A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra).

The major histocompatibility complex (MHC) acts as an interface between the immune system and infectious diseases. Accurate characterization and genotyping of the extremely variable MHC loci are challenging especially without a reference sequence. We designed a combination of long-range PCR, Illumina short-reads, and Oxford Nanopore MinION long-reads approaches to capture the genetic variation of the MHC II DRB locus in an Italian population of the Alpine chamois (Rupicapra rupicapra). We utilized long-range PCR to generate a 9 Kb fragment of the DRB locus. Amplicons from six different individuals were fragmented, tagged, and simultaneously sequenced with Illumina MiSeq. One of these amplicons was sequenced with the MinION device, which produced long reads covering the entire amplified fragment. A pipeline that combines short and long reads resolved several short tandem repeats and homopolymers and produced a de novo reference, which was then used to map and genotype the short reads from all individuals. The assembled DRB locus showed a high level of polymorphism and the presence of a recombination breakpoint. Our results suggest that an amplicon-based NGS approach coupled with single-molecule MinION nanopore sequencing can efficiently achieve both the assembly and the genotyping of complex genomic regions in multiple individuals in the absence of a reference sequence.

Evidence for past and present hybridization in three Antarctic icefish species provides new perspectives on an evolutionary radiation.

Determining the timing, extent and underlying causes of interspecific gene exchange during or following speciation is central to understanding species' evolution. Antarctic notothenioid fish, thanks to the acquisition of antifreeze glycoproteins during Oligocene transition to polar conditions, experienced a spectacular radiation to >100 species during Late Miocene cooling events. The impact of recent glacial cycles on this group is poorly known, but alternating warming and cooling periods may have affected species' distributions, promoted ecological divergence into recurrently opening niches and/or possibly brought allopatric species into contact. Using microsatellite markers and statistical methods including Approximate Bayesian Computation, we investigated genetic differentiation, hybridization and the possible influence of the last glaciation/deglaciation events in three icefish species of the genus Chionodraco. Our results provide strong evidence of contemporary and past introgression by showing that: (i) a substantial fraction of contemporary individuals in each species has mixed ancestry, (ii) evolutionary scenarios excluding hybridization or including it only in ancient times have small or zero posterior probabilities, (iii) the data support a scenario of interspecific gene flow associated with the two most recent interglacial periods. Glacial cycles might therefore have had a profound impact on the genetic composition of Antarctic fauna, as newly available shelf areas during the warmer intervals might have favoured secondary contacts and hybridization between diversified groups. If our findings are confirmed in other notothenioids, they offer new perspectives for understanding evolutionary dynamics of Antarctic fish and suggest a need for new predictions on the effects of global warming in this group.

ABC as a flexible framework to estimate demography over space and time: some cons, many pros.

The analysis of genetic variation to estimate demographic and historical parameters and to quantitatively compare alternative scenarios recently gained a powerful and flexible approach: the Approximate Bayesian Computation (ABC). The likelihood functions does not need to be theoretically specified, but posterior distributions can be approximated by simulation even assuming very complex population models including both natural and human-induced processes. Prior information can be easily incorporated and the quality of the results can be analysed with rather limited additional effort. ABC is not a statistical analysis per se, but rather a statistical framework and any specific application is a sort of hybrid between a simulation and a data-analysis study. Complete software packages performing the necessary steps under a set of models and for specific genetic markers are already available, but the flexibility of the method is better exploited combining different programs. Many questions relevant in ecology can be addressed using ABC, but adequate amount of time should be dedicated to decide among alternative options and to evaluate the results. In this paper we will describe and critically comment on the different steps of an ABC analysis, analyse some of the published applications of ABC and provide user guidelines.

The role of human demographic history in determining the distribution and frequency of transferase-deficient galactosaemia mutations.

Classical or transferase-deficient galactosaemia is an inherited metabolic disorder caused by mutation in the human Galactose-1-phosphate uridyl transferase (GALT) gene. Of some 170 causative mutations reported, fewer than 10% are observed in more than one geographic region or ethnic group. To better understand the population history of the common GALT mutations, we have established a haplotyping system for the GALT locus incorporating eight single nucleotide polymorphisms and three short tandem repeat markers. We analysed haplotypes associated with the three most frequent GALT gene mutations, Q188R, K285N and Duarte-2 (D2), and estimated their age. Haplotype diversity, in conjunction with measures of genetic diversity and of linkage disequilibrium, indicated that Q188R and K285N are European mutations. The Q188R mutation arose in central Europe within the last 20 000 years, with its observed east-west cline of increasing relative allele frequency possibly being due to population expansion during the re-colonization of Europe by Homo sapiens in the Mesolithic age. K285N was found to be a younger mutation that originated in Eastern Europe and is probably more geographically restricted as it arose after all major European population expansions. The D2 variant was found to be an ancient mutation that originated before the expansion of Homo sapiens out of Africa.

Ancient vs. recent processes as factors shaping the genetic variation of the European wild boar: are the effects of the last glaciation still detectable?

The European wild boar is an important game species, subjected to local extinctions and translocations in the past, and currently enormously and worryingly expanding in some areas where management is urgently required. Understanding the relative roles of ancient and recent events in shaping the genetic structure of this species is therefore not only an interesting scientific issue, but it represents also the basis for addressing future management strategies. In addition, several pig breeds descend from the European wild boar, but the geographical location of the domestication area(s) and the possible introgression of pig genomes into wild populations are still open questions. Here, we analysed the genetic variation in different wild boar populations in Europe. Ten polymorphic microsatellites were typed in 252 wild boars and the mtDNA control region was sequenced in a subset of 145 individuals. Some samples from different pig breeds were also analysed. Our results, which were obtained considering also 612 published mtDNA sequences, suggest that (i) most populations are similarly differentiated, but the major discontinuity is found along the Alps; (ii) except for the Italian populations, European wild boars show the signature of a postglacial demographic expansion; (iii) Italian populations seem to preserve a high proportion of preglaciation diversity; (iv) the demographic decline which occurred in some areas in the last few centuries did not produce a noticeable reduction of genetic variation; (v) signs of human-mediated gene flow among populations are weak, although in some regions the effects of translocations are detectable and a low degree of pig introgression can be identified; (vi) the hypothesis of an independent domestication centre in Italy is not supported by our data, which in turn confirm that Central European wild boar might have represented an important source for domestic breeds. We can therefore conclude that recent human activities had a limited effect on the wild boar genetic structure. It follows that areas with high variation and differentiation represent natural reservoirs of genetic diversity to be protected avoiding translocations. In this context controlling some populations by hunting is not expected to affect significantly genetic variation in this species.

Analysis of DNA diversity by spatial autocorrelation.

Two statistics are proposed for summarizing spatial patterns of DNA diversity. These autocorrelation indices for DNA analysis, or AIDAs, can be applied to RFLP and sequence data; the resulting set of autocorrelation coefficients, or correlogram, measures whether, and to what extent, individual DNA sequences or haplotypes resemble the haplotypes sampled at arbitrarily chosen spatial distances. Analyses of computer-generated sets of data, and of RFLP data from two natural populations, show that AIDAs allow one to objectively and simply identify basic patterns in the spatial distribution of haplotypes. These statistics, therefore, seem to be a useful tool both to explore the genetic structure of a population and to suggest hypotheses on the evolutionary processes that shaped the observed patterns.

The use of intraallelic variability for testing neutrality and estimating population growth rate.

To better understand the forces affecting individual alleles, we introduce a method for finding the joint distribution of the frequency of a neutral allele and the extent of variability at closely linked marker loci (the intraallelic variability). We model three types of intraallelic variability: (a) the number of nonrecombinants at a linked biallelic marker locus, (b) the length of a conserved haplotype, and (c) the number of mutations at a linked marker locus. If the population growth rate is known, the joint distribution provides the basis for a test of neutrality by testing whether the observed level of intraallelic variability is consistent with the observed allele frequency. If the population growth rate is unknown but neutrality can be assumed, the joint distribution provides the likelihood of the growth rate and leads to a maximum-likelihood estimate. We apply the method to data from published data sets for four loci in humans. We conclude that the Delta32 allele at CCR5 and a disease-associated allele at MLH1 arose recently and have been subject to strong selection. Alleles at PAH appear to be neutral and we estimate the recent growth rate of the European population to be approximately 0.027 per generation with a support interval of (0.017-0.037). Four of the relatively common alleles at CFTR also appear to be neutral but DeltaF508 appears to be significantly advantageous to heterozygous carriers.

Using linked markers to infer the age of a mutation.

Advances in sequencing and genotyping technologies over the last decade have enabled geneticists to easily characterize genetic variation at the nucleotide level. Hundreds of genes harboring mutations associated with genetic disease have now been identified by positional cloning. Using variation at closely linked genetic markers, it is possible to predict the times in the past at which particular mutations arose. Such studies suggest that many of the rare mutations underlying human genetic disorders are relatively young. Studies of variation at genetic markers linked to particular mutations can provide insights into human geographic history, and historical patterns of natural selection and disease, that are not available from other sources. We review two approaches for estimating allele age using variation at linked genetic markers. A phylogenetic approach aims to reconstruct the gene tree underlying a sample of chromosomes carrying a particular mutation, obtaining a "direct" estimate of allele age from the age of the root of this tree. A population genetic approach relies on models of demography, mutation, and/or recombination to estimate allele age without explicitly reconstructing the gene tree. Phylogenetic methods are best suited for studies of ancient mutations, while population genetic methods are better suited for studies of recent mutations. Methods that rely on recombination to infer the ages of alleles can be fine-tuned by choosing linked markers at optimal map distances to maximize the information available about allele age. A limitation of methods that rely on recombination is the frequent lack of a fine-scale linkage map. Maximum likelihood and Bayesian methods for estimating allele age that rely on intensive numerical computation are described, as well as "composite" likelihood and moment-based methods that lead to simple estimators. The former provide more accurate estimates (particularly for large samples of chromosomes) and should be employed if computationally practical.

Do Basque- and Caucasian-speaking populations share non-Indo-European ancestors?

Genetic evidence is consistent with the view that the Indo-European languages were propagated in Europe by the diffusion of early farmers. The existence of phylogenetic relationships between European populations speaking other languages has been proposed on linguistic and archaeological grounds, and is here tested by analyzing allele frequencies at ten polymorphic protein and blood group loci. Genetic distances between speakers of Basque and Caucasian languages are compared with those between controls, i.e. contiguous populations speaking Indo-European and Altaic. Although some statistical tests show an excess of genetic similarity between Basque and South Caucasian speakers, most results do not support their common origin. If the Basques and the Caucasian-speaking populations share common ancestors, recent evolutionary phenomena must have caused divergence between them, so that their gene frequencies do not appear more similar now than those of random pairs of populations separated by the same geographic distance.

Disentangling the effects of recombination, selection, and demography on the genetic variation at a major histocompatibility complex class II gene in the alpine chamois.

The major histocompatibility complex (MHC) harbours some of the most polymorphic loci in vertebrate genomes. MHC genes are thought to be subject to some form of balancing selection, most likely pathogen-mediated selection. Hence, MHC genes are excellent candidates for exploring adaptive processes. In this study, we investigated the genetic variation at exon 2 of the DRB class II MHC locus in 191 alpine chamois (Rupicapra rupicapra) from 10 populations in the eastern Alps of Italy. In particular, we were interested in distinguishing and estimating the relative impact of selective and demographic factors, while taking into account the confounding effect of recombination. The extremely high d(n)/d(s) ratio and the presence of trans-species polymorphisms suggest that a strong long-term balancing selection effect has been operating at this locus throughout the evolutionary history of this species. We analysed patterns of genetic variation within and between populations, and the mitochondrial D-loop polymorphism patterns were analysed to provide a baseline indicator of the effects of demographic processes. These analyses showed that (i) the chamois experienced a demographic decline in the last 5000-30 000 years, most likely related to the postglacial elevation in temperature; (ii) this demographic process can explain the results of neutrality tests applied to MHC variation within populations, but cannot justify the much weaker divergence between populations implied by MHC as opposed to mitochondrial DNA; (iii) similar sets of divergent alleles are probably maintained with similar frequencies by balancing selection in different populations, and this mechanism is also operating in small isolated populations, which are strongly affected by drift.

Expected effects of mass screening policies on the frequency of cystic fibrosis homozygotes.

We evaluated, by deterministic computer simulation, some effects of a screening programme for carriers of cystic fibrosis mutations. Two different selective regimes (heterozygote advantage and directional selection against recessive homozygotes) and three kinds of response to the screening were simulated. The curves describing the expected decline in the frequency of CF homozygotes allow one to predict some benefits of a screening campaign. In addition, it is shown that a strategy aimed at testing couples, rather than individuals, may become less expensive after only two generations of screening. The main source of uncertainty for a screening programme remains the selection mechanism, namely the existence of some sort of biological advantage for heterozygous carriers of CF mutations.

Inferring admixture proportions from molecular data.

We derive here two new estimators of admixture proportions based on a coalescent approach that explicitly takes into account molecular information as well as gene frequencies. These estimators can be applied to any type of molecular data (such as DNA sequences, restriction fragment length polymorphisms [RFLPs], or microsatellite data) for which the extent of molecular diversity is related to coalescent times. Monte Carlo simulation studies are used to analyze the behavior of our estimators. We show that one of them (mY) appears suitable for estimating admixture from molecular data because of its absence of bias and relatively low variance. We then compare it to two conventional estimators that are based on gene frequencies. mY proves to be less biased than conventional estimators over a wide range of situations and especially for microsatellite data. However, its variance is larger than that of conventional estimators when parental populations are not very differentiated. The variance of mY becomes smaller than that of conventional estimators only if parental populations have been kept separated for about N generations and if the mutation rate is high. Simulations also show that several loci should always be studied to achieve a drastic reduction of variance and that, for microsatellite data, the mean square error of mY rapidly becomes smaller than that of conventional estimators if enough loci are surveyed. We apply our new estimator to the case of admixed wolflike Canid populations tested for microsatellite data.

Inferring admixture proportions from molecular data: extension to any number of parental populations.

The relative contribution of two parental populations to a hybrid group (the admixture proportions) can be estimated using not only the frequencies of different alleles, but also the degree of molecular divergence between them. In this paper, we extend this possibility to the case of any number of parental populations. The newly derived multiparental estimator is tested by Monte Carlo simulations and by generating artificial hybrid groups by pooling mtDNA samples from human populations. The general properties (including the variance) of the two-parental estimator seem to be retained by the multiparental estimator. When mixed human populations are considered and hypervariable single-locus data are analyzed (mtDNA control region), errors in the estimated contributions appear reasonably low only when highly differentiated parental populations are involved. Finally, the method applied to the hybrid Canary Island population points to a much lower female contribution from Spain than has previously been estimated.

Genetics and the population history of Europe.

Analysis of genetic variation among modern individuals is providing insight into prehistoric events. Comparisons of levels and patterns of genetic diversity with the predictions of models based on archeological evidence suggest that the spread of early farmers from the Levant was probably the main episode in the European population history, but that both older and more recent processes have left recognizable traces in the current gene pool.

The number of segregating sites in expanding human populations, with implications for estimates of demographic parameters.

The frequency distribution of pairwise differences between sequences of mtDNA has recently been used to estimate the size of human populations before and after a hypothetical episode of rapid population growth and the time at which the population grew. To test the internal consistency of this method, we used three different sets of human mtDNA data and the corresponding demographic parameters estimated from the distribution of pairwise differences to determine by simulation the expected number of segregating sites, S, and its empirical distribution. The results indicate that the observed values of S are significantly lower than expected in two of three cases under the assumption of the infinite-sites model. Further simulations in which mutations were allowed to occur more than once at the same site and in which there was variation in mutation rate among sites show that the expected number of segregating sites can be much lower than under the infinite-site assumption. Nevertheless, the observed value of S is still significantly different from the value expected under the expansion hypothesis in two of three cases.

Using rare mutations to estimate population divergence times: a maximum likelihood approach.

In this paper we propose a method to estimate by maximum likelihood the divergence time between two populations, specifically designed for the analysis of nonrecurrent rare mutations. Given the rapidly growing amount of data, rare disease mutations affecting humans seem the most suitable candidates for this method. The estimator RD, and its conditional version RDc, were derived, assuming that the population dynamics of rare alleles can be described by using a birth-death process approximation and that each mutation arose before the split of a common ancestral population into the two diverging populations. The RD estimator seems more suitable for large sample sizes and few alleles, whose age can be approximated, whereas the RDc estimator appears preferable when this is not the case. When applied to three cystic fibrosis mutations, the estimator RD could not exclude a very recent time of divergence among three Mediterranean populations. On the other hand, the divergence time between these populations and the Danish population was estimated to be, on the average, 4,500 or 15,000 years, assuming or not a selective advantage for cystic fibrosis carriers, respectively. Confidence intervals are large, however, and can probably be reduced only by analyzing more alleles or loci.

Geographical structuring in the mtDNA of Italians.

Geographical patterns of mtDNA variation were studied in 12 Italian samples (1072 individuals) by two different spatial autocorrelation methods. Separate analyses of the frequencies of 12 restriction morphs show North-South clines, differences between Sardinia and the mainland populations, and the effects of isolation by distance. A recently developed autocorrelation statistic summarizing molecular similarity at all sites (AIDA; autocorrelation index for DNA analysis) confirms the presence of a clinical pattern; differences between random pairs of haplotypes tend to increase with their geographical distance. The partition of gene diversity, however, reveals that most variability occurs within populations, whereas differences between populations are minor (GST = 0.057). When the data from the 12 samples are pooled, two descriptors of genetic variability (number of polymorphic sites and average sequence difference between pairs of individuals) do not behave as expected under neutrality. The presence of clinal patterns, Tajima's tests, and a simulation experiment agree in suggesting that population sizes increased rapidly in Italy and Sicily but not necessarily so in Sardinia. The distribution of pairwise sequence differences in the Italian peninsula (excluding Sardinia) permits a tentative location of the demographic increase between 8000 and 20,500 years ago. These dates are consistent with archaeological estimates of two distinct expansion processes, occurring, respectively, in the Neolithic and after the last glacial maximum in the Paleolithic. Conversely, there is no genetic evidence that such processes have had a major impact on the Sardinian population.

DNA fingerprinting data and the analysis of population genetic structure by comparing band-sharing patterns.

Genetic isolation among populations can be effectively investigated by multilocus DNA fingerprinting. If populations have diverged, it is expected that the mean proportion of bands shared by individuals from the same population, Bw, exceeds the corresponding mean, Bb, calculated from pairs of individuals from distinct populations. A problem arises in deciding whether any difference between Bw and Bb is statistically significant. In fact, any two band-sharing data (bij), contributing to Bw or Bb, are not independent if they share a common individual (like bij and bjl). This prevents a correct application of parametric tests, such as the Student's t-test. Recently, a modification of this test has been proposed that should avoid the independence problem. Using a large number of samples of fingerprints, simulated from an appropriate 'genetic' model, under a wide range of conditions, we compared the performances of the Student's t-test, the modified t-test and five new permutation tests, where individuals, rather than bij values, are permuted. We found that: (i) the Student's t-test can be very permissive, rejecting too often the null hypothesis when true, but is correct or conservative in certain cases; (ii) the modified t-test is extremely conservative when the null hypothesis is true and very inefficient otherwise; (iii) all five permutation tests are strictly correct, provided that individuals are ordered randomly on gels; and (iv) in this case, the permutation tests are equally efficient, and are not inferior to the Student's t-test when the latter is approximately correct and provides a fair benchmark.

Testing hypotheses on processes of genetic and linguistic change in the Caucasus.

Extensive genetic diversity exists in the populations of the Caucasus. Various hypotheses on its origin and evolution were tested by comparing genetic, geographic, and linguistic distances. Seventeen polymorphic loci and 107 localities were considered, and Mantel tests of matrix association were carried out. Genetic differences correlate more with linguistic than with geographic distances; but when populations are grouped by the language spoken, this correlation loses significance, whereas genetic and geographic distances between groups appear significantly associated. Hypotheses that classify North and South Caucasian languages into distinct families or that treat all North Caucasian languages as independent linguistic entities fail to account for genetic variation better than simpler models. We interpret these results as evidence for an evolutionary process in which linguistic and genetic divergence has resulted from population subdivision and from processes of elite dominance, that is, language replacement not associated with major migratory movements.