled amplicons. megasat reads sequence files and automatically scores microsatellite genotypes. It uses fuzzy matches to allow for sequencing errors and applies decision rules to account for amplification artefacts, including nontarget amplification products, replication slippage during PCR (amplification stutter) and differential amplification of alleles. An important feature of megasat is the generation of histograms of the length–frequency distributions of amplification products for each locus and each individual. These histograms, analogous to electropherograms traditionally used to score microsatellite genotypes, enable rapid evaluation and editing of automatically scored genotypes. megasat is written in Perl, runs on Windows, Mac OS X and Linux systems, and includes a simple graphical user interface. We demonstrate megasat using data from guppy, Poecilia reticulata. We genotype 1024 guppies at 43 microsatellites per run on an Illumina MiSeq sequencer. We evaluated the accuracy of automatically called genotypes using two methods, based on pedigree and repeat genotyping data, and obtained estimates of mean genotyping error rates of 0.021 and 0.012. In both estimates, three loci accounted for a disproportionate fraction of genotyping errors; conversely, 26 loci were scored with 0–1 detected error (error rate ≤0.007). Our results show that with appropriate selection of loci, automated genotyping of microsatellite loci can be achieved with very high throughput, low genotyping error and very low genotyping costs.     

中国西南地区特有水生植物海菜花微卫星引物开发

水鳖科(Hydrocharitaceae)海菜花(Ottelia acuminata)是中国西南地区特有的水生单子叶植物。基于AFLP技术的磁珠富集快速分离技术(Fast Isolation by AFLP of Sequences Containing Repeats,FIASCO),共筛选出9对多态性引物并对3个居群45个个体进行分析。结果表明:三个居群的等位基因数目为1~3个,观测杂合度从0.000~0.933,期望杂合度从0.000~0.605。这些筛选出的微卫星引物将用于海菜花后续的谱系地理学和生态遗传学研究。     

Cross-amplification of ungulate microsatellite markers in the endemic Indian antelope or blackbuck (Antilope cervicapra) for population monitoring and conservation genetics studies in south Asia

The Indian antelope or blackbuck (Antilope cervicapra) is endemic to the Indian subcontinent, inhabiting scrublands and dry grasslands. Most of the blackbuck populations are small, isolated, and threatened by habitat fragmentation and degradation. Management of such disjunct populations requires genetic characterization, which is critical for assessing hazards of stochastic events and inbreeding. Addressing the scarcity of such information on the blackbuck, we describe a novel panel of microsatellite markers that could be used to monitor blackbuck demography and population genetic parameters using non-invasive faecal sampling. We screened microsatellites (n?=?40) that had been reported to amplify in bovid and cervid species using faecal samples of the blackbuck collected from Kaimoor Wildlife Sanctuary, Uttar Pradesh, India and its vicinities. We selected 12 markers for amplification using faecal DNA extracts (n?=?140) in three multiplex reactions. We observed a mean amplification success rate of 72.4% across loci (92.1–25.7%) with high allele diversity (mean number of alleles/locus?=?8.67?±?1.03). Mean genotyping error rates across the markers were low to moderate (allelic drop-out rate?=?0.09; false allele rate?=?0.11). The proportions of first- and second-order relatives in the study population were 0.69% and 6.21%, respectively. Based on amplification success, genotyping error rates and the probability of identity (P_ID), we suggest (i) a panel of five microsatellite markers (cumulative P_ID?=?1.24?×?10^–5) for individual identification and population monitoring and (ii) seven additional markers for conservation genetics studies. This study provides essential tools capable of augmenting blackbuck conservation strategies at the landscape level, integral to protecting the scrubland-grassland ecosystem.

     

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.     

A set of plastid microsatellite loci for the genus Dyckia (Bromeliaceae) derived from 454 pyrosequencing

• Premise of the study: Phylogeographical analyses of Dyckia (Bromeliaceae) suffer from low levels of sequence variation. Plastid microsatellite markers were developed to achieve a better-resolved genus-wide plastid genealogy of Dyckia. • Methods and Results: Approximately 84% of the D. marnier-lapostollei plastome was sequenced using 454 technology. Flanking primer pairs were designed for 34 out of 36 chloroplast simple sequence repeats (cpSSRs) detected, and 12 loci were further characterized by genotyping Dyckia samples at the level of populations and species. Three, five, and six cpSSRs were polymorphic among four individuals of D. limae, 12 individuals of D. dissitiflora, and 12 of D. pernambucana, respectively, with two to three alleles per locus and species. All loci were polymorphic among 19 different Dyckia species, with three to 10 alleles per locus. Ten primer pairs cross-amplified with bromeliad genera from five subfamilies. • Conclusions: The set of 12 cpSSR markers provides a toolbox to analyze phylogeographical patterns of Dyckia species.     

Isolation and characterization of 20 polymorphic microsatellite loci for Scaptodrosophila hibisci

Scaptodrosophila hibisci is an endemic Australian Drosophilidae that breeds in the flowers of native Hibiscus . Here we report the isolation and amplification of 20 polymorphic microsatellite loci . We cloned these microsatellites because loci developed for Drosophila melanogaster failed to amplify in S. hibisci . Null alleles were detected at six loci, and five were X‐linked. Two of the primer pairs amplified an unlinked ‘bonus’ locus. One locus containing juxtaposed microsatellite loci was suitable for designing an additional set of primers. Mean number of alleles per locus was 10, mean H _O and H _E per locus were 0.532 and 0.636, respectively.     

Population structure of the olive flounder (Paralichthys olivaceus) in Korea inferred from microsatellite marker analysis

The population structure of olive flounder Paralichthys olivaceus was estimated using nine polymorphic microsatellite (MS) loci in 459 individuals collected from eight populations, including five wild and three hatchery populations in Korea. Genetic variation in hatchery (mean number of alleles per locus, A = 10·2–12·1; allelic richness, A_R = 9·3–10·1; observed heterozygosity, H_O = 0·766–0·805) and wild (mean number of alleles per locus, A = 11·8–19·6; allelic richness, A_R = 10·9–16·1; observed heterozygosity, H_O = 0·820–0·888) samples did not differ significantly, suggesting a sufficient level of genetic variation in these well‐managed hatchery populations, which have not lost a substantial amount of genetic diversity. Neighbour‐joining tree and principal component analyses showed that genetic separation between eastern and pooled western and southern wild populations in Korea was probably influenced by restricted gene flow between regional populations due to the barrier effects of sea currents. The pooled western and southern populations are genetically close, perhaps because larval dispersal may depend on warm currents. One wild population (sample from Wando) was genetically divergent from the main distribution, but it was genetically close to hatchery populations, indicating that the genetic composition of the studied populations may be affected by hydrographic conditions and the release of fish stocks. The estimated genetic population structure and potential applications of MS markers may aid in the proper management of P. olivaceus populations.     

Genetic diversity within and among Atlantic cod (Gadus morhua) farmed in marine cages: a proof-of-concept study for the identification of escapees

This study presents a molecular genetic characterization of Atlantic cod reared in commercial marine farms. Samples consisted of approximately 47 fish collected from nine cages located on four farms throughout Norway. In addition, 28 farmed escapees were recaptured in the sea (443 fish in total). Nine microsatellite loci and the Pan I gene were analysed, revealing a total of 181 alleles. Each sample contained 43–63% of total allelic variation. Comparing variation with published data for wild cod indicates that lower genetic variation exists within single cages than in wild populations. Significant linkage disequilibrium was observed amongst pairs of loci in all samples, suggesting a low number of contributing parental fish. Global F_ST was 0.049, and the highest pairwise F_ST value (pooled loci) was 0.085. For single loci, the Pan I gene was the most diagnostic, displaying a global F_ST of 0.203. Simulations amongst the samples collected on farms revealed an overall correct self-assignment percentage of 75%, demonstrating a high probability of identifying individuals to their farm of origin. Identification of the 28 escapees revealed a single cage as the most likely source of origin for half of the escapees, whilst the remaining fish were assigned to a mixture of samples, suggesting more than one source of escapees.     

Isolation and characterization of microsatellite markers in Japanese pear (Pyrus pyrifolia Nakai)

Fourteen microsatellite markers were developed from an enriched genomic library of Japanese pear (Pyrus pyrifolia) by selective hybridization. They were characterized using 17 Japanese pear cultivars. The expected heterozygosity and observed heterozygosity ranged from 0.21 to 0.74 and from 0 to 0.88, respectively. Two to 11 alleles were detected per locus, with IPPN09 and IPPN15 judged to amplify multiple loci. IPPN17 was the most informative locus with the lowest probability of identity (0.19). These primers exhibited a high cross‐species transferability between species and genera.     

Characterization of 11 microsatellite loci derived from genomic sequences of polychaete Capitella capitata complex

Eleven microsatellite loci derived from the genomic sequence data of Capitella capitata were characterized using 30 samples. The observed number of alleles per locus ranged from four to 36. The levels of observed and expected heterozygosities for polymorphic loci were from 0.10 to 0.87 and from 0.37 to 0.98, averaging 0.52 and 0.77, respectively. Analyses of Hardy–Weinberg equilibrium and genotypic linkage disequilibrium suggest the possible presence of both null alleles and Wahlund effect. One of the 11 loci was difficult to amplify for genotyping. Therefore, the rest 10 loci are good molecular markers for population genetic analysis.     

Genetic diversity and molecular differentiation of Chinese toad based on microsatellite markers

Genetic diversity and population structure of 9 populations of Bufo gargarizans with total 111 samples in China were assessed using seven microsatellite loci. The analysed microsatellite markers produced 161 alleles, varied from 9 to 38 alleles each locus. The number of alleles per population per locus ranged from 4.43 to 10.29. Polymorphic information content showed that all seven loci were highly informative (mean = 0.810 ± 0.071). The average observed heterozygosity was less than the expected (0.353 ± 0.051 and 0.828 ± 0.067, respectively). All tested populations gave significant departures from Hardy–Weinberg equilibrium. Genetic differentiation among the populations was considerably high with the overall and pairwise F _ST values (mean = 0.160 ± 0.039), and showed fairly high level of inbreeding (indicated by a mean F _IS value of 0.504 ± 0.051) and global heterozygote deficit. In comparison to other amphibian studies; however, our results suggested that the level of genetic structuring in B. gargarizans was relatively low in the geographical scale of the study area. Interestingly, the speculated population bottleneck was found to be absent and the analyses provide only weak evidence for a recent contraction in size even though there was severe inbreeding (indicated by the F _IS value) in the Chinese toad populations.     

Development of microsatellite markers for the dwarf bamboo species Sasa cernua and Sasa kurilensis (Poaceae) in northern Japan

Ten polymorphic microsatellite markers were isolated from the dwarf bamboo species Sasa cernua and Sasa kurilensis. The applicability of these markers was confirmed by genotyping of open‐pollinated seeds and leaf samples from natural populations. Genotypes of seeds from each culm shared at least one allele from the maternal parent without contradiction. All 10 loci were polymorphic in S. cernua with 2–15 alleles (average H_E = 0.532). Eight loci were polymorphic in S. kurilensis with 2–10 alleles (average H_E = 0.532). These markers will be useful in detailing the extent of clonal and sexual reproduction in these species.     

A first set of gene-derived polymorphic microsatellite markers in half-smooth tongue sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

Half-smooth tongue sole (Cynoglossus semilaevis) is a commercially important marine fish species in China. A set of type I microsatellite markers were identified through bioinformatic mining of the GenBank database. Thirteen of these markers showed polymorphisms through genotyping a sample of 30 individuals. A total of 47 alleles were detected, with an average of 3.6 alleles per locus. The number of alleles, observed and expected heterozygosity per locus ranged from two to five, from 0.14 to 0.93 and from 0.27 to 0.77, respectively. Three loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0038) and no significant linkage disequilibrum between pairs of loci was found. The markers identified in this study will contribute to construction of genetic linkage maps and mapping of quantitative trait loci (QTL) of C. semilaevis.     

Characterization of polymorphic microsatellite loci in the aphid species Macrosiphum euphorbiae (Hemiptera: Aphididae)

Fourteen microsatellite loci were isolated and characterized in the potato aphid, Macrosiphum euphorbiae Thomas, by screening a genomic library with the oligonucleotide probes (GA)₁₀ (GT)₁₀ and (GATA)₄. Allelic diversity was estimated in samples collected from potato fields in Tunisia. Ten loci displayed polymorphism that ranged from two to four alleles per locus and the observed heterozygosity ranged from zero to one. These markers could be used to study the population genetic structure of this polyphagous aphid species.     

Isolation and characterization of ten novel microsatellite loci in the red-winged tinamou (<Emphasis Type="Italic">Rhynchotus rufescens</Emphasis>, Tinamiformes,Aves) and cross-amplification in other tinamous

We describe the isolation and characterization of ten microsatellite loci from the red-winged tinamou (Rhynchotus rufescens) and also evaluated the cross-amplification of these loci and other ten loci previously developed for the great tinamou (Tinamus major) in other tinamous. Genetic variability was assessed using 24 individuals. Six loci were polymorphic with moderate to high number of alleles per locus (2–12 alleles) and showed expected heterozygosity (HE) ranging from 0.267 to 0.860. All loci conformed to the Hardy–Weinberg expectation and linkage disequilibrium was not significant for any pair of loci. This battery of polymorphic loci showed high paternity exclusion probability (0.986) and low genetic identity probability (4.95 × 10⁻⁵), proving to be helpful for parentage tests and population analyses in the red-winged tinamou. The cross-amplification was moderate where of the 160 locus/taxon combinations, 46 (28.75%) successfully amplified.