AFLP utility for population assignment studies: analytical investigation and empirical comparison with microsatellites

Individual-based population assignment tests have thus far mainly relied on the use of microsatellite loci. However, the logistic difficulty of screening large numbers of loci required to reach sufficient statistical power hampers the usefulness of microsatellites in situations of weak population structuring. Amplified fragment length polymorphisms (AFLP) represents an alternative for overcoming this logistical issue as the technique allows the user to characterize a much larger number of loci with a comparable analytical effort. In this study, an assignment test based on maximum likelihood for dominant markers was used to investigate the potential usefulness of AFLP for population assignment. We also compared assignment success achieved with AFLP with that obtained using microsatellites in a case study of low population differentiation involving whitefish (Coregonus clupeaformis) sympatric ecotypes. The analytical investigation showed that the minimum number of AFLP loci required to reach an assignment success of 95% stood within values that are easily achievable in many situations. This also showed how assignment success varied according to the number of AFLP loci used, their absolute frequency and their frequency differential and sampling errors, as well as the number of putative source populations. The case study showed that given a comparable analytical effort in the laboratory, AFLP were much more efficient than the microsatellite loci in discriminating the source of an individual among putative populations. AFLP resulted in higher assignment success at all levels of stringency and the log-likelihood differences between populations obtained with AFLP for each individual were much larger than those obtained with microsatellites. These results indicate that research involving individual-based population assignment methods should benefit importantly from the use of AFLP markers, especially in systems characterized by weak population structuring.     

The effects of locus number,genetic divergence,and genotyping error on the utility of dominant markers for hybrid identification

In surveys of hybrid zones, dominant genetic markers are often used to identify individuals of hybrid origin and assign these individuals to one of several potential hybrid classes. Quantitative analyses that address the statistical power of dominant markers in such inference are scarce. In this study, dominant genotype data were simulated to evaluate the effects of, first, the number of loci analyzed, second, the magnitude of differentiation between the markers scored in the groups that are hybridizing, and third, the level of genotyping error associated with the data when assigning individuals to various parental and hybrid categories. The overall performance of the assignment methods was relatively modest at the lowest level of divergence examined (F_st ~ 0.4), but improved substantially at higher levels of differentiation (F_st ~ 0.67 or 0.8). The effect of genotyping error was dependent on the level of divergence between parental taxa, with larger divergences tempering the effects of genotyping error. These results highlight the importance of considering the effects of each of the variables when assigning individuals to various parental and hybrid categories, and can help guide decisions regarding the number of loci employed in future hybridization studies to achieve the power and level of resolution desired.     

Differentiation among Spanish sheep breeds using microsatellites

Genetic variability at 18 microsatellites was analysed on the basis of individual genotypes in five Spanish breeds of sheep – Churra, Latxa, Castellana, Rasa-Aragonesa and Merino -, with Awassi also being studied as a reference breed. The degree of population subdivision calculated between Spanish breeds from F_ST diversity indices was around 7% of total variability. A high degree of reliability was obtained for individual-breed assignment from the 18 loci by using different approaches among which the Bayesian method provided to be the most efficient, with an accuracy for nine microsatellites of over 99%. Analysis of the Bayesian assignment criterion illustrated the divergence between any one breed and the others, which was highest for Awassi sheep, while no great differences were evident among the Spanish breeds. Relationships between individuals were analysed from the proportion of shared alleles. The resulting dendrogram showed a remarkable breed structure, with the highest level of clustering among members of the Spanish breeds in Latxa and the lowest in Merino sheep, the latter breed exhibiting a peculiar pattern of clustering, with animals grouped into several closely set nodes. Analysis of individual genotypes provided valuable information for understanding intra- and inter-population genetic differences and allowed for a discussion with previously reported results using populations as taxonomic units.     

How useful is DNA extracted from the legs of archived insects for microsatellite-based population genetic analyses?

DNA obtained from museum specimens provides a historical perspective on levels of genetic diversity. Archived samples are irreplaceable so it is desirable that only parts of the specimens are used, which constrains the amount of DNA obtained from small taxa. However, at present there are no quantitative data on yields of DNA from such samples. In this paper we determine the amount of DNA that may be extracted from the legs of museum-archived specimens of the damselfly Coenagrion mercuriale (Charpentier) and the suitability of this DNA for PCR-amplification of nuclear genetic loci (microsatellites). We find that (i) the yield of DNA correlates with the genotyping success rate and (ii) the amount of DNA obtained from the legs decreases with time since sample collection until 1954, before which no DNA could be detected (although DNA may be present in very low quantities). This cut-off point for successful DNA extraction corresponds with the date until reliable genotypes could be obtained by routine PCR. Thus, air-dried insect legs more than 50 years old appear to have limited usefulness for studies that seek to amplify many nuclear loci without the use of other techniques that may be used to increase the possible low-quantities of template DNA present.     

Isolation and characterization of microsatellite loci from the endangered New Zealand takahe (Gruiformes; Rallidae; Porphyrio hochstetteri)

Nineteen polymorphic microsatellite loci were characterized from the endangered takahe (Porphyrio hochstetteri). Like many of New Zealand's other native avian species, levels of polymorphism were low, with variation detected at only 19 of 110 (17.3%) loci, and most polymorphic loci (78.9%) were diallelic (mean number of alleles = 2.3). Despite these low levels of variation, the microsatellites developed here will be useful for parentage assignment for confirming pedigrees, and investigating relationships between genetic variation, pedigree‐based inbreeding and reproductive success in this highly endangered species.     

Widespread amplification of amplified fragment length polymorphisms (AFLPs) in marine Antarctic animals

Although recent years have witnessed a rapid growth in the number of genetic studies of Antarctic organisms, relatively few studies have so far used nuclear markers, possibly due to the perceived cost and difficulty of isolating markers such as microsatellites. However, an often overlooked alternative is to use amplified fragment length polymorphisms (AFLPs), a versatile and low-cost method capable of generating large numbers of predominantly nuclear loci in virtually any organism. We conducted a literature review of population genetic studies of Antarctic organisms, finding that fewer than 10% used AFLPs. Moreover, a strong taxonomic bias was found, with studies employing mitochondrial DNA or microsatellites focussing predominantly on animals, while those using AFLPs were mostly of plants or lower organisms. Consequently, we explored the extent to which AFLPs amplify across a range of Antarctic marine animal taxa by genotyping eight individuals each of twelve different species, ranging from echinoderms through soft corals to pelagic fish, at four selective primer combinations. AFLPs readily amplified across all of the taxa, generating between 32 and 84 loci per species, with on average 56.5% of these being polymorphic. In general, levels of polymorphism bore little relationship with expectations based on larger populations of broadcast-spawning species being more variable, though we did find a tentative positive correlation between the number of AFLP bands amplified and a measure of effective population size. Our study lends further support for the utility and ease of use of AFLPs and their suitability for studies of Antarctic species across a wide range of taxa.     

New microsatellite loci in the dwarf yams Dioscorea group Epipetrum (Dioscoreaceae)

? Premise of the study: Microsatellite loci were isolated and characterized from enriched genomic libraries of two taxa of the Chilean Epipetrum group of Dioscorea to assess their levels of genetic diversity and population differentiation. ? Methods and Results: Eleven microsatellite loci were identified. Six out of nine microsatellites from D. biloba amplified in D. humilis, and the two microsatellites from D. humilis amplified in both taxa. Two different sets of eight loci amplified in each of the two tested taxa, D. biloba and D. humilis. The average number of alleles was 5.75 and 5 for D. biloba and D. humilis, respectively. Higher levels of mean genetic diversity were found in D. biloba (H(E) = 0.639) than in D. humilis (H(E) = 0.414). ? Conclusions: These microsatellite primers will be useful in population genetic studies and to establish conservation strategies in the endangered taxa of the Epipetrum group of Dioscorea.     

Molecular identification of a cryptic species in the Amazonian predatory catfish genus Pseudoplatystoma (Bleeker, 1962) from Peru

Pseudoplatystoma species are highly prized South American Pimelodid migratory catfishes. Until recently, their taxonomy was not clearly established, with discrepancies between morphological and molecular analyses. Here, Pseudoplatystoma species from the Peruvian Amazon were characterized at the molecular level from a sample representing the observed range of their color pattern variations in the study area. Analyses were performed using seven microsatellite loci for 103 specimens and, for part of them (52), using sequences of two regions of their mitochondrial genome [Cytochrome Oxidase subunit I (COI) and Control Region (CR)]. Factorial correspondence analysis and assignment tests based on microsatellite polymorphism showed that the specimens originally identified as P. punctifer belonged to two different gene pools highly differentiated from P. tigrinum. Morphological examination identified two different morphotypes (with and without black stripes), suggesting the existence of two distinct taxa within P. punctifer. This result was corroborated by the ML tree based on CR sequences, where all individuals but four clustered in a similar way as in the FCA and Bayesian assignment tests. For these four individuals, mitochondrial introgression or retention of ancestral polymorphism was likely. In contrast, the ML tree based on COI sequences showed that reciprocal monophyly was not yet achieved for this marker for the two P. punctifer taxa. The existence of three sympatric species of Pseudoplatystoma in the Peruvian Amazon is discussed in relation to their molecular characteristics, color patterns and ecology. Evolutionary scenarios regarding their divergence are hypothesized.     

Breed demarcation and potential for breed allocation of horses assessed by microsatellite markers

Population demarcation of eight horse breeds was investigated using genotype information of 306 horses from 26 microsatellite loci. The breeds include the indigenous Norwegian breeds Fjord Horse, Nordland/Lyngen Horse, Døle Horse and Coldblooded Trotter together with Icelandic Horse, Shetland Pony, Standardbred and Thoroughbred. Both phylogenetic analysis and a maximum likelihood method were applied to examine the potential for breed allocation of individual animals. The phylogenetic analysis utilizing simple allele sharing statistics revealed clear demarcation among the breeds; 95% of the individuals clustered together with animals of the same breed in the phylogenetic tree. Even breeds with a short history of divergence like Døle Horse and Coldblooded Trotter formed distinct clusters. Implementing the maximum likelihood method allocated 96% of the individuals to their source population, applying an assignment stringency of a log of the odds ratio larger than 2. Lower allocation stringency assigned nearly all the horses. Only three individuals were wrongly allocated a breed by both methods. In conclusion, the study demonstrates clear distinction among horse breeds, and by combining the two assignment methods breed allocation could be determined for more than 99% of the individuals.     

The application of SSRs characterized for grape (Vitis vinifera) to conservation studies in Vitaceae

The use of microsatellite loci developed from a single plant species across a number of related taxa is becoming increasingly widespread. This approach can provide highly informative markers even for species for which microsatellites have not been characterized. As a number of expressed sequence tag (EST)-derived and enrichment-derived microsatellites are available for grape (Vitis vinifera), this study set out to assess transferability of nine such loci across 25 species from five different Vitaceae genera. Intergeneric transfer success in Vitaceae was high (51.1%) and EST-derived loci performed better than enrichment-derived loci. Six loci were further tested across two Australian native species, Cissus hypoglauca and C. sterculiifolia, in order to assess the conservation of microsatellite repeats and their flanking sequences. Polymorphism of these selected loci was successfully tested for each species across a small, isolated rain forest population from northern New South Wales (Australia). Results from this preliminary investigation suggest that it is possible to use grape-derived simple sequence repeats (SSR) loci for population studies on Vitaceae. As Vitaceae are an important component of rain forest flora, the availability of such highly informative loci will be beneficial to future studies aimed at determining the genetic consequences of rain forest fragmentation.     

Characterization of long transcribed microsatellites in Helianthus annuus (Asteraceae)

? Premise of the study: Research on the evolutionary role of exonic microsatellites currently lacks an understanding of the evolutionary pressures that promote or limit their expansion. Contrasting levels of variability and genetic structures at anonymous and transcribed microsatellite loci of varying lengths are likely to provide useful insights regarding the relative strength of selection acting on different classes of microsatellites. We have developed primers for long transcribed microsatellites in Helianthus annuus to make these comparisons. ? Methods and Results: Eight relatively long microsatellites from sequences in the expressed sequence tag database of H. annuus were characterized. A total of 63 individuals from three populations in Kansas were genotyped. The number of alleles per locus ranged from four to 11 with an average observed heterozygosity of 0.723. ? Conclusions: Our study has generated suitable tools for studying the population genetics of long transcribed microsatellites that are potentially influenced by selection.     

Molecular signatures of divergence and selection in closely related pine taxa

Efforts to detect loci under selection in plants have mostly focussed on single species. However, assuming that intraspecific divergence may lead to speciation, comparisons of genetic variation within and among recently diverged taxa can help to locate such genes. In this study, coalescent and outlier detection methods were used to assess nucleotide polymorphism and divergence at 79 nuclear gene fragments (1212 SNPs) in 16 populations (153 individuals) of the closely related, but phenotypically and ecologically distinct, pine taxa Pinus mugo, P. uliginosa and P. uncinata across their European distributions. Simultaneously, mitochondrial DNA markers, which are maternally inherited in pines and distributed by seeds at short geographic distance, were used to assess genetic relationships of the focal populations and taxa. The majority of nuclear loci showed homogenous patterns of variation between the taxa due to a high number of shared SNPs and haplotypes, similar levels of polymorphism, and low net divergence. However, against this common genetic background and an overall low population structure within taxa at mitochondrial markers, we identified several genes showing signatures of selection, accompanied by significant intra- and interspecific divergence. Our results indicate that loci involved in species divergence may be involved in intraspecific local adaptation.     

Conservation genomics of anadromous Atlantic salmon across its North American range: outlier loci identify the same patterns of population structure as neutral loci

Anadromous Atlantic salmon (Salmo salar) is a species of major conservation and management concern in North America, where population abundance has been declining over the past 30 years. Effective conservation actions require the delineation of conservation units to appropriately reflect the spatial scale of intraspecific variation and local adaptation. Towards this goal, we used the most comprehensive genetic and genomic database for Atlantic salmon to date, covering the entire North American range of the species. The database included microsatellite data from 9142 individuals from 149 sampling locations and data from a medium‐density SNP array providing genotypes for >3000 SNPs for 50 sampling locations. We used neutral and putatively selected loci to integrate adaptive information in the definition of conservation units. Bayesian clustering with the microsatellite data set and with neutral SNPs identified regional groupings largely consistent with previously published regional assessments. The use of outlier SNPs did not result in major differences in the regional groupings, suggesting that neutral markers can reflect the geographic scale of local adaptation despite not being under selection. We also performed assignment tests to compare power obtained from microsatellites, neutral SNPs and outlier SNPs. Using SNP data substantially improved power compared to microsatellites, and an assignment success of 97% to the population of origin and of 100% to the region of origin was achieved when all SNP loci were used. Using outlier SNPs only resulted in minor improvements to assignment success to the population of origin but improved regional assignment. We discuss the implications of these new genetic resources for the conservation and management of Atlantic salmon in North America.     

Biodiversity of 20 chicken breeds assessed by SNPs located in gene regions

Twenty-five single nucleotide polymorphisms (SNPs) were analyzed in 20 distinct chicken breeds. The SNPs, each located in a different gene and mostly on different chromosomes, were chosen to examine the use of SNPs in or close to genes (g-SNPs), for biodiversity studies. Phylogenetic trees were constructed from these data. When bootstrap values were used as a criterion for the tree repeatability, doubling the number of SNPs from 12 to 25 improved tree repeatability more than doubling the number of individuals per population, from five to ten. Clustering results of these 20 populations, based on the software STRUCTURE, are in agreement with those previously obtained from the analysis of microsatellites. When the number of clusters was similar to the number of populations, affiliation of birds to their original populations was correct (>95%) only when at least the 22 most polymorphic SNP loci (out of 25) were included. When ten populations were clustered into five groups based on STRUCTURE, we used membership coefficient (Q) of the major cluster at each population as an indicator for clustering success level. This value was used to compare between three marker types; microsatellites, SNPs in or close to genes (g-SNPs) and SNPs in random fragments (r-SNPs). In this comparison, the same individuals were used (five to ten birds per population) and the same number of loci (14) used for each of the marker types. The average membership coefficients (Q) of the major cluster for microsatellites, g-SNPs and r-SNPs were 0.85, 0.7, and 0.64, respectively. Analysis based on microsatellites resulted in significantly higher clustering success due to their multi-allelic nature. Nevertheless, SNPs have obvious advantages, and are an efficient and cost-effective genetic tool, providing broader genome coverage and reliable estimates of genetic relatedness.     

Isolation and characterization of microsatellite loci in Ainsliaea faurieana (Asteraceae), an endemic plant species on Yakushima Island, Japan, and cross-species amplification in closely related taxa

Fourteen microsatellite loci were isolated and characterized for Ainsliaea faurieana, an endemic perennial plant species on Yakushima Island. In an analysis of 37 individuals from Yakushima Island, the observed number of alleles ranged from two to six. The expected and observed heterozygosities were 0.109-0.811 and 0.000-0.27, respectively. The inbreeding coefficient was 0.489-1.000. The interspecific applicability of these microsatellites was evaluated by analysing three closely related taxa from the Ryukyu Islands. All primer pairs for the 14 loci tested successfully amplified in all taxa.     

Sampling Issues Affecting Accuracy of Likelihood-based Classification Using Genetical Data

We demonstrate the effectiveness of a genetic algorithm for discovering multi-locus combinations that provide accurate individual assignment decisions and estimates of mixture composition based on likelihood classification. Using simulated data representing different levels of inter-population differentiation (F_st～ 0.01 and 0.10), genetic diversities (four or eight alleles per locus), and population sizes (20, 40, 100 individuals in baseline populations), we show that subsets of loci can be identified that provide comparable levels of accuracy in classification decisions relative to entire multi-locus data sets, where 5, 10, or 20 loci were considered. Microsatellite data sets from hatchery strains of lake trout, Salvelinus namaycush, representing a comparable range of inter-population levels of differentiation in allele frequencies confirmed simulation results. For both simulated and empirical data sets, assignment accuracy was achieved using fewer loci (e.g., three or four loci out of eight for empirical lake trout studies). Simulation results were used to investigate properties of the ‘leave-one-out’ (L1O) method for estimating assignment error rates. Accuracy of population assignments based on L1O methods should be viewed with caution under certain conditions, particularly when baseline population sample sizes are low (<50).     

New polymorphic microsatellite loci for the Greek smooth newt, Lissotriton vulgaris graecus, and their utility in the nominotypical subspecies

Eight polymorphic microsatellites were isolated from the Greek smooth newt (Lissotriton vulgaris graecus) using a microsatellite enrichment protocol and selective hybridization with a biotinylated (AC)(11) probe. The loci showed different variation patterns in a single breeding population (32 individuals) with mean number of alleles at 5.0 and mean observed heterozygosity at 0.520. The amplification success also in the nominotypical subspecies favours the use of these microsatellite loci in population genetic analyses as well as in the study of contact zones between smooth newt subspecies.     

New methods employing multilocus genotypes to select or exclude populations as origins of individuals

A new method for assigning individuals of unknown origin to populations, based on the genetic distance between individuals and populations, was compared to two existing methods based on the likelihood of multilocus genotypes. The distribution of the assignment criterion (genetic distance or genotype likelihood) for individuals of a given population was used to define the probability that an individual belongs to the population. Using this definition, it becomes possible to exclude a population as the origin of an individual, a useful extension of the currently available assignment methods. Using simulated data based on the coalescent process, the different methods were evaluated, varying the time of divergence of populations, the mutation model, the sample size, and the number of loci. Likelihood-based methods (especially the Bayesian method) always performed better than distance methods. Other things being equal, genetic markers were always more efficient when evolving under the infinite allele model than under the stepwise mutation model, even for equal values of the differentiation parameter F(st). Using the Bayesian method, a 100% correct assignment rate can be achieved by scoring ca. 10 microsatellite loci (H approximately 0.6) on 30-50 individuals from each of 10 populations when the F(st) is near 0.1.     

Evaluating the utility of microsatellites for investigations of autopolyploid taxa

Autopolyploid taxa present numerous challenges for population genetic analyses due to difficulties determining allele dosage. Dosage ambiguity hinders accurate assessment of allele frequencies, multilocus genotypes (MLGTs), as well as levels and patterns of clonality. The pervasiveness of polyploidy in the evolutionary history of plant taxa makes this a recurring problem. Whereas diploidization of loci may occur over time, duplication of at least some loci is still frequently evident. Fortunately, with high-quality allozyme gels, it is possible to accurately infer allele dosage and, thus, determine exact MLGTs. However, accurately assessing dosage of microsatellite peaks is nearly impossible when studying wild populations with a large number of alleles per locus. Even if precise knowledge of genotypes is not required, for comparable numbers of alleles per locus and loci, the number of "phenotypes" is always lower with microsatellites than allozymes due to the inability to assess allele dosage. Microsatellite loci typically have more alleles per locus relative to allozymes although fewer loci are generally employed. Here, we present a mathematical model for comparing the relative utility of simple sequence repeat (SSR) versus allozyme markers to discriminate MLGTs. For example, the average plant allozyme study (2.6 alleles per locus, 10 polymorphic loci) has better discriminating power than SSR markers with 10 alleles at each of 3 loci, 9 alleles at 4 loci, 6 alleles at 5 loci, 5 alleles at 6 loci, and 4 alleles at 8 loci, demonstrating the value of assessing the relative discriminating power of these markers.