The accuracy of a heritability estimator using molecular information

The heritability of a quantitative trait is a key parameter to quantify the genetic variation present in a population. Although estimates of heritability require accurate information on the genetic relationship among individuals, pedigree data is generally lacking in natural populations. Nowadays, the increasing availability of DNA markers is making possible the estimation of coancestries from neutral molecular information. In 1996, K. Ritland developed an approach to estimate heritability from the regression of the phenotypic similarity on the marker-based coancestry. We carried out simulations to analyze the accuracy of the estimates of heritability obtained by this method using information from a variable number of neutral codominant markers. Because the main application of the estimator is on populations with no family structure, such as natural populations, its accuracy was tested under this scenario. However, the method was also investigated under other scenarios, in order to test the influence of different factors (family structure, assortative mating and phenotypic selection) on the precision. Our results suggest that the main factor causing a directional bias in the estimated heritability is the presence of phenotypic selection, and that very noisy estimates are obtained in the absence of a familiar structure and for small population sizes. The estimated heritabilities from marker-based coancestries showed lower accuracy than the estimated heritabilities from genealogical coancestries. However, a large amount of bias occurred even in the most favourable situation where genealogical coancestries are known. The results also indicate that the molecular markers are more suitable to infer coancestry than inbreeding.     

Estimating genetic drift and effective population size from temporal shifts in dominant gene marker frequencies

Measurement of temporal change in allele frequencies represents an indirect method for estimating the genetically effective size of populations. When allele frequencies are estimated for gene markers that display dominant gene expression, such as, e.g. random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers, the estimates can be seriously biased. We quantify bias for previous allele frequency estimators and present a new expression that is generally less biased and provides a more precise assessment of temporal allele frequency change. We further develop an estimator for effective population size that is appropriate when dealing with dominant gene markers. Comparison with estimates based on codominantly expressed genes, such as allozymes or microsatellites, indicates that about twice as many loci or sampled individuals are required when using dominant markers to achieve the same precision.     

Empirical evaluation of genetic clustering methods using multilocus genotypes from 20 chicken breeds

We tested the utility of genetic cluster analysis in ascertaining population structure of a large data set for which population structure was previously known. Each of 600 individuals representing 20 distinct chicken breeds was genotyped for 27 microsatellite loci, and individual multilocus genotypes were used to infer genetic clusters. Individuals from each breed were inferred to belong mostly to the same cluster. The clustering success rate, measuring the fraction of individuals that were properly inferred to belong to their correct breeds, was consistently approximately 98%. When markers of highest expected heterozygosity were used, genotypes that included at least 8-10 highly variable markers from among the 27 markers genotyped also achieved >95% clustering success. When 12-15 highly variable markers and only 15-20 of the 30 individuals per breed were used, clustering success was at least 90%. We suggest that in species for which population structure is of interest, databases of multilocus genotypes at highly variable markers should be compiled. These genotypes could then be used as training samples for genetic cluster analysis and to facilitate assignments of individuals of unknown origin to populations. The clustering algorithm has potential applications in defining the within-species genetic units that are useful in problems of conservation.     

Microsatellite analysis supports mitochondrial phylogeography of the hellbender (Cryptobranchus alleganiensis)

We investigated genetic diversity of the hellbender (Cryptobranchus alleganiensis) throughout its range in the eastern US using nuclear markers and compared our results to a previously published mitochondrial analysis. A variety of nuclear markers, including protein-coding gene introns and microsatellites were tested but only microsatellites were variable enough for population level analysis. Microsatellite loci showed moderate among population sharing of alleles, in contrast to the reciprocal monophyly exhibited by mitochondrial DNA. However, analyses using F-statistics and Bayesian clustering algorithms showed considerable population subdivision and clustered hellbender populations into the same major groups as the mtDNA. The microsatellites combined with the mtDNA data suggest that gene flow is severely restricted or non-existent among eight major groups, and potentially among populations (rivers) within groups. The combined mtDNA and microsatellite data suggest that the currently recognized hellbender subspecies are paraphyletic. We suggest that the eight independent groups identified in our study should be managed as such, rather than basing conservation decisions on the two named subspecies of hellbender.     

Isolation and characterization of microsatellite loci in Babylonia areolata and cross-species amplification in Babylonia formosae habei

Nine novel microsatellite primer pairs were presented for Babylonia areolata, representing the first microsatellite markers available for this genus. Levels of polymorphism were variable with 2 to 11 alleles per locus and expected heterozygosities ranging from 0.073 to 0.907 in 27 individuals of the population from which the loci were isolated. We found significant heterozygote deficit at one locus that might be attributable to null alleles. We were successful at cross-amplifying six loci in the congeneric B. formosae habei. These markers are therefore potentially useful for conservation studies, population structure assessment, ecological analyses and linkage map construction.     

The Limits of Individual Identification from Sample Allele Frequencies: Theory and Statistical Analysis

It was shown recently using experimental data that it is possible under certain conditions to determine whether a person with known genotypes at a number of markers was part of a sample from which only allele frequencies are known. Using population genetic and statistical theory, we show that the power of such identification is, approximately, proportional to the number of independent SNPs divided by the size of the sample from which the allele frequencies are available. We quantify the limits of identification and propose likelihood and regression analysis methods for the analysis of data. We show that these methods have similar statistical properties and have more desirable properties, in terms of type-I error rate and statistical power, than test statistics suggested in the literature.     

Bayesian biomarker identification based on marker-expression proteomics data

We are studying variable selection in multiple regression models in which molecular markers and/or gene-expression measurements as well as intensity measurements from protein spectra serve as predictors for the outcome variable (i.e., trait or disease state). Finding genetic biomarkers and searching genetic–epidemiological factors can be formulated as a statistical problem of variable selection, in which, from a large set of candidates, a small number of trait-associated predictors are identified. We illustrate our approach by analyzing the data available for chronic fatigue syndrome (CFS). CFS is a complex disease from several aspects, e.g., it is difficult to diagnose and difficult to quantify. To identify biomarkers we used microarray data and SELDI-TOF-based proteomics data. We also analyzed genetic marker information for a large number of SNPs for an overlapping set of individuals. The objectives of the analyses were to identify markers specific to fatigue that are also possibly exclusive to CFS. The use of such models can be motivated, for example, by the search for new biomarkers for the diagnosis and prognosis of cancer and measures of response to therapy. Generally, for this we use Bayesian hierarchical modeling and Markov Chain Monte Carlo computation.     

Linkage disequilibrium and signatures of selection on chromosomes 19 and 29 in beef and dairy cattle

The objective of this study was to quantify the extent of linkage disequilibrium (LD) on bovine chromosomes 19 and 29 and to study the pattern of selection signatures in beef and dairy breeds (Angus and Holstein) of Bos taurus. The extent of LD was estimated for 370 and 186 single nucleotide polymorphism markers on BTA19 and 29 respectively using the square of the correlation coefficient (r(2)) among alleles at pairs of loci. A comparison of the extent of LD found that the decline of LD followed a similar pattern in both breeds. We observed long-range LD and found that LD dissipates to background levels at a locus separation of about 20 Mb on both chromosomes. Along each chromosome, patterns of LD were variable in both breeds. We find that a minimum of 30 000 informative and evenly spaced markers would be required for whole-genome association studies in cattle. In addition, we have identified chromosomal regions that show some evidence of selection for economically important traits in Angus and Holstein cattle. The results of this study are of importance for the design and application of association studies.     

Microsatellite analysis of genetic variation in black bear populations

Measuring levels of genetic variation is an important aspect of conservation genetics The informativeness of such measurements is related to the variability of the genetic markers used; a particular concern in species, such as bears, which are characterized by low levels of genetic variation resulting from low population densities and small effective population sizes We describe the development of microsatellite analysis in bears and its use in assessing interpopulation differences in genetic variation in black bears from three Canadian National Parks These markers are highly variable and allowed identification of dramatic differences in both distribution and amount of variation between populations Low levels of variation were observed in a population from the Island of Newfoundland The significance of interpopulation differences in variability was tested using a likelihood ratio test of estimates of θ= 4 N_eu.     

Characterizing Uncertainty in High-Density Maps from Multiparental Populations

Multiparental populations are of considerable interest in high-density genetic mapping due to their increased levels of polymorphism and recombination relative to biparental populations. However, errors in map construction can have significant impact on QTL discovery in later stages of analysis, and few methods have been developed to quantify the uncertainty attached to the reported order of markers or intermarker distances. Current methods are computationally intensive or limited to assessing uncertainty only for order or distance, but not both simultaneously. We derive the asymptotic joint distribution of maximum composite likelihood estimators for intermarker distances. This approach allows us to construct hypothesis tests and confidence intervals for simultaneously assessing marker-order instability and distance uncertainty. We investigate the effects of marker density, population size, and founder distribution patterns on map confidence in multiparental populations through simulations. Using these data, we provide guidelines on sample sizes necessary to map markers at sub-centimorgan densities with high certainty. We apply these approaches to data from a bread wheat Multiparent Advanced Generation Inter-Cross (MAGIC) population genotyped using the Illumina 9K SNP chip to assess regions of uncertainty and validate them against the recently released pseudomolecule for the wheat chromosome 3B.     

Identification of individuals by analysis of biallelic DNA markers, using PCR and solid-phase minisequencing.

We have developed a new method for forensic identification of individuals, in which a panel of biallelic DNA markers are amplified by the PCR, and the variable nucleotides are detected in the amplified DNA fragments by the solid-phase minisequencing method. A panel of 12 common polymorphic nucleotides located on different chromosomes with reported allele frequencies close to .5 were chosen for the test. The allele frequencies for most of the markers were found to be similar in the Finnish and other Caucasian populations. We also introduce a novel approach for rapid determination of the population frequencies of biallelic markers. By this approach we were able to determine the allele frequencies of the markers in the Finnish population, by quantitative analysis of three pooled DNA samples representing 3,000 individuals. The power of discrimination and exclusion of the solid-phase minisequencing typing test with 12 markers was similar to that of three VNTR markers that are routinely used in forensic analyses at our institute. The solid-phase minisequencing method was successfully applied to type paternity and forensic case samples. We also show that the quantitative nature of our method allows typing of mixed samples.     

Using molecular markers for pedigree reconstruction of the greater amberjack (Seriola dumerili) in the absence of parental information

Ensuring appropriate levels of genetic diversity in captive populations is essential to avoid inbreeding and loss of rare alleles by genetic drift. Pedigree reconstruction and parentage analysis in the absence of parental genotypes can be a challenging task that relies in the assignment of sibship relationships among the offspring. Here, we used eight highly variable microsatellite markers and three different assignment methods to reconstruct the most likely genotypes of a parental group of wild Seriola dumerili fish based on the genotypes of six cohorts of their offspring, to assess their relative contributions to the offspring. We found that a combination of the four most variable microsatellites was enough to identify the number of parents and their contribution to the offspring, suggesting that the variability of the markers can be more critical than the number of markers. Estimated effective population sizes were lower than the number of breeders and variable among years. The results suggest unequal parental contribution that should be accounted for breeding programs in the future.     

Twenty-three new microsatellite loci in the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae)

The stable fly, Stomoxys calcitrans (L.), is a significant pest of cattle. Twenty-three microsatellite markers were isolated from a repeat-enriched genomic library of S. calcitrans. We characterized variation at these markers and found that 17 loci were polymorphic in two fly populations from Florida. Two to nine alleles were observed among the variable microsatellite loci and expected heterozygosities ranged from 0.03704 to 0.85115. These markers will be useful for characterizing population genetic differentiation and for tracking the migration patterns of stable flies in the USA and worldwide.     

Genetic and morphological patterns show variation in frequency of hybrids between Ipomopsis (Polemoniaceae) zones of sympatry

Variation in rates of hybridization among zones of sympatry between a pair of species provides a useful window into the effect of local conditions on the evolution of reproductive isolation. We employed floral morphological traits and neutral genetic markers to quantify the frequency of individuals intermediate to the two parental species in two zones of sympatry between Ipomopsis aggregata and I. tenuituba, using clustering methods that make no a priori assumptions about population structure. The sites differed not only in the frequency of intermediate individuals, but also in climate, pollinator abundance and behavior and spatial structure of plant populations. Both floral traits, which are likely to be under natural selection and molecular markers, which are quasi-neutral, indicated more population structure at one site than the other, the pattern being more pronounced for floral morphology. One likely explanation for this difference between sites is that local ecological conditions, particularly pollinator choice of flowers, have promoted different rates of hybridization between these species. Hence, the evolution of reproductive isolation might depend in part on local conditions, and thus differ among populations of the same pair of species.     

Color and genomic ancestry in Brazilians: a study with forensic microsatellites

The population of Brazil, formed by extensive admixture between Amerindians, Europeans and Africans, is one of the most variable in the world. We have recently published a study that used ancestry-informative markers to conclude that in Brazil, at an individual level, color, as determined by physical evaluation, was a poor predictor of genomic ancestry, estimated by molecular markers. To corroborate these findings we undertook the present investigation based on data from 12 commercially available forensic microsatellites that were utilized to estimate the personal genomic origin for each of 752 individuals from the city of S?o Paulo, belonging to different Brazilian color categories (275 Whites, 192 Intermediates and 285 Blacks). The genotypes permitted the calculation of a personal likelihood-ratio estimator of African or European ancestry. Although the 12 marker set proved capable of discriminating between European and African individuals, we observed very significant overlaps among the three color categories of Brazilians. This was confirmed quantitatively using a Bayesian analysis of population structure that did not demonstrate significant genetic differentiation between the three color groups. These results corroborate and validate our previous conclusions using ancestry-informative markers that in Brazil at the individual level there is significant dissociation of color and genomic ancestry.     

Untangling individual variation in natural populations: ecological, genetic and epigenetic correlates of long-term inequality in herbivory

Individual variation in ecologically important features of organisms is a crucial element in ecology and evolution, yet disentangling its underlying causes is difficult in natural populations. We applied a genomic scan approach using amplified fragment length polymorphism (AFLP) markers to quantify the genetic basis of long‐term individual differences in herbivory by mammals at a wild population of the violet Viola cazorlensis monitored for two decades. In addition, methylation‐sensitive amplified polymorphism (MSAP) analyses were used to investigate the association between browsing damage and epigenetic characteristics of individuals, an aspect that has been not previously explored for any wild plant. Structural equation modelling was used to identify likely causal structures linking genotypes, epigenotypes and herbivory. Individuals of V. cazorlensis differed widely in the incidence of browsing mammals over the 20‐year study period. Six AFLP markers (1.6% of total) were significantly related to herbivory, accounting altogether for 44% of population‐wide variance in herbivory levels. MSAP analyses revealed considerable epigenetic variation among individuals, and differential browsing damage was significantly related to variation in multilocus epigenotypes. In addition, variation across plants in epigenetic characteristics was related to variation in several herbivory‐related AFLP markers. Statistical comparison of alternative causal models suggested that individual differences in herbivory are the outcome of a complex causal structure where genotypes and epigenotypes are interconnected and have direct and indirect effects on herbivory. Insofar as methylation states of MSAP markers influential on herbivory are transgenerationally heritable, herbivore‐driven evolutionary changes at the study population will involve correlated changes in genotypic and epigenotypic distributions.     

Characterization of polymorphic microsatellite loci in Yucca filamentosa

Yuccas and their pollinator moths are a textbook example of mutualism, yet we lack sufficiently variable markers to properly study the population genetics of the plants. We characterized 13 polymorphic microsatellite loci for Yucca filamentosa by screening primers derived from an expressed sequence tag database. We found four to 13 alleles per locus and the observed heterozygosity ranged from 0.31 to 1. These markers will be useful in future ecological studies of Y. filamentosa.     

Microsatellites for the mangrove tree Avicennia germinans (Acanthaceae): Tools for hybridization and mating system studies

? Premise of the study: We developed a new set of microsatellite markers for the black mangrove Avicennia germinans, to provide new informative tools for further studies of the mating system, interspecific hybridization, and population genetics. ? Methods and Results: We used the microsatellite-enriched library approach to isolate and characterize 25 new primer pairs. Sixteen of them are polymorphic, showing a variable degree of variation in A. germinans, while nine were monomorphic in the samples examined. Eight exhibited private alleles in A. schaueriana. ? Conclusions: These results indicate that these new microsatellite markers will be useful molecular tools for further studies of A. germinans and A. schaueriana population genetics, mating systems, and hybridization.     

The utility of fast evolving molecular markers for studying speciation in the Antarctic benthos

The Southern Ocean is surprisingly rich in species that coexist in one of the most extreme environments on Earth yet the processes leading to speciation in this ecosystem are not well understood. To remedy this, tools that measure the genetic connectedness within a species are needed. Although useful for phylogenetic purposes, the readily available mitochondrial markers (e.g. 16S, COI) suffer from numerous shortcomings for population genetics. Therefore, molecular markers are needed that are sufficiently variable, unlinked, biparentally inherited, and distributed over the whole genome. We argue that microsatellites are suitable markers that have not been widely used in exploratory studies due to their difficult initial set-up. Working with the Ceratoserolis trilobitoides species complex (Isopoda), we demonstrate that using a novel protocol many microsatellites can be identified quickly. An increased availability of these highly sensitive markers will be useful for studies addressing the origin of species in the Southern Ocean and their response to future climate change. Christoph Held and Florian Leese contributed equally to this paper.