Quantifying the genetic component of phenotypic variation in unpedigreed wild plants: tailoring genomic scan for within-population use

The study of adaptive genetic variation in natural populations is central to evolutionary biology. Quantitative genetics methods, however, are hardly applicable to long-lived organisms, and current knowledge on adaptive genetic variation in wild plants mostly refers to annuals and short-lived perennials. Studies on long-lived species are essential to explore possible life-history correlates of genetic variation, selection, and trait heritability. In this paper, we propose a method based on molecular markers to quantify the genetic basis of individual phenotypic differences in wild plants under natural conditions. Rather than focusing on inferring individual relatedness to estimate the heritability of phenotypic traits, we directly estimate the proportion of observed phenotypic variance that is statistically accounted for by genotypic differences between individuals. This is achieved by (i) identifying loci that are correlated across individuals with the phenotypic trait of interest by means of an amplified fragment length polymorphism (AFLP)-based explorative genomic scan, and (ii) fitting multiple regression and linear random effect models to estimate the effects of genotype, environment and genotype × environment on phenotypes. We apply this method to estimate genotypic and environmental effects on cumulative maternal fecundity in a wild population of the long-lived Viola cazorlensis monitored for 20 years. Results show that between 56–63% (depending on estimation method) of phenotypic variance in fecundity is accounted for by genotypic differences in 11 AFLP loci that are significantly related to fecundity. Genotype × environment effects accounted for 38% of fecundity variance, which may help to explain the unexpectedly high levels of genetic variance for fecundity found.     

The expansion of conservation genetics

The 'crisis discipline' of conservation biology has voraciously incorporated many technologies to speed up and increase the accuracy of conservation decision-making. Genetic approaches to characterizing endangered species or areas that contain endangered species are prime examples of this. Technical advances in areas such as high-throughput sequencing, microsatellite analysis and non-invasive DNA sampling have led to a much-expanded role for genetics in conservation. Such expansion will allow for more precise conservation decisions to be made and, more importantly, will allow conservation genetics to contribute to area- and landscape-based decision-making processes.     

Non-invasive genetic sampling and individual identification

Individual identification via non-invasive sampling is of prime importance in conservation genetics and in behavioural ecology. This approach allows for genetics studies of wild animals without having to catch them, or even to observe them. The material used as a source of DNA is usually faeces, shed hairs, or shed feathers. It has been recendy shown that this material may lead to genotyping errors, mainly due to allelic dropout. In addition to these technical errors, there are problems with accurately estimating the probability of identity (PI, or the probability of two individuals having identical genotypes) because of the presence of close relatives in natural populations. As a consequence, before initiating an extensive study involving non-invasive sampling, we strongly suggest conducting a pilot study to assess both the technical difficulties and the PI for the genetic markers to be used. This pilot study could be carried out in three steps: (i) estimation of the PI using preliminary genetic data; (ii) simulations taking into account the PI and choosing the technical error rate mat is sufficiently low for assessing the scientific question; (iii) polymerase chain reaction (PCR) experiments to check if it is technically possible to achieve this error rate.     

High through-put characterization of insect morphocryptic entities by a non-invasive method using direct-PCR of fecal DNA

The development of non-invasive molecular techniques is currently increasing, particularly in the fields of behavioural ecology and conservation genetics of mammals. Surprisingly, genetic studies of Arthropods and particularly the insects have not benefited yet from the contributions that non-invasive methods have made. Here, we outline a strategy for identifying phytophagous insect genetic entities based on direct-PCR of fecal DNA combined with double strand conformation polymorphism (DSCP) typing. This allows the differentiation of morphocryptic entities within the species Ceutorhynchus assimilis (Coleoptera: Curculionidae), a candidate biocontrol agent of a noxious weed. The results obtained clearly demonstrate the potential for this method to provide a valuable means for genetic and ecological studies of Arthropods.     

Molecular characterization and evolutionary pattern of the 9- cis -epoxycarotenoid dioxygenase NCED1 gene in grapevine

This study provides the first analysis of the level and patterns of nucleotide polymorphism of the NCED1 gene in grapevine (Vitis vinifera L.). A total of 123 sequences of the gene were analyzed to give a sample of 50 wild accessions and 73 cultivars. A high single nucleotide polymorphism and haplotype diversity was revealed in the cultivars studied, especially Tunisian germplasms which present an important and diverse reservoir of genetic diversity for grape breeding and conservation. The haplotype distribution highlights two origins of the cultivars studied: one may be related to primary grapevine gene pool domestication while the second seems to be more recent. Thus, besides domestication, gene introgression has also played a role in shaping the current varietal landscape of grape cultivars. Higher nucleotide and haplotype polymorphism was recorded for cultivars. This was accompanied by a higher recombination rate in cultivated grapevines for this gene, a recent selective sweep in wild samples and a balancing selection in cultivars. The conservation of genetic diversity of the endangered wild germplasm is important to ensure that the wild population can be used in future breeding programs of the domesticated cultivars. The high number of alleles discovered can be used as a valuable source for association studies between allele frequencies and phenotypic variations in this gene. In addition to natural selection, molecular evidence shows that genetic variation in this locus appears to be shaped by a combination of mutation and recombination events.     

Development and characterization of 43 microsatellite markers for the critically endangered primrose Primula reinii using MiSeq sequencing

Primula reinii (Primulaceae), a perennial herb belonging to the Primula section Reinii, occurs on wet, shaded rocky cliffs in the mountains of Japan. This threatened species comprises four varieties; these plants are very localized and rare in the wild. In this study, 43 microsatellite markers were developed using MiSeq sequencing to facilitate conservation genetics of these critically endangered primroses. We developed novel microsatellite markers for three varieties of P. reinii, and tested its polymorphism and genetic diversity using natural populations. These novel markers displayed relatively high polymorphism; the number of alleles and expected heterozygosities ranged from 2 to 6 (mean=3.2) and 0.13 to 0.82 (mean=0.45), respectively. All loci were in HardyeWeinberg equilibrium. These microsatellite markers will be powerful tools to assess P. reinii genetic diversity and develop effective conservation and management strategies.     

The fate of balanced,phenotypic polymorphisms in fragmented metapopulations

In large populations, genetically distinct phenotypic morphs can be maintained in equilibrium (at a 1 : 1 ratio in the simplest case) by frequency‐dependent selection, as shown by Sewall Wright. The consequences of population fragmentation on this equilibrium are not widely appreciated. Here, I use a simple computational model to emphasize that severe fragmentation biases the morph ratio towards the homozygous recessive genotype through drift in very small populations favouring the more common recessive allele. This model generalizes those developed elsewhere for heterostylous plants and major histocompatibility complex alleles, emphasizes one particular outcome and avoids the restricting assumptions of more analytical models. There are important implications for both fundamental evolutionary biology and conservation genetics. I illustrate this with a range of examples but refer particularly to shell polymorphism in snails. These examples show how habitat fragmentation could have a direct and often unappreciated effect on species at the level of their population genetics.     

Conservation genetics in a globally changing environment: present problems,paradoxes and future challenges

Despite recent advances in conservation genetics and related disciplines and the growing impact that conservation genetics is having in conservation biology, our knowledge on several key issues in the field is still insufficient. Here we identify some of these issues together with addressing several paradoxes which have to be solved before conservation genetics can face new challenges that are appearing in the transitory phase from the population genetics into the population genomics era. Most of these issues, paradoxes and challenges, like the central dogma of conservation genetics, the computational, theoretical and laboratory experiment achievements and limitations in the conservation genetics field have been discussed. Further knowledge on the consequences of inbreeding and outbreeding depression in wild populations as well as the capacity of small populations to adapt to local environmental conditions is also urgently needed. The integration of experimental, theoretical and applied conservation genetics will contribute to improve our understanding of methodological and applied aspects of conservation genetics.     

Characterization of polymorphic microsatellite loci in a traditional Chinese medicinal plant,Gastrodia elata

Genetic characterization of germplasm resource is essential for the conservation and efficient utilization of a traditional Chinese medicinal plant, Gastrodia elata. Thirty‐two primer pairs flanking microsatellite repeats were designed and tested using 32 individuals from eight wild populations. A total of 13 microsatellite loci were found highly polymorphic, with three to 10 alleles per locus and gene diversity ranging from 0.400 to 0.841. These microsatellites have been directly applied to the ongoing population and conservation genetics studies.     

Perspectives of genomics for genetic conservation of livestock

Genomics provides new opportunities for conservation genetics. Conservation genetics in livestock is based on estimating diversity by pedigree relatedness and managing diversity by choosing those animals that maximize genetic diversity. Animals can be chosen as parents for the next generation, as donors of material to a gene bank, or as breeds for targeting conservation efforts. Genomics provides opportunities to estimate diversity for specific parts of the genome, such as neutral and adaptive diversity and genetic diversity underlying specific traits. This enables us to choose candidates for conservation based on specific genetic diversity (e.g. diversity of traits or adaptive diversity) or to monitor the loss of diversity without conservation. In wild animals direct genetic management, by choosing candidates for conservation as in livestock, is generally not practiced. With dense marker maps opportunities exist for monitoring relatedness and genetic diversity in wild populations, thus enabling a more active management of diversity.     

Genetic Analysis of Copper Resistance in PARAMECIUM AURELIA Syngen 4

Measurement of the median tolerance limit to cupric ion of 28 wild stocks of Paramecium aurelia syngen 4 revealed a phenotypic polymorphism: two stocks showed a much greater resistance than the others. Genetic analysis showed the resistant phenotype was produced by a recessive allele, cur, identical in both resistant wild stocks. No modification of the phenotypic expression of this allele could be detected after backcrossing into different genetic backgrounds. The results are interpreted to support the importance of mutational adaptation in this inbreeding species. Possible patterns of internal genetic organization of species which rely on mutation for adaptation are discussed.     

Applications and techniques for non-invasive faecal genetics research in felid conservation

Non-invasive genetic techniques utilising DNA extracted from faeces hold great promise for felid conservation research. These methods can be used to establish species distributions, model habitat requirements, analyse diet, estimate abundance and population density, and form the basis for population, landscape and conservation genetic analyses. Due to the elusive nature of most felid species, non-invasive genetic methods have the potential to provide valuable data that cannot be obtained with traditional observational or capture techniques. Thus, these methods are particularly valuable for research and conservation of endangered felid species. Here, we review recent studies that use non-invasive faecal genetic techniques to survey or study wild felids; provide an overview of field, laboratory and analysis techniques; and offer suggestions on how future non-invasive genetic studies can be expanded or improved to more effectively support conservation.     

Altered wing phenotypes of captive-bred migratory birds lower post-release fitness

Captive breeding and release to the wild is a globally important conservation tool. However, captivity can result in phenotypic changes that incur post-release fitness costs, especially if they affect strenuous or risky behaviours. Bird wing shape is critical for migration success and suboptimal phenotypes are strongly selected against. In this study, I demonstrate surprising plasticity of bird wing phenotypes in captivity for 4/16 studied species. In a model species, captive-born juveniles with wild wing phenotypes (a 1-mm longer distal primary flight feather) survived post-release at 2.7 times the rate of those with captive phenotypes (i.e. a shorter distal feather). Subtle phenotypic changes and their fitness impacts are more common than widely realised because they are easily overlooked. To improve captive breeding for conservation, practitioners must surveil phenotypic changes and find ways to mitigate them.     

Population structure, demographic history, and evolutionary patterns of a green-fruited tomato, Solanum peruvianum (Solanaceae), revealed by spatial genetics analyses

? Premise of the study: Wild relatives of crop species have long been viewed as an important genetic resource for crop improvement, but basic information about the population biology of these species is often lacking. This study investigated the population structure, demographic history, and evolutionary patterns of a green-fruited relative of the cultivated tomato, Solanum peruvianum. ? Methods: We investigated spatial genetics of S. peruvianum and screened for loci potentially under natural selection by integrating amplified fragment length polymorphism (AFLP) genotypes, phenotypic data, geography, and geographic information system (GIS)-derived climate data of 19 natural populations. ? Key results: Solanum peruvianum had a moderate degree of population differentiation, likely reflecting partial geographic isolation between species. Populations had a distribution pattern consistent with north-to-south "stepping-stone" dispersal with significant isolation by distance (IBD), similar to other tomato species. Several AFLP loci showed evidence of selection and associated with climate variables. However, phenotypic traits generally did not correlate with climate variables. ? Conclusions: Geographic features of the coastal Andes is likely an important factor that determines the migration pattern and population structure of S. peruvianum, but climatic factors do not appear to be critical for its phenotypic evolution, perhaps due to a high degree of phenotypic plasticity. Spatial genetics of wild relatives of crop species is a powerful approach to understand their evolutionary patterns and to accelerate the discovery of their potential for crop improvements.     

Color phenotypes are under similar genetic control in two distantly related species of Timema stick insect

Ecology and genetics are both of general interest to evolutionary biologists as they can influence the phenotypic and genetic response to selection. The stick insects Timema podura and Timema cristinae exhibit a green/melanistic body color polymorphism that is subject to different ecologically based selective regimes in the two species. Here, we describe aspects of the genetics of this color polymorphism in T. podura, and compare this to previous results in T. cristinae. We first show that similar color phenotypes of the two species cluster in phenotypic space. We then use genome‐wide association mapping to show that in both species, color is controlled by few loci, dominance relationships between color alleles are the same, and SNPs associated with color phenotypes colocalize to the same linkage group. Regions within this linkage group that harbor genetic variants associated with color exhibit elevated linkage disequilibrium relative to genome wide expectations, but more strongly so in T. cristinae. We use these results to discuss predictions regarding how the genetics of color could influence levels of phenotypic and genetic variation that segregate within and between populations of T. podura and T. cristinae, drawing parallels with other organisms.     

Genetic variation in the endangered wild apple (Malus sylvestris (L.) Mill.) in Belgium as revealed by amplified fragment length polymorphism and microsatellite markers

The genetic variation within and between wild apple samples (Malus sylvestris) and cultivated apple trees was investigated with amplified fragment length polymorphisms (AFLP) and microsatellite markers to develop a conservation genetics programme for the endangered wild apple in Belgium. In total, 76 putative wild apples (originating from Belgium and Germany), six presumed hybrids and 39 cultivars were typed at 12 simple sequence repeats (SSR) and 139 amplified fragment length polymorphism (AFLP) loci. Principal co-ordinate analysis and a model-based clustering method classified the apples into three major gene pools: wild Malus sylvestris genotypes, edible cultivars and ornamental cultivars. All presumed hybrids and two individuals (one Belgian, one German) sampled as M. sylvestris were assigned completely to the edible cultivar gene pool, revealing that cultivated genotypes are present in the wild. However, gene flow between wild and cultivated gene pools is shown to be almost absent, with only three genotypes that showed evidence of admixture between the wild and edible cultivar gene pools. Wild apples sampled in Belgium and Germany constitute gene pools that are clearly differentiated from cultivars and although some geographical pattern of genetic differentiation among wild apple populations exists, most variation is concentrated within samples. Concordant conclusions were obtained from AFLP and SSR markers, which showed highly significant correlations in both among-genotypes and among-samples genetic distances.     

Single QTL mapping and nucleotide-level resolution of a physiologic trait in wine Saccharomyces cerevisiae strains

Natural Saccharomyces cerevisiae yeast strains exhibit very large genotypic and phenotypic diversity. However, the link between phenotype variation and genetic determinism is still difficult to identify, especially in wild populations. Using genome hybridization on DNA microarrays, it is now possible to identify single-feature polymorphisms among divergent yeast strains. This tool offers the possibility of applying quantitative genetics to wild yeast strains. In this instance, we studied the genetic basis for variations in acetic acid production using progeny derived from two strains from grape must isolates. The trait was quantified during alcoholic fermentation of the two strains and 108 segregants derived from their crossing. A genetic map of 2212 markers was generated using oligonucleotide microarrays, and a major quantitative trait locus (QTL) was mapped with high significance. Further investigations showed that this QTL was due to a nonsynonymous single-nucleotide polymorphism that targeted the catalytic core of asparaginase type I (ASP1) and abolished its activity. This QTL was only effective when asparagine was used as a major nitrogen source. Our results link nitrogen assimilation and CO(2) production rate to acetic acid production, as well as, on a broader scale, illustrating the specific problem of quantitative genetics when working with nonlaboratory microorganisms.     

Cross-species microsatellite amplification in Vasconcellea and related genera and their use in germplasm classification.

To generate inexpensive and efficient DNA markers for addressing a number of population genetics problems and identification of wild hybrids in Vasconcellea, we have evaluated the use of simple sequence repeat (SSR) primers previously developed for other species. A set of 103 Vasconcellea accessions and some individuals of the related genera Carica and Jacaratia were analyzed with 10 primer pairs directing amplification of chloroplast microsatellites in Nicotiana tabacum and 9 nuclear SSR primer pairs recently identified in Vasconcellea x heilbornii. Heterologous amplification of chloroplast SSRs was successful for 8 of the 10 loci, of which 6 showed polymorphism. Seven of the 9 nuclear SSR primer pairs were useful in Vasconcellea and often also in Jacaratia and Carica, all revealing polymorphism. Exclusive haplotypes for each described taxon were identified based on chloroplast microsatellite data. Clustering based on separate nuclear and chloroplast data resulted in a clear grouping per taxon, but only low resolution was obtained above species level. The codominancy of nuclear SSRs and the general high polymorphism rate of SSR markers will make them more useful in future population genetics studies and diversity assessment in conservation programs.     

Development and use of single nucleotide polymorphism markers for candidate resistance genes in wild peanuts (Arachis spp)

The cultivated peanut (Arachis hypogaea L.) is an allotetraploid of recent origin, with an AABB genome and low genetic diversity. Perhaps because of its limited genetic diversity, this species lacks resistance to a number of important pests and diseases. In contrast, wild species of Arachis are genetically diverse and are rich sources of disease resistance genes. Consequently, a study of wild peanut relatives is attractive from two points of view: to help understand peanut genetics and to characterize wild alleles that could confer disease resistance. With this in mind, a diploid population from a cross between two wild peanut relatives was developed, in order to make a dense genetic map that could serve as a reference for peanut genetics and in order to characterize the regions of the Arachis genome that code for disease resistance. We tested two methods for developing and genotyping single nucleotide polymorphisms in candidate genes for disease resistance; one is based on single-base primer extension methods and the other is based on amplification refractory mutation system-polymerase chain reaction. We found single-base pair extension to be an efficient method, suitable for high-throughput, single-nucleotide polymorphism mapping; it allowed us to locate five candidate genes for resistance on our genetic map.     

Genetic variance for behavioural ‘predictability’ of stress response

Genetic factors underpinning phenotypic variation are required if natural selection is to result in adaptive evolution. However, evolutionary and behavioural ecologists typically focus on variation among individuals in their average trait values and seek to characterize genetic contributions to this. As a result, less attention has been paid to if and how genes could contribute towards within‐individual variance or trait ‘predictability’. In fact, phenotypic ‘predictability’ can vary among individuals, and emerging evidence from livestock genetics suggests this can be due to genetic factors. Here, we test this empirically using repeated measures of a behavioural stress response trait in a pedigreed population of wild‐type guppies. We ask (a) whether individuals differ in behavioural predictability and (b) whether this variation is heritable and so evolvable under selection. Using statistical methodology from the field of quantitative genetics, we find support for both hypotheses and also show evidence of a genetic correlation structure between the behavioural trait mean and individual predictability. We show that investigating sources of variability in trait predictability is statistically tractable and can yield useful biological interpretation. We conclude that, if widespread, genetic variance for ‘predictability’ will have major implications for the evolutionary causes and consequences of phenotypic variation.