Dynamic management of genetic resources: maintenance of outcrossing in experimental metapopulations of a predominantly inbreeding species

Dynamic management of genetic resources inpredominantly inbreeding species requiresincreased levels of outcrossing to limit theloss of genetic variation due to smallereffective sizes and to favour the emergence ofnew genetic combinations. Here, we show thatoutcrossing rates can artificially bepermanently increased in experimental evolvingplant metapopulations, using Arabidopsisthaliana as a model. We introducedmale-sterility genes and used an adequatemanagement of the resulting female plants tomodify the outcrossing rates. As expected, theincrease in outcrossing resulted in lowerlevels of heterozygote deficiency (F _is) than observed in natural populationsof A. thaliana and therefore in themaintenance of potentially higher levels ofgenetic variation. An additional selectiveadvantage for females' offspring, due to theproduction of larger seeds by females and apossible heterosis effect, furthermore led tosmaller F _is than expected from therealized outcrossing rate. This selectiveadvantage also resulted in an increase infemale frequency, especially in metapopulationswith large population sizes, creating anon-causal negative correlation between femalefrequency and heterozygote deficiency.     

A quantitative genetic model for analyzing species differences in outcrossing species

A genetic model based on a two-level intra- and interspecific mating design is proposed to estimate the genetic architecture of species differences and heterosis for outcrossing species. The underlying genetic analyses make use of classical quantitative genetic theories and recent results from molecular genetic studies. Gene effects across different quantitative trait loci (QTL) can be approximated by a geometric series. Under natural selection, gene effects are often associated with allele frequencies in a particular way, which can be approximated by the gamma distribution. By incorporating these approximations into family structural analyses in the mating design, we are able to estimate a number of genetic parameters that contribute to quantitative genetic variation based on a nonlinear optimization approach. These parameters include the number of QTL, their gene effects, and their allele frequencies in the parental populations. We perform simulation studies and illustrate an example to demonstrate the statistical property and procedure of the method.     

OneMap: software for genetic mapping in outcrossing species

OneMap is an environment for constructing linkage maps of outcrossing plant species, using full-sib families derived from two outbred parents. The analyses are performed using a novel methodology based on the maximum likelihood approach for simultaneous estimation of linkage and linkage phases (Wu et al. 2002), which has been successfully applied to sugarcane (Garcia et al. 2006). It is implemented as a set of functions for the freely distributed software R, and handles pairwise marker analysis, marker ordering and map refinement. The software is freely available at http://www.ciagri.usp.br/ approximately aafgarci/OneMap/.     

Variation in outcrossing rate and population genetic structure of Clarkia tembloriensis (Onagraceae)

Summary Outcrossing rate estimates for eight accessions of Clarkia tembloriensis indicate that this annual plant species has a wide interpopulational range of outcrossing rate ( ). Populations' t estimates were significantly correlated with observed heterozygosity and mean number of alleles per locus. Estimated fixation indices, , for most populations were very close to their expected values, F_eq, for a given Nei's gene diversity statistics showed that the group of outcrossing populations have more total genetic variation and less differentiation among populations than does the group of selfing populations. These results indicate that the breeding system of C. tembloriensis has had a strong influence on the amount and distribution of genetic variation within and among its populations.     

Evolutionary genetic consequences of facultative sex and outcrossing

Explaining the selective forces that underlie different reproductive modes forms a major part of evolution research. Many organisms are facultative sexuals, with the ability to reproduce both sexually and asexually. Reduced sequencing costs means it is now possible to start investigating genome sequences of a wider number of these organisms in depth, but teasing apart the genetic forces underlying the maintenance of facultative sexual reproduction remains a challenge. An analogous problem exists when determining the genetic consequences of a degree of outcrossing (and recombination) in otherwise self‐fertilizing organisms. Here, I provide an overview of existing research on the evolutionary basis behind different reproductive modes, with a focus on explaining the population genetic effects favouring low outcrossing rates in either partially selfing or asexual species. I review the outcomes that both self‐fertilization and asexuality have on either purging deleterious mutations or fixing beneficial alleles, and what empirical data exist to support these theories. In particular, a greater application of mathematical models to genomic data has provided insight into the numerous effects that transitions to self‐fertilization from outcrossing have on genetic architecture. Similar modelling approaches could be used to determine the forces shaping genetic diversity of facultative sexual species. Hence, a further unification of mathematical models with next‐generation sequence data will prove important in exploring the genetic influences on reproductive system evolution.     

Spatial genetic analyses reveal strong genetic structure in two populations of the outcrossing tree fern Alsophila firma (Cyatheaceae)

The development of spatial genetic structure (SGS) in seed plants has been linked to several biological attributes of species, such as breeding system and life form. However, little is known about SGS in ferns, which together with lycopods are unique among land plants in having two free‐living life stages. We combined spatial aggregation statistics and spatial genetic autocorrelation analyses using five plastid microsatellites and one nuclear gene to investigate SGS in two populations of the outcrossing tree fern Alsophila firma (Cyatheaceae). We assessed how the observed patterns compare with those estimated for other ferns and seed plants. Populations of A. firma exhibited strong SGS, spatial clustering of individuals, substantial clonal diversity and no inbreeding. SGS in ferns appears to be higher than in most seed plants analysed to date. Contrary to our expectations, an outcrossing breeding system, wind dispersal and an arborescent life form did not translate into weak or no SGS. In ferns, SGS is probably being affected by the life cycle with two free‐living life stages. The reproductive biology of ferns appears to be more complex than previously thought. This implies that SGS in ferns is affected by some factors that cannot be inferred from the study of flowering plants. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 439–449.     

A new genetic locus for self-compatibility in the outcrossing grass species perennial ryegrass (Lolium perenne)

BackgroundSelf-incompatibility (SI) is a physiological mechanism that many flowering plants employ to prevent self-fertilization and maintain heterozygosity. In the grass family this is known to be controlled by a two locus (S-Z) system; however, the SI system is intrinsically leaky. Modifier genes of both the S and Z loci and a further locus, T, are known to override SI leading to self-fertilization and self-seed production. This has implications for the ecological and evolutionary success as well as the commercial breeding of grasses. Here we report a study where the genetic control of self-compatibility (SC) was determined from the results of self-pollinating an F₂ population of perennial ryegrass from two independently derived inbred lines produced by single-seed descent.Methods In vitro self-pollinations of 73 fertile plants were analysed. A genetic association analysis was made with a panel of 1863 single-nucleotide polymorphism (SNP) markers, generated through genotype-by-sequencing methodology. Markers were placed on a recombination map of seven linkage groups (LGs) created using Joinmap v.5. The seed set on self- and open-pollinated inflorescences was determined on 143 plants, including the 73 plants analysed for self-pollination response.Key ResultsSelf-pollinations revealed a bimodal distribution of percentage SC with peaks at 50 and 100 %. A single quantitative trait locus (QTL) was identified with peak association for marker 6S14665z17875_11873 that mapped to LG 6. Peak position was associated with maximum marker segregation distortion. The self-compatible plants were equally fecund after self- and open pollination.ConclusionsThis is the first report in the Poaceae family of an SC locus located on LG 6. This new SC QTL discovery, as well as indicating the complex nature of the pollen–stigma recognition process and its evolutionary significance, provides an additional source of SC for breeding perennial ryegrass.     

Effects of epistasis on tests for linkage in self-pollinated species

Summary Tests for linkage based on covariances among relatives in self-pollinated species are usually based upon an assumption that epistasis is not important. This study was conducted to determine the impact of epistasis on, and to investigate the sensitivity of, such tests. Thirty covariances were calculated for each of ten non-epistatic and ten epistatic genetic models with varying probabilities of recombination between two coupling or repulsion loci. Each set of covariances was tested for linkage by comparing covariances calculated for the model with those expected for an additive-dominance model with no linkage. Results showed that the test for linkage is quite insensitive to the effects of linkage due to the disproportionate influence of inbreeding. Repulsion linkages should be easier to detect than coupling linkages for all models. Epistasis was found to mimic or counteract the effects of linkage. Tests for linkage based on covariances within a hierarchical mating design appear to be insensitive to linkage and may confuse the effects of linkage and epistasis.     

Diversity, genetic structure and evidence of outcrossing in British populations of the rock fern Adiantum capillus-veneris using microsatellites

Microsatellites were isolated and a marker system was developed in the fern Adiantum capillus-veneris. Polymorphic markers were then used to study the genetic diversity and structure of populations within the UK and Ireland where this species grows at the northern edge of its range, requiring a specific rock habitat and limited to a few scattered populations. Three dinucleotide loci detected a high level of diversity (23 alleles and 28 multilocus genotypes) across the UK and Ireland, with nearly all variation partitioned among rather than within populations. Of 17 populations represented by multiple samples, all except four were monomorphic. Heterozygosity was detected in three populations, all within Glamorgan, Wales (UK), showing evidence of outcrossing. We make inferences on the factors determining the observed levels and patterns of genetic variation and the possible evolutionary history of the populations.     

Comparison of three genetic similarity coefficients based on dominant markers from predominantly self-pollinating species

Three genetic similarity coefficients were estimated and compared for their usefulness: simple matching (S _SM), Jaccard’s (S _J) and Dice’s (S _D), all based on dominant markers data from individuals representing predominantly self-pollinating species. AFLP markers were used to analyze 139 Phaseolus vulgaris L. (common bean) and 67 Lactuca saligna L. (least lettuce) accessions, and RAPD markers were used to analyze 110 Triticum dicoccoides Koern. (wild emmer wheat) accessions. Similar discriminating structure and power based on the three genetic similarity coefficients was found for each of the three species. This discriminating power was high for both P. vulgaris and L. saligna but moderate for T. dicoccoides. With closely related individuals, as in our study, the absence of a band in two individuals should be due to an identical cause inherited from the same ancestor. Accordingly we propose the use of S _SM, which alone out of the three examined coefficients involved shared absence of DNA bands, as contributing to genetic similarity. When RAPDs are employed, inferences about population structure and nucleotide divergence should be made with prudence as the nature of genetic variation uncovered by RAPDs is often unclear.     

Characterization and practical use of self-compatibility in outcrossing grass species

BackgroundSelf-incompatibility (SI) systems prevent self-fertilization in several species of Poaceae, many of which are economically important forage, bioenergy and turf grasses. Self-incompatibility ensures cross-pollination and genetic diversity but restricts the ability to fix useful genetic variation. In most inbred crops it is possible to develop high-performing homozygous parental lines by self-pollination, which then enables the creation of F₁ hybrid varieties with higher performance, a phenomenon known as heterosis. The inability to fully exploit heterosis in outcrossing grasses is partially responsible for lower levels of improvement in breeding programmes compared with inbred crops. However, SI can be overcome in forage grasses to create self-compatible populations. This is generating interest in understanding the genetical basis of self-compatibility (SC), its significance for reproductive strategies and its exploitation for crop improvement, especially in the context of F₁ hybrid breeding.ScopeWe review the literature on SI and SC in outcrossing grass species. We review the currently available genomic tools and approaches used to discover and characterize novel SC sources. We discuss opportunities barely explored for outcrossing grasses that SC facilitates. Specifically, we discuss strategies for wide SC introgression in the context of the Lolium–Festuca complex and the use of SC to develop immortalized mapping populations for the dissection of a wide range of agronomically important traits. The germplasm available is a valuable practical resource and will aid understanding the basis of inbreeding depression and hybrid vigour in key temperate forage grass species.ConclusionsA better understanding of the genetic control of additional SC loci offers new insight into SI systems, their evolutionary origins and their reproductive significance. Heterozygous outcrossing grass species that can be readily selfed facilitate studies of heterosis. Moreover, SC introduction into a range of grass species will enable heterosis to be exploited in innovative ways in genetic improvement programmes.     

Comparison of the fine-scale genetic structure of three dipterocarp species

We investigated the fine-scale genetic structure of three tropical-rainforest trees, Hopea dryobalanoides, Shorea parvifolia and S. acuminata (Dipterocarpaceae), in Peninsular Malaysia, all of which cooccurred within a 6-ha plot in Pasoh Forest Reserve. A significant genetic structure was found in H. dryobalanoides, weaker (but still significant) genetic structure in S. parvifolia and nonsignificant structure in S. acuminata. Seeds of all three species are wind dispersed, and their flowers are thought to be insect pollinated. The most obvious difference among these species is their height: S. parvifolia and S. acuminata are canopy species, whereas H. dryobalanoides is a subcanopy species. Clear differences were also found among these species in their range of seed dispersal, which depends on the height of the release point; so taller trees disperse their seed more extensively. The estimates of seed dispersal area were consistent with the degree of genetic structure found in the three species. Therefore, tree height probably had a strong influence on the fine-scale genetic structure of the three species.     

Increased selfing and correlated paternity in a small population of a predominantly outcrossing conifer, Pinus sylvestris

Outcrossing rate, the rates of ovule and seed abortion, and levels of correlated paternity were estimated in a small population of Pinus sylvestris, a predominantly outcrossing conifer, and were compared with estimates from two widely dispersed woodlands of the same species, showing a range of densities. On average, seed trees of the small population showed an eight-fold higher selfing rate (25 vs. 3%) and a 100-fold greater incidence of correlated paternity (19.6 vs. 0.2%) than did trees from the large populations. No evidence was found of pollen limitation within the remnant stand, as suggested by ovule abortion rates. Investigation of the mating patterns in the small population, based on the unambiguous genealogy of 778 open-pollinated seeds, showed a large departure from random mating. Only 8% of the possible mating pairs within the stand were observed. Correlated paternity rate within a maternal sibship was negatively associated (rs = -0.398, P < 0.050) with the distance to the nearest neighbour, and shared paternity among maternal sibships was negatively correlated (rs = -0.704, P < 0.001) with the distance between seed trees. Numerical simulations, based on the estimated individual pollen dispersal kernel, suggest that restricted dispersal might have been the key factor affecting mating patterns in the small population and, together with low population density, may account for the observed mating system variation between the small and the large populations. The results of this study show that a severe size reduction may substantially affect the mating system of a wind-pollinated, typically outcrossed plant species.     

Internal genetic structure and outcrossing rate in a natural population of Araucaria angustifolia (Bert.) O. Kuntze

The internal genetic structure and outcrossing rate of a population of Araucaria angustifolia (Bert.) O. Kuntze were investigated using 16 allozyme loci. Estimates of the mean number of alleles per loci (1.6), percentage of polymorphic loci (43.8%), and expected genetic diversity (0.170) were similar to those obtained for other gymnosperms. The analysis of spatial autocorrelation demonstrated the presence of internal structure in the first distance classes (up to 70 m), suggesting the presence of family structure. The outcrossing rate was high (0.956), as expected for a dioecious species. However, it was different from unity, indicating outcrossings between related individuals and corroborating the presence of internal genetic structure. The results of this study have implications for the methodologies used in conservation collections and for the use or analysis of this forest species.     

Patterns of genetic diversity in outcrossing and selfing populations of Arabidopsis lyrata

Arabidopsis lyrata is normally considered an obligately outcrossing species with a strong self-incompatibility system, but a shift in mating system towards inbreeding has been found in some North American populations (subspecies A. lyrata ssp. lyrata). This study provides a survey of the Great Lakes region of Canada to determine the extent of this mating system variation and how outcrossing rates are related to current population density, geographical distribution, and genetic diversity. Based on variation at microsatellite markers (progeny arrays to estimate multilocus outcrossing rates and population samples to estimate diversity measures) and controlled greenhouse pollinations, populations can be divided into two groups: (i) group A, consisting of individuals capable of setting selfed seed (including autogamous fruit set in the absence of pollinators), showing depressed outcrossing rates (T(m) = 0.2-0.6), heterozygosity (H(O) = 0.02-0.06) and genetic diversity (H(E) = 0.08-0.10); and (ii) group B, consisting of individuals that are predominantly self-incompatible (T(m) > 0.8), require pollinators for seeds set, and showing higher levels of heterozygosity (H(O) = 0.13-0.31) and diversity (H(E) = 0.19-0.410). Current population density is not related to the shift in mating system but does vary with latitude. Restricted gene flow among populations was evident among all but two populations (F(ST) = 0.11-0.8). Group A populations were more differentiated from one another (F(ST) = 0.78) than they were from group B populations (F(ST) = 0.59), with 41% of the variation partitioned within populations, 47% between populations, and 12% between groups. No significant relationship was found between genetic and geographical distance. Results are discussed in the context of possible postglacial expansion scenarios in relation to loss of self-incompatibility.     

High outcrossing in the annual colonizing species Ambrosia artemisiifolia (Asteraceae)

Background and Aims

Variation in mating patterns may be particularly evident in colonizing species because they commonly experience wide variation in plant density. Here, the role of density for the mating system of Ambrosia artemisiifolia (common ragweed), a wind-pollinated annual colonizing species previously reported as self-compatible, is explored.

Methods

The effect of population density on the proportion of self- and cross-fertilized seeds was examined using allozyme markers and experimental arrays conducted over two seasons in the field. Also the reproductive success of isolated plants located in diverse habitats was measured. The potential occurrence of a physiological mechanism preventing self-fertilization, i.e. self-incompatibility, following controlled self- and cross-pollinations in the glasshouse was examined.

Key Results

Outcrossing rates estimated using allozyme markers were uniformly high, regardless of the spacing between plants. However, when single plants were isolated from congeners they set few seeds. Observations of pollen-tube growth and seed set following controlled pollinations demonstrated that plants of A. artemisiifolia possess a strong self-incompatibility mechanism, contrary to earlier reports and assumptions.

Conclusions

The maintenance of high outcrossing rates in colonizing populations of A. artemisiifolia is likely to be facilitated by the prodigious production of wind-borne pollen, high seed production and extended seed dormancy.Key words: Self-incompatibility, outcrossing rate, density dependence, colonization, wind-pollination, Ambrosia artemisiifolia (ragweed), Asteraceae     

Phylogeography of the crown-of-thorns starfish: genetic structure within the Pacific species

Previous studies of the population genetic structure of the corallivorous crown-of-thorns starfish (COTS) Acanthaster planci in the Pacific Ocean showed high levels of gene flow that were assumed to reflect a high dispersal potential. However, the phylogeographic analyses of the Pacific crown-of-thorns starfish species of this study, using the highly variable mitochondrial control region and the most complete geographic coverage to date, contradict this view. Results show high levels of overall genetic structure (Φ_ST = 0.198), suggesting a complex history of range restrictions and expansions, a pattern that we hypothesize results from changes in topography and oceanography associated with sea-level changes. However, results also show signatures of ongoing gene flow between populations isolated in the past and high levels of genetic connectivity even among distant populations. Combined, these results indicate that while there are significant limits to genetic exchange among populations among Pacific Ocean populations of the crown-of-thorns starfish, the high larval dispersal potential of this species is often achieved as well.     

Simultaneous maximum likelihood estimation of linkage and linkage phases in outcrossing species

With the advent of new molecular marker technologies, it is now feasible to initiate genome projects for outcrossing plant species, which have not received much attention in genetic research, despite their great agricultural and environmental value. Because outcrossing species typically have heterogeneous genomes, data structure for molecular markers representing an entire genome is complex: some markers may have more alleles than others, some markers are codominant whereas others are dominant, and some markers are heterozygous in one parent but fixed in the other parent whereas the opposite can be true for other markers. A major difficulty in analyzing these different types of marker at the same time arises from uncertainty about parental linkage phases over markers. In this paper, we present a general maximum-likelihood-based algorithm for simultaneously estimating linkage and linkage phases for a mixed set of different marker types containing fully informative markers (segregating 1:1:1:1) and partially informative markers (or missing markers, segregating 1:2:1, 3:1, and 1:1) in a full-sib family derived from two outbred parent plants. The characterization of linkage phases is based on the posterior probability distribution of the assignment of alternative alleles at given markers to two homologous chromosomes of each parent, conditional on the observed phenotypes of the markers. Two- and multi-point analyses are performed to estimate the recombination fraction and determine the most likely linkage phase between different types of markers. A numerical example is presented to demonstrate the statistical properties of the model for characterizing the linkage phase between markers.     

Population history and genetic structure of a circumpolar species: the arctic fox

The circumpolar arctic fox Alopex lagopus thrives in cold climates and has a high migration rate involving long-distance movements. Thus, it differs from many temperate taxa that were subjected to cyclical restriction in glacial refugia during the Ice Ages. We investigated population history and genetic structure through mitochondrial control region variation in 191 arctic foxes from throughout the arctic. Several haplotypes had a Holarctic distribution and no phylogeographical structure was found. Furthermore, there was no difference in haplotype diversity between populations inhabiting previously glaciated and unglaciated regions. This suggests current gene flow among the studied populations, with the exception of those in Iceland, which is surrounded by year-round open water. Arctic foxes have often been separated into two ecotypes: 'lemming' and 'coastal'. An analysis of molecular variance suggested particularly high gene flow among populations of the 'lemming' ecotype. This could be explained by their higher migration rate and reduced fitness in migrants between ecotypes. A mismatch analysis indicated a sudden expansion in population size around 118 000 BP, which coincides with the last interglacial. We propose that glacial cycles affected the arctic fox in a way opposite to their effect on temperate species, with interglacials leading to short-term isolation in northern refugia.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 79–89.     

Balance between inbreeding and outcrossing in a nannandrous species,the moss Homalothecium lutescens

Epiphytic dwarf males on the females present a possible solution to the problem of short fertilization distances in mosses. However, leptokurtic spore dispersal makes dwarf males likely to be closely related to their host shoot, with an accompanying risk of inbreeding. The capacity of a female to harbour a high number of different dwarf males suggests that there may be mechanisms in place that counteract inbreeding, such as polyandry and post-fertilization selection. We have genotyped sporophytes, female host shoots and dwarf males in four populations of the moss Homalothecium lutescens. We found no evidence of selective sporophyte abortion based on level of heterozygosity. The occurrence of entirely homozygous sporophytes together with significantly positive inbreeding coefficients in three of the populations (mean F_IS between 0.48 and 0.64) suggest frequent mother–son mating events. However, 23% of all sampled sporophytes had a higher level of heterozygosity compared with the mean expected heterozygosity at the population level. Polyandry was frequent, on average 59% of the sporophytes on a female shoot were sired by distinct fathers. In conclusion, sporadic fertilizations by dwarf males originating from nonhost female shoots appear to counteract strong inbreeding.