The potential costs of accounting for genotypic errors in molecular parentage analyses

Genotypic errors, whether due to mutation or laboratory error, can cause the genotypes of parents and their offspring to appear inconsistent with Mendelian inheritance. As a result, molecular parentage analyses are expected to benefit when allowances are made for the presence of genotypic errors. However, a cost of allowing for genotypic errors might also be expected under some analytical conditions, primarily because parentage analyses that assume nonzero genotypic error rates can neither assign nor exclude parentage with certainty. The goal of this work was therefore to determine whether or not such costs might be important under conditions relevant to parentage analyses, particularly in natural populations. Simulation results indicate that the costs may often outweigh the benefits of accounting for nonzero error rates, except in situations where data are available for many marker loci. Consequently, the most powerful approach to handling genotypic errors in parentage analyses might be to apply likelihood equations with error rates set to values substantially lower than the rates at which genotypic errors occur. When applying molecular parentage analyses to natural populations, we advocate an increased consideration of optimal strategies for handling genotypic errors. Currently available software packages contain procedures that can be used for this purpose.     

Patterns and levels of gene flow in Rhododendron metternichii var. hondoense revealed by microsatellite analysis

Parentage analysis was conducted to elucidate the patterns and levels of gene flow in Rhododendron metternichii Sieb. et Zucc. var. hondoense Nakai in a 150 x 70 m quadrant in Hiroshima Prefecture, western Japan. The population of R. metternichii occurred as three subpopulations at the study site. Seventy seedlings were randomly collected from each of three 10 x 10 m plots (S1, S2, and S3) on the forest floor of each subpopulation (A1, A2, and A3). Almost all parents (93.8%) of the 70 seedlings were unambiguously identified by using 12 pairs of microsatellite markers. Within the quadrant, adult trees less than 5 m from the centre of the seedling bank (plots S1, S2, and S3) produced large numbers of seedlings. The effects of tree height and distance from the seedling bank on the relative fertilities of adult trees were highly variable among subpopulations because of the differences in population structure near the seedling bank: neither distance nor tree height had any significant effect in subpopulation A1; distance from the seedling bank had a significant effect in subpopulation A2; and tree height had a significant effect in subpopulation A3. Although gene flow within each subpopulation was highly restricted to less than 25 m and gene flow among the three subpopulations was extremely small (0-2%), long-distance gene flow from outside the quadrant reached 50%. This long-distance gene flow may be caused by a combination of topographical and vegetational heterogeneity, differences in flowering phenology, and genetic substructuring within subpopulations.     

Parentage versus two-generation analyses for estimating pollen-mediated gene flow in plant populations

Assessment of contemporary pollen-mediated gene flow in plants is important for various aspects of plant population biology, genetic conservation and breeding. Here, through simulations we compare the two alternative approaches for measuring pollen-mediated gene flow: (i) the NEIGHBORHOOD model--a representative of parentage analyses, and (ii) the recently developed TWOGENER analysis of pollen pool structure. We investigate their properties in estimating the effective number of pollen parents (N(ep)) and the mean pollen dispersal distance (delta). We demonstrate that both methods provide very congruent estimates of N(ep) and delta, when the methods' assumptions considering the shape of pollen dispersal curve and the mating system follow those used in data simulations, although the NEIGHBORHOOD model exhibits generally lower variances of the estimates. The violations of the assumptions, especially increased selfing or long-distance pollen dispersal, affect the two methods to a different degree; however, they are still capable to provide comparable estimates of N(ep). The NEIGHBORHOOD model inherently allows to estimate both self-fertilization and outcrossing due to the long-distance pollen dispersal; however, the TWOGENER method is particularly sensitive to inflated selfing levels, which in turn may confound and suppress the effects of distant pollen movement. As a solution we demonstrate that in case of TWOGENER it is possible to extract the fraction of intraclass correlation that results from outcrossing only, which seems to be very relevant for measuring pollen-mediated gene flow. The two approaches differ in estimation precision and experimental efforts but they seem to be complementary depending on the main research focus and type of a population studied.     

A method for the estimation of gene flow parameters from a population structure caused by restricted gene flow and genetic drift

Summary A method has been developed which enables the estimation of the plant gene flow parameters _p (pollen dispersal), _s (seed dispersal) and t (outcrossing rate) from a selection-free continuously structured population in equilibrium. The method uses Wright's F-coefficients and introduces a new F-function which describes the genetic similarity as a function of the spatial distance. The method has been elaborated for wind pollinated plant species but can be modified for insect pollination and for animal species. In practice allozymes will provide for the necessary neutral genetic variation. The more loci used and the more intermediate the gene frequencies, the more reliable the results. For the estimation of _p and t together (when the outcrossing rate is not known) at least two chromosomally unlinked loci are required. The method for estimating _s depends on whether the plant species is annual or perennial. The mechanism of selfing has been analysed by the explanation of the value of t by three components: population density (d), pollen flow (_p) and relative fertilization potential of own pollen (Z). The concepts of neighbourhood size and isolation by distance, developed by Wright, who used a single gene flow parameter , have been extended to the situation which is realistic for seed plants, using all three parameters _p, _s and t. When _p is large with respect to _s, _s largely determines the value of the neighbourhood size, whereas _p is the most dominating factor in isolation by distance. The use of local effective population size and mean gene transport per generation instead of neighbourhood size and neighbourhood area, respectively, is proposed to avoid confusion. Computer simulations have been carried out to check the validity and the reliability of the method. Populations of 200 plants, using two or three loci with intermediate allele frequencies, gave good results in the calculation of _p with known value of t and of _s and N_e. With unknown t, especially with lower values of t, larger populations of at least 1,000 plants are necessary to obtain reasonably accurate results for _p and mean gene transport per generation M.Grassland Species Research Group Publication No. 81     

植物交配系统及其在植物保护中的应用

植物交配系统是影响植物居群遗传结构最关键的生物学因素之一。在具体实践中,对遗传育种方法的选择和植物保护策略的制定具有指导意义。本文回顾了植物交配系统的研究历史,分析了相关概念,讨论了植物交配系统的变异与进化,介绍了相关的基本原理和研究方法,对研究中使用的分析工具作了比较。对国内外植物交配系统研究的进展,讨论了在实际研究中的取样及针对不同植物的保护策略问题,强调了植物交配系统信息在植物保护中的重要性,认为在植物保护中应加强植物交配系统的研究。     

Methods of parentage analysis in natural populations

The recent proliferation of hypervariable molecular markers has ushered in a surge of techniques for the analysis of parentage in natural and experimental populations. Consequently, the potential for meaningful studies of paternity and maternity is at an all-time high. However, the details and implementation of the multifarious techniques often differ in subtle ways that can influence the results of parentage analyses. Now is a good time to reflect on the available techniques and to consider their strengths and weaknesses. Here, we review the leading techniques in parentage analysis, with a particular emphasis on those that have been implemented in readily useable software packages. Our survey leads to some important insights with respect to the utility of the different approaches. This review should serve as a useful guide to anyone who wishes to embark on the study of parentage.     

Extensive pollen flow in two ecologically contrasting populations of Populus trichocarpa

Pollen-mediated gene flow was measured in two populations of black cottonwood using direct (paternity analysis) and indirect (correlated paternity) methods. The Marchel site was an area with an approximate radius of 250 m in a large continuous stand growing in a mesic habitat in western Oregon. In contrast, the Vinson site was an area with a radius of approximately 10 km and consisted of small, disjunct and isolated stands in the high desert of eastern Oregon. Pollen immigration was extensive in both populations, and was higher in the Marchel site (0.54 ± 0.02) than in the substantially larger and more isolated Vinson site (0.32 ± 0.02). Pollen pool differentiation among mothers was approximately five times stronger in the Vinson population (Φ_FT = 0.253, N  = 27 mothers) than in the Marchel population (Φ_FT = 0.052, N  = 5 mothers). Pollen dispersal was modelled using a mixed dispersal curve that incorporated pollen immigration. Predicted pollination frequencies generated based on this curve were substantially more accurate than those based on the widely used exponential power dispersal curve. Male neighbourhood sizes ( sensu Wright 1946 ) estimated using paternity analysis and pollen pool differentiation were remarkably similar. They were three to five times smaller in the Vinson population, which reflected the substantial ecological and demographic differences between the two populations. When the same mathematical function was used, applying direct and indirect methods resulted in similar pollen dispersal curves, thus confirming the value of indirect methods as a viable lower-cost alternative to paternity analysis.     

Comparing direct vs. indirect estimates of gene flow within a population of a scattered tree species

The comparison between historical estimates of gene flow, using variance in allelic frequencies, and contemporary estimates of gene flow, using parentage assignment, is expected to provide insights into ecological and evolutionary processes at work within and among populations. Genetic variation at six microsatellite loci was used to quantify genetic structure in the insect-pollinated, animal-dispersed, low-density tree Sorbus torminalis L. Crantz, and to derive historical estimates of gene flow. The neighbourhood size and root-mean-squared dispersal distance inferred from seedling genotypes ( N _b  = 70 individuals, σ _e  = 417 m) were similar to those inferred from adult genotypes ( N _b  = 114 individuals, σ _e  = 472 m). We also used parentage analyses and a neighbourhood model approach after an evaluation of the statistical properties of this method on simulated data. From our data, we estimated even contributions of seed- and pollen-mediated dispersal to the genetic composition of established seedlings, with both fat-tailed pollen and seed dispersal kernels, and slightly higher mean distance of pollen dispersal (248 m) as compared to seed dispersal (135 m). The resulting contemporary estimate of gene dispersal distance (σ _c  = 211 m) was ∼twofold smaller than the historical estimates. Besides different assumptions and statistical nuances of both approaches, this discrepancy is likely to reflect a recent restriction in the scale of gene flow which requires manager's attention in a context of increasing forest fragmentation.     

Power of exclusion for parentage verification and probability of match for identity in American Kennel Club breeds using 17 canine microsatellite markers

DNA analysis of microsatellite markers has become a common tool for verifying parentage in breed registries and identifying individual animals that are linked to a database or owner. Panels of markers have been developed in canines, but their utility across and within a wide range of breeds has not been reported. The American Kennel Club (AKC) authorized a study to determine the power to exclude non-parents and identify individuals using DNA genotypes of 17 microsatellite markers in two panels. Cheek swab samples were voluntarily collected at Parent Breed Club National Specialty dog shows and 9561 samples representing 108 breeds were collected, averaging 88.5 dogs per breed. The primary panel of 10 markers exceeded 99% power of exclusion for canine parentage verification of 61% of the breeds. In combination with the secondary panel of seven markers, 100% of the tested breeds exceeded 99% power of exclusion. The minimum probability match rate of the first panel was 3.6 x 10(-5) averaged across breeds, and with the addition of the second panel, the probability match rate was 3.2 x 10(-8); thus the probability of another random, unrelated dog with the same genotype is very low. The results of this analysis indicated that, on average, the primary panel meets the AKC's needs for routine parentage testing, but that a combination of 10-15 genetic markers from the two panels could yield a universal canine panel with enhanced processing efficiency, reliability and informativeness.     

Do marker‐based paternity assignments favour heterozygous and unrelated males?

Genetic marker‐based parentage analyses are widely applied to studies of natural populations in the fields of evolutionary biology, conservation biology and ecology. When the same markers used in a parentage analysis are used together with the inferred parentage in a downstream analysis, such as the analysis of mate choice in terms of heterozygosity or relatedness, a bias may be incurred because a subset of the genotypes are favoured in parentage assignments or non‐exclusions. A previous simulation study shows that exclusion‐based paternity analyses are biased in favour of heterozygous males, and males less related to the mothers than expected under random mating. In this study, I investigated the biases of genetic paternity analyses achieved by both exclusion‐ and likelihood‐based methods, using both analytical and simulation approaches. It is concluded that while both exclusion‐ and likelihood‐based methods can lead to biased paternity assignments or non‐exclusions in favour of a subset of genotypes, the bias is not consistently towards heterozygous males or males apparently less related to mothers. Both the direction and extent of the bias depend heavily on the allele frequency distribution and the number of markers, the methods used for paternity assignments, and the estimators of relatedness. There exist important differences in the patterns of the biases between exclusion‐ and likelihood‐based paternity analysis methods. It is concluded that the markers, except when they are highly informative to yield accurate paternity assignments or exclusions, should be split into two subsets which are used separately in the paternity and downstream analyses.     

Mate choice, sexual coercion and gene flow in guppy populations

The role of behaviour in gene flow in Trinidadian guppies Poecilia reticulata was assessed using fish from an upstream and downstream pair of populations that differ in predation regime. High-predation (downstream) females preferred males from the corresponding low-predation population, but high-predation males achieved greater reproductive success under competition. This suggests that post-copulatory as well as pre-copulatory events are important in determining rates of gene flow.     

Bayesian quantification of ecological determinants of outcrossing in natural plant populations: Computer simulations and the case study of biparental inbreeding in English yew

The mating system is a central parameter of plant biology because it shapes their ecological and evolutionary properties. Therefore, determining ecological variables that influence the mating system is important for a deeper understanding of the functioning of plant populations. Here, using old concepts and recent statistical developments, we propose a new statistical tool to make inferences about ecological determinants of outcrossing in natural plant populations. The method requires codominant genotypes of seeds collected from maternal plants within different locations. Using extensive computer simulations, we demonstrated that the method is robust to the issues expected for real‐world data, including the Wahlund effect, inbreeding and genotyping errors such as allele dropout and allele misclassification. Furthermore, we showed that the estimates of ecological effects and outcrossing rates can be severely biased if genotyping errors and genetic differentiation are not treated explicitly. Application of the new method to the case study of a dioecious tree (Taxus baccata) allowed revealing that female trees that grow in lower local densities have a greater tendency towards mating with relatives. Moreover, we also demonstrated that biparental inbreeding is higher in populations that are characterized by a longer mean distance between trees and a smaller mean trunk perimeter. We found these results to agree with both the theoretical predictions and the history of English yew.     

Effects of genotyping errors on parentage exclusion analysis

Genetic markers are widely used to determine the parentage of individuals in studies of mating systems, reproductive success, dispersals, quantitative genetic parameters and in the management of conservation populations. These markers are, however, imperfect for parentage analyses because of the presence of genotyping errors and undetectable alleles, which may cause incompatible genotypes (mismatches) between parents and offspring and thus result in false exclusions of true parentage. Highly polymorphic markers widely used in parentage analyses, such as microsatellites, are especially prone to genotyping errors. In this investigation, I derived the probabilities of excluding a random (related) individual from parentage and the probabilities of Mendelian-inconsistent errors (mismatches) and Mendelian-consistent errors (which do not cause mismatches) in parent-offspring dyads, when a marker having null alleles, allelic dropouts and false alleles is used in a parentage analysis. These probabilities are useful in evaluating the impact of various types of genotyping errors on the information content of a set of markers in and thus the power of a parentage analysis, in determining the threshold number of genetic mismatches that is appropriate for a parentage exclusion analysis and in estimating the rates of genotyping errors and frequencies of null alleles from observed mismatches between known parent-offspring dyads. These applications are demonstrated by numerical examples using both hypothetical and empirical data sets and discussed in the context of practical parentage exclusion analyses.     

Phylogeography of mitochondrial DNA and Y-chromosome haplogroups reveal asymmetric gene flow in populations of Eastern India

Polymorphisms in mitochondrial (mt) DNA and Y-chromosomes of seven socially and linguistically diverse castes and tribes of Eastern India were examined to determine their genetic relationships, their origin, and the influence of demographic factors on population structure. Samples from the Orissa Brahmin, Karan, Khandayat, Gope, Juang, Saora, and Paroja were analyzed for mtDNA hypervariable sequence (HVS) I and II, eight Y-chromosome short tandem repeats (Y-STRs), and lineage-defining mutations diagnostic for Indian- and Eurasian-specific haplogroups. Our results reveal that haplotype diversity and mean pairwise differences (MPD) was higher in caste groups of the region (>0.998, for both systems) compared to tribes (0.917-0.996 for Y-STRs, and 0.958-0.988 for mtDNA haplotypes). The majority of paternal lineages belong to the R1a1, O2a, and H haplogroups (62.7%), while 73.2% of maternal lineages comprise the Indian-specific M*, M5, M30, and R* mtDNA haplogroups, with a sporadic occurrence of West Eurasian lineages. Our study reveals that Orissa Brahmins (a higher caste population) have a genetic affinity with Indo-European speakers of Eastern Europe, although the Y-chromosome data show that the genetic distances of populations are not correlated to their position in the caste hierarchy. The high frequency of the O2a haplogroup and absence of East Asian-specific mtDNA lineages in the Juang and Saora suggest that a migration of Austro-Asiatic tribes to mainland India was exclusively male-mediated which occurred during the demographic expansion of Neolithic farmers in southern China. The phylogeographic analysis of mtDNA and Y-chromosomes revealed varied ancestral sources for the diverse genetic components of the populations of Eastern India.     

Dams and canyons disrupt gene flow among populations of a threatened riparian plant

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Complex patterns of mating revealed in a Eucalyptus regnans seed orchard using allozyme markers and the neighbourhood model

The neighbourhood model apportions offspring of individual mother plants to self-fertilization, outcrossing to males within a circumscribed area around the mother plant (the neighbourhood), and outcrossing to males outside the neighbourhood. Formerly the model was applied only to haploid pollen gametes in the offspring of conifers, but is extended so that it can be used with genotypic data from diploid offspring of both angiosperms and gymnosperms. In addition, it is shown that the mating parameters can be estimated without independent estimates of allele frequencies in the pollen pools outside the neighbourhood; thus the model might be applied effectively to natural populations exposed to unknown external pollen sources. Parameters of the neighbourhood mating model were estimated for a 10-year-old seed orchard population of the insect-pollinated tree, Eucalyptus regnans, in southeast Australia, which contained a mixture of two geographical provenances (Victoria and Tasmania). The mating patterns revealed were complex. Crosses between trees of the same provenance occurred three times more often than crosses between trees of different provenances. Levels of self-fertilization and patterns of mating within neighbourhoods were influenced by provenance origin, crop fecundity and orchard position (central vs. edge) of mother trees. Gene dispersal, however, was extensive, with approximately 50% of effective pollen gametes coming from males more than 40 m away from mother trees (average distance between neighbouring trees was 7.4 m). Thus, insect pollinators are efficient promoters of cross-fertilization in this orchard, with the result that the effective number of males mating with each female is large.     

Genetic consequences of habitat fragmentation in plant populations: susceptible signals in plant traits and methodological approaches

Conservation of genetic diversity, one of the three main forms of biodiversity, is a fundamental concern in conservation biology as it provides the raw material for evolutionary change and thus the potential to adapt to changing environments. By means of meta‐analyses, we tested the generality of the hypotheses that habitat fragmentation affects genetic diversity of plant populations and that certain life history and ecological traits of plants can determine differential susceptibility to genetic erosion in fragmented habitats. Additionally, we assessed whether certain methodological approaches used by authors influence the ability to detect fragmentation effects on plant genetic diversity. We found overall large and negative effects of fragmentation on genetic diversity and outcrossing rates but no effects on inbreeding coefficients. Significant increases in inbreeding coefficient in fragmented habitats were only observed in studies analyzing progenies. The mating system and the rarity status of plants explained the highest proportion of variation in the effect sizes among species. The age of the fragment was also decisive in explaining variability among effect sizes: the larger the number of generations elapsed in fragmentation conditions, the larger the negative magnitude of effect sizes on heterozygosity. Our results also suggest that fragmentation is shifting mating patterns towards increased selfing. We conclude that current conservation efforts in fragmented habitats should be focused on common or recently rare species and mainly outcrossing species and outline important issues that need to be addressed in future research on this area.     

栖息地片断化对动物种群间基因流的影响及其测定方法

栖息地片断化指在人为活动和自然干扰下,大面积连续分布的自然栖息地被其它非适宜栖息地分隔成许多面积较小的斑块(岛屿)的过程。栖息地片断化是导致生物多样性丧失和物种绝灭的主要因素,也是威胁自然界生物生存的重要因素之一。栖息地片断化将影响基因在种群间和种群内的运动(基因流),开展栖息地片断化研究对保持物种间遗传多样性具有重要意义。本文介绍了片断化程度和基因流的测定方法。回顾了国内外的研究现状,并探讨了片断化对种群间基因流的影响和对濒危物种保护的意义。     

Pollen and seed flow patterns of Carapa guianensis Aublet. (Meliaceae) in two types of Amazonian forest

Various factors affect spatial genetic structure in plant populations, including adult density and primary and secondary seed dispersal mechanisms. We evaluated pollen and seed dispersal distances and spatial genetic structure of Carapa guianensis Aublet. (Meliaceae) in occasionally inundated and terra firme forest environments that differed in tree densities and secondary seed dispersal agents. We used parentage analysis to obtain contemporary gene flow estimates and assessed the spatial genetic structure of adults and juveniles. Despite the higher density of adults (diameter at breast height ≥ 25 cm) and spatial aggregation in occasionally inundated forest, the average pollen dispersal distance was similar in both types of forest (195 ± 106 m in terra firme and 175 ± 87 m in occasionally inundated plots). Higher seed flow rates (36.7% of juveniles were from outside the plot) and distances (155 ± 84 m) were found in terra firme compared to the occasionally inundated plot (25.4% and 114 ± 69 m). There was a weak spatial genetic structure in juveniles and in terra firme adults. These results indicate that inundation may not have had a significant role in seed dispersal in the occasionally inundated plot, probably because of the higher levels of seedling mortality.     

Unbiased relatedness estimation in structured populations

Knowledge of the genetic relatedness between individuals is important in many research areas in quantitative genetics, conservation genetics, forensics, evolution, and ecology. In the absence of pedigree records, relatedness can be estimated from genetic marker data using a number of estimators. These estimators, however, make the critical assumption of a large random mating population without genetic structures. The assumption is frequently violated in the real world where geographic/social structures or nonrandom mating usually lead to genetic structures. In this study, I investigated two approaches to the estimation of relatedness between a pair of individuals from a subpopulation due to recent common ancestors (i.e., relatedness is defined and measured with the current focal subpopulation as reference). The indirect approach uses the allele frequencies of the entire population with and without accounting for the population structure, and the direct approach uses the allele frequencies of the current focal subpopulation. I found by simulations that currently widely applied relatedness estimators are upwardly biased under the indirect approach, but can be modified to become unbiased and more accurate by using Wright's F(st) to account for population structures. However, the modified unbiased estimators under the indirect approach are clearly inferior to the unmodified original estimators under the direct approach, even when small samples are used in estimating both allele frequencies and relatedness.