The Analysis of Polyploid Genetic Data.

Though polyploidy is an important aspect of the evolutionary genetics of both plants and animals, the development of population genetic theory of polyploids has seriously lagged behind that of diploids. This is unfortunate since the analysis of polyploid genetic data-and the interpretation of the results-requires even more scrutiny than with diploid data. This is because of several polyploidy-specific complications in segregation and genotyping such as tetrasomy, double reduction, and missing dosage information. Here, we review the theoretical and statistical aspects of the population genetics of polyploids. We discuss several widely used types of inferences, including genetic diversity, Hardy-Weinberg equilibrium, population differentiation, genetic distance, and detecting population structure. For each, we point out how the statistical approach, expected result, and interpretation differ between different ploidy levels. We also discuss for each type of inference what biases may arise from the polyploid-specific complications and how these biases can be overcome. From our overview, it is clear that the statistical toolbox that is available for the analysis of genetic data is flexible and still expanding. Modern sequencing techniques will soon be able to overcome some of the current limitations to the analysis of polyploid data, though the techniques are lagging behind those available for diploids. Furthermore, the availability of more data may aggravate the biases that can arise, and increase the risk of false inferences. Therefore, simulations such as we used throughout this review are an important tool to verify the results of analyses of polyploid genetic data.

Identification of the Submergence Tolerance QTL Come Quick Drowning1 (CQD1) in Arabidopsis thaliana.

Global climate change is predicted to increase water precipitation fluctuations and lead to localized prolonged floods in agricultural fields and natural plant communities. Thus, understanding the genetic basis of submergence tolerance is crucial in order to improve plant survival under these conditions. In this study, we performed a quantitative trait locus (QTL) analysis in Arabidopsis to identify novel candidate genes for increased submergence tolerance by using Kas-1 and Col (gl1) parental accessions and their derived recombinant inbred lines (RILs). We measured survival after submergence in dark for a 13-day period and used median lethal time, LT50 values for the QTL analysis. A major QTL, the Come Quick, Drowning (CQD1) locus, was detected in 2 independent experiments on the lower arm of chromosome 5 involved in higher submergence tolerance in the parental accession Kas-1. For fine-mapping, we then constructed near isogenic lines (NILs) by backcrossing the CQD1 QTL region. We also analyzed QTL regions related to size, leaf number, flowering, or survival in darkness and none of the QTL related to these traits overlapped with CQD1. The submergence tolerance QTL, CQD1, region detected in this study includes genes that have potential to be novel candidates effecting submergence tolerance such as trehalose-6-phosphate phosphatase and respiratory burst oxidase protein D. Gene expression and functional analysis for these genes under submergence would reveal the significance of these novel candidates and provide new perspectives for understanding genetic basis of submergence tolerance.

Abiotic stress QTL in lettuce crop-wild hybrids: comparing greenhouse and field experiments.

The development of stress-tolerant crops is an increasingly important goal of current crop breeding. A higher abiotic stress tolerance could increase the probability of introgression of genes from crops to wild relatives. This is particularly relevant to the discussion on the risks of new GM crops that may be engineered to increase abiotic stress resistance. We investigated abiotic stress QTL in greenhouse and field experiments in which we subjected recombinant inbred lines from a cross between cultivated Lactuca sativa cv. Salinas and its wild relative L. serriola to drought, low nutrients, salt stress, and aboveground competition. Aboveground biomass at the end of the rosette stage was used as a proxy for the performance of plants under a particular stress. We detected a mosaic of abiotic stress QTL over the entire genome with little overlap between QTL from different stresses. The two QTL clusters that were identified reflected general growth rather than specific stress responses and colocated with clusters found in earlier studies for leaf shape and flowering time. Genetic correlations across treatments were often higher among different stress treatments within the same experiment (greenhouse or field), than among the same type of stress applied in different experiments. Moreover, the effects of the field stress treatments were more correlated with those of the greenhouse competition treatments than to those of the other greenhouse stress experiments, suggesting that competition rather than abiotic stress is a major factor in the field. In conclusion, the introgression risk of stress tolerance (trans-)genes under field conditions cannot easily be predicted based on genomic background selection patterns from controlled QTL experiments in greenhouses, especially field data will be needed to assess potential (negative) ecological effects of introgression of these transgenes into wild relatives.

Identification of quantitative trait loci and a candidate locus for freezing tolerance in controlled and outdoor environments in the overwintering crucifer Boechera stricta.

Development of chilling and freezing tolerance is complex and can be affected by photoperiod, temperature and photosynthetic performance; however, there has been limited research on the interaction of these three factors. We evaluated 108 recombinant inbred lines of Boechera stricta, derived from a cross between lines originating from Montana and Colorado, under controlled long day (LD), short-day (SD) and in an outdoor environment (OE). We measured maximum quantum yield of photosystem II, lethal temperature for 50% survival and electrolyte leakage of leaves. Our results revealed significant variation for chilling and freezing tolerance and photosynthetic performance in different environments. Using both single- and multi-trait analyses, three main-effect quantitative trait loci (QTL) were identified. QTL on linkage group (LG)3 were SD specific, whereas QTL on LG4 were found under both LD and SD. Under all conditions, QTL on LG7 were identified, but were particularly predictive for the outdoor experiment. The co-localization of photosynthetic performance and freezing tolerance effects supports these traits being co-regulated. Finally, the major QTL on LG7 is syntenic to the Arabidopsis C-repeat binding factor locus, known regulators of chilling and freezing responses in Arabidopsis thaliana and other species.

Root transcript profiling of two Rorippa species reveals gene clusters associated with extreme submergence tolerance.

Complete submergence represses photosynthesis and aerobic respiration, causing rapid mortality in most terrestrial plants. However, some plants have evolved traits allowing them to survive prolonged flooding, such as species of the genus Rorippa, close relatives of Arabidopsis (Arabidopsis thaliana). We studied plant survival, changes in carbohydrate and metabolite concentrations, and transcriptome responses to submergence of two species, Rorippa sylvestris and Rorippa amphibia. We exploited the close relationship between Rorippa species and the model species Arabidopsis by using Arabidopsis GeneChip microarrays for whole-genome transcript profiling of roots of young plants exposed to a 24-h submergence treatment or air. A probe mask was used based on hybridization of genomic DNA of both species to the arrays, so that weak probe signals due to Rorippa species/Arabidopsis mismatches were removed. Furthermore, we compared Rorippa species microarray results with those obtained for roots of submerged Arabidopsis plants. Both Rorippa species could tolerate deep submergence, with R. sylvestris surviving much longer than R. amphibia. Submergence resulted in the induction of genes involved in glycolysis and fermentation and the repression of many energy-consuming pathways, similar to the low-oxygen and submergence response of Arabidopsis and rice (Oryza sativa). The qualitative responses of both Rorippa species to submergence appeared roughly similar but differed quantitatively. Notably, glycolysis and fermentation genes and a gene encoding sucrose synthase were more strongly induced in the less tolerant R. amphibia than in R. sylvestris. A comparison with Arabidopsis microarray studies on submerged roots revealed some interesting differences and potential tolerance-related genes in Rorippa species.

Genomic and environmental selection patterns in two distinct lettuce crop-wild hybrid crosses.

Genomic selection patterns and hybrid performance influence the chance that crop (trans)genes can spread to wild relatives. We measured fitness(-related) traits in two different field environments employing two different crop-wild crosses of lettuce. We performed quantitative trait loci (QTL) analyses and estimated the fitness distribution of early- and late-generation hybrids. We detected consistent results across field sites and crosses for a fitness QTL at linkage group 7, where a selective advantage was conferred by the wild allele. Two fitness QTL were detected on linkage group 5 and 6, which were unique to one of the crop-wild crosses. Average hybrid fitness was lower than the fitness of the wild parent, but several hybrid lineages outperformed the wild parent, especially in a novel habitat for the wild type. In early-generation hybrids, this may partly be due to heterosis effects, whereas in late-generation hybrids transgressive segregation played a major role. The study of genomic selection patterns can identify crop genomic regions under negative selection across multiple environments and cultivar-wild crosses that might be applicable in transgene mitigation strategies. At the same time, results were cultivar-specific, so that a case-by-case environmental risk assessment is still necessary, decreasing its general applicability.

Genomic regions in crop-wild hybrids of lettuce are affected differently in different environments: implications for crop breeding.

Many crops contain domestication genes that are generally considered to lower fitness of crop-wild hybrids in the wild environment. Transgenes placed in close linkage with such genes would be less likely to spread into a wild population. Therefore, for environmental risk assessment of GM crops, it is important to know whether genomic regions with such genes exist, and how they affect fitness. We performed quantitative trait loci (QTL) analyses on fitness(-related) traits in two different field environments employing recombinant inbred lines from a cross between cultivated Lactuca sativa and its wild relative Lactuca serriola. We identified a region on linkage group 5 where the crop allele consistently conferred a selective advantage (increasing fitness to 212% and 214%), whereas on linkage group 7, a region conferred a selective disadvantage (reducing fitness to 26% and 5%), mainly through delaying flowering. The probability for a putative transgene spreading would therefore depend strongly on the insertion location. Comparison of these field results with greenhouse data from a previous study using the same lines showed considerable differences in QTL patterns. This indicates that care should be taken when extrapolating experiments from the greenhouse, and that the impact of domestication genes has to be assessed under field conditions.

Does insect netting affect the containment of airborne pollen from (GM-) plants in greenhouses?

Greenhouses are a well-accepted containment strategy to grow and study genetically modified plants (GM) before release into the environment. Various containment levels are requested by national regulations to minimize GM pollen escape. We tested the amount of pollen escaping from a standard greenhouse, which can be used for EU containment classes 1 and 2. More specifically, we investigated the hypothesis whether pollen escape could be minimized by insect-proof netting in front of the roof windows, since the turbulent airflow around the mesh wiring could avoid pollen from escaping. We studied the pollen flow out of greenhouses with and without insect netting of two non-transgenic crops, Ryegrass (Loliummultiflorum) and Corn (Zea Mays). Pollen flow was assessed with Rotorod(®) pollen samplers positioned inside and outside the greenhouse' roof windows. A significant proportion of airborne pollen inside the greenhouse leaves through roof windows. Moreover, the lighter pollen of Lolium escaped more readily than the heavier pollen of Maize. In contrast to our expectations, we did not identify any reduction in pollen flow with insect netting in front of open windows, even under induced airflow conditions. We conclude that insect netting, often present by default in greenhouses, is not effective in preventing pollen escape from greenhouses of wind-pollinated plants for containment classes 1 or 2. Further research would be needed to investigate whether other alternative strategies, including biotic ones, are more effective. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10453-011-9237-8) contains supplementary material, which is available to authorized users.

Hybridization between crops and wild relatives: the contribution of cultivated lettuce to the vigour of crop-wild hybrids under drought, salinity and nutrient deficiency conditions.

With the development of transgenic crop varieties, crop-wild hybridization has received considerable consideration with regard to the potential of transgenes to be transferred to wild species. Although many studies have shown that crops can hybridize with their wild relatives and that the resulting hybrids may show improved fitness over the wild parents, little is still known on the genetic contribution of the crop parent to the performance of the hybrids. In this study, we investigated the vigour of lettuce hybrids using 98 F(2:3) families from a cross between cultivated lettuce and its wild relative Lactuca serriola under non-stress conditions and under drought, salinity and nutrient deficiency. Using single nucleotide polymorphism markers, we mapped quantitative trait loci associated with plant vigour in the F(2:3) families and determined the allelic contribution of the two parents. Seventeen QTLs (quantitative trait loci) associated with vigour and six QTLs associated with the accumulation of ions (Na(+), Cl(-) and K(+)) were mapped on the nine linkage groups of lettuce. Seven of the vigour QTLs had a positive effect from the crop allele and six had a positive effect from the wild allele across treatments, and four QTLs had a positive effect from the crop allele in one treatment and from the wild allele in another treatment. Based on the allelic effect of the QTLs and their location on the genetic map, we could suggest genomic locations where transgene integration should be avoided when aiming at the mitigation of its persistence once crop-wild hybridization takes place.

Wait or escape? Contrasting submergence tolerance strategies of Rorippa amphibia, Rorippa sylvestris and their hybrid.

BACKGROUND AND AIMS: Differential responses of closely related species to submergence can provide insight into the evolution and mechanisms of submergence tolerance. Several traits of two wetland species from habitats with contrasting flooding regimes, Rorippa amphibia and Rorippa sylvestris, as well as F(1) hybrid Rorippa × anceps were analysed to unravel mechanisms underlying submergence tolerance. METHODS: In the first submergence experiment (lasting 20 d) we analysed biomass, stem elongation and carbohydrate content. In the second submergence experiment (lasting 3 months) we analysed survival and the effect of re-establishment of air contact on biomass and carbohydrate content. In a separate experiment we analysed expression of two carbohydrate catabolism genes, ADH1 and SUS1, upon re-establishment of air contact following submergence. KEY RESULTS: All plants had low mortality even after 3 months of submergence. Rorippa sylvestris was characterized by 100 % survival and higher carbohydrate levels coupled with lower ADH1 gene expression as well as reduced growth compared with R. amphibia. Rorippa amphibia and the hybrid elongated their stems but this did not pay-off in higher survival when plants remained submerged. Only R. amphibia and the hybrid benefited in terms of increased biomass and carbohydrate accumulation upon re-establishing air contact. CONCLUSIONS: Results demonstrate contrasting 'escape' and 'quiescence' strategies between Rorippa species. Being a close relative of arabidopsis, Rorippa is an excellent model for future studies on the molecular mechanism(s) controlling these strategies.

Crop to wild introgression in lettuce: following the fate of crop genome segments in backcross populations.

BACKGROUND: After crop-wild hybridization, some of the crop genomic segments may become established in wild populations through selfing of the hybrids or through backcrosses to the wild parent. This constitutes a possible route through which crop (trans)genes could become established in natural populations. The likelihood of introgression of transgenes will not only be determined by fitness effects from the transgene itself but also by the crop genes linked to it. Although lettuce is generally regarded as self-pollinating, outbreeding does occur at a low frequency. Backcrossing to wild lettuce is a likely pathway to introgression along with selfing, due to the high frequency of wild individuals relative to the rarely occurring crop-wild hybrids. To test the effect of backcrossing on the vigour of inter-specific hybrids, Lactuca serriola, the closest wild relative of cultivated lettuce, was crossed with L. sativa and the F(1) hybrid was backcrossed to L. serriola to generate BC(1) and BC(2) populations. Experiments were conducted on progeny from selfed plants of the backcrossing families (BC(1)S(1) and BC(2)S(1)). Plant vigour of these two backcrossing populations was determined in the greenhouse under non-stress and abiotic stress conditions (salinity, drought, and nutrient deficiency). RESULTS: Despite the decreasing contribution of crop genomic blocks in the backcross populations, the BC(1)S(1) and BC(2)S(1) hybrids were characterized by a substantial genetic variation under both non-stress and stress conditions. Hybrids were identified that performed equally or better than the wild genotypes, indicating that two backcrossing events did not eliminate the effect of the crop genomic segments that contributed to the vigour of the BC(1) and BC(2) hybrids. QTLs for plant vigour under non-stress and the various stress conditions were detected in the two populations with positive as well as negative effects from the crop. CONCLUSION: As it was shown that the crop contributed QTLs with either a positive or a negative effect on plant vigour, we hypothesize that genomic regions exist where transgenes could preferentially be located in order to mitigate their persistence in natural populations through genetic hitchhiking.

Other tetraploid species and conspecific diploids as sources of genetic variation for an autotetraploid.

PREMISE OF THE STUDY: Most plants are polyploid and have more than two copies of the genome. The evolutionary success of polyploids is often attributed to their potential to harbor increased genetic variation, but it is poorly understood how polyploids can attain such variation. Because of their formation bottleneck, newly formed tetraploids start out with little variation. Tetraploids may attain genetic variation through a combination of new mutations, recurrent formation, and gene exchange with diploid ancestors or related tetraploid species. We explore the role of gene exchange and introgression in autotetraploid Rorippa amphibia, a species that harbors more genetic variation than its diploid ancestors. • METHODS: We crossed autotetraploid R. amphibia to diploid conspecifics and tetraploid R. sylvestris and backcrossed resulting F(1) hybrids. We used flow cytometry to determine the ploidy of all progeny. • KEY RESULTS: Tetraploids of R. amphibia and R. sylvestris were interfertile; F(1) hybrids were fertile and could backcross. Crosses between diploids and tetraploids yielded a small number of viable, often tetraploid progeny. This indicates that unreduced gametes can facilitate gene flow from diploids to tetraploids. We detected a frequency of unreduced gametes of around 2.7 per 1000, which was comparable between diploids and tetraploids. • CONCLUSIONS: Introgression from tetraploid R. sylvestris provides a realistic source of variation in autotetraploid R. amphibia. Only in a scenario where other compatible partners are absent, for example immediately after tetraploidization, gene flow through unreduced gametes from diploids could be an important source of genetic variation for tetraploids.

Segregation models for disomic, tetrasomic and intermediate inheritance in tetraploids: a general procedure applied to Rorippa (yellow cress) microsatellite data.

Tetraploid inheritance has two extremes: disomic in allotetraploids and tetrasomic in autotetraploids. The possibility of mixed, or intermediate, inheritance models has generally been neglected. These could well apply to newly formed hybrids or to diploidizing (auto)tetraploids. We present a simple likelihood-based approach that is able to incorporate disomic, tetrasomic, and intermediate inheritance models and estimates the double-reduction rate. Our model shows that inheritance of microsatellite markers in natural tetraploids of Rorippa amphibia and R. sylvestris is tetrasomic, confirming their autotetraploid origin. However, in F(1) hybrids inheritance was intermediate to disomic and tetrasomic inheritance. Apparently, in meiosis, chromosomes paired preferentially with the homolog from the same parental species, but not strictly so. Detected double-reduction rates were low. We tested the general applicability of our model, using published segregation data. In two cases, an intermediate inheritance model gave a better fit to the data than the tetrasomic model advocated by the authors. The existence of inheritance intermediate to disomic and tetrasomic has important implications for linkage mapping and population genetics and hence breeding programs of tetraploids. Methods that have been developed for either disomic or tetrasomic tetraploids may not be generally applicable, particularly in systems where hybridization is common.

Different flooding responses in Rorippa amphibia and Rorippa sylvestris, and their modes of expression in F1 hybrids.

The river floodplain species Rorippa amphibia, Rorippa sylvestris, and their hybrid Rorippa x anceps were studied here, with the aim of identifying potential species differences with respect to flooding tolerance, and of assessing their expression in F1 hybrids. Parents and their F1 hybrids were subjected to three flooding treatments mimicking natural conditions, and growth-related and leaf morphological traits were compared. In contrast to R. sylvestris, R. amphibia responded to waterlogging by forming specialized roots, and its growth was not reduced. These traits were dominantly expressed in hybrids. Both species and the hybrids established shoot growth over 2 wk of complete submergence. Only in R. sylvestris was this not at the expense of root biomass, suggesting that R. sylvestris can photosynthesize underwater. Rorippa sylvestris also showed a hyponastic response. Hybrids were intermediate to the parents in this respect. This study shows that phenotypic expression of parental traits in F1 hybrids is mostly additive, but can also be dominant. This suggests that a large overlap in habitat use of parents and hybrids is likely. If such an overlap occurs, the main evolutionary consequences of hybridization in Rorippa will be the introgression of genes, as the hybrids are fully fertile.

Genetic diversity in diploid vs. tetraploid Rorippa amphibia (Brassicaceae).

The frequency of polyploidy increases with latitude in the Northern Hemisphere, especially in deglaciated, recently colonized areas. The cause or causes of this pattern are largely unknown, but a greater genetic diversity of individual polyploid plants due to a doubled genome and/or a hybrid origin is seen as a likely factor underlying selective advantages related to life in extreme climates and/or colonization ability. A history of colonization in itself, as well as a recent origin, and possibly a limited number of polyploidization events would all predict less genetic diversity in polyploids than in diploids. The null hypothesis of higher gene diversity in polyploids has to date hardly been quantified and is here tested in self-incompatible Rorippa amphibia (Brassicaceae). The species occurs in diploid and tetraploid forms and displays clear geographical polyploidy in Europe. On the basis of eight microsatellite loci it can be concluded that the level of gene diversity is higher in tetraploids than in diploids, to an extent that is expected under neutral evolution when taking into account the larger effective population size in the doubled cytotype. There is thus no evidence for reduced genetic diversity in the tetraploids. The evidence presented here may mean that the tetraploids' origin is not recent, has not been affected by bottlenecks and/or that tetraploids were formed multiple times while an effect of introgression may also play a role.

GENERALISTS, SPECIALISTS, AND THE EVOLUTION OF PHENOTYPIC PLASTICITY IN SYMPATRIC POPULATIONS OF DISTINCT SPECIES.

The evolution of phenotypic plasticity is studied in a model with two reproductively isolated "species" in a coarse-grained environment, consisting of two types of habitats. A quantitative genetic model for selection was constructed, in which habitats differ in the optimal value for a focal trait, and with random dispersal among habitats. The main interest was to study the effects of different selection regimes. Three cases were investigated: (1) without any limits to plasticity; (2) without genetic variation for plasticity; and (3) with a fitness cost for phenotypically plastic reactions. In almost all cases a generalist strategy to exploit both habitats emerged. Without any limits to plasticity, optimal adaptive reactions evolved. Without any genetic variation for plasticity, a compromise strategy with an intermediate, fixed phenotype evolved, whereas in the presence of costs a plastic compromise between the demands of the habitats and the costs associated with plasticity was found. Specialization and phenotypic differentiation was only found when selection within habitats was severe and optimal phenotypes for different habitats were widely different. Under soft selection (local regulation of population numbers in each habitat) the specialists coexisted; under hard selection (global regulation of population numbers) one specialist outcompeted the other. The prevalent evolutionary outcome of compromises rather than specialization implies that costs or constraints are not necessarily detectable as local adaptation in transplantation or translocation experiments.

EVOLUTION OF GENERALISTS AND SPECIALISTS IN SPATIALLY HETEROGENEOUS ENVIRONMENTS.

Quantitative genetic models are used to investigate the evolution of generalists and specialists in a coarse-grained environment with two habitat types when there are costs attached to being a generalist. The outcomes for soft and hard selection models are qualitatively different. Under soft selection (e.g., for juvenile or male-reproductive traits) the population evolves towards the single peak in the adaptive landscape. At equilibrium, the population mean phenotype is a compromise between the reaction that would be optimal in both habitats and the reaction with the lowest cost. Furthermore, the equilibrium is closer to the optimal phenotype in the most frequent habitat, or the habitat in which selection on the focal trait is stronger. A specialist genotype always has a lower fitness than a generalist, even when the costs are high. In contrast, under hard selection (e.g., for adult or female-reproductive traits) the adaptive landscape can have one, two, or three peaks; a peak represents a population specialized to one habitat, equally adapted to both habitats, or an intermediate. One peak is always found when the reaction with the lowest cost is not much different from the optimal reaction, and this situation is similar to the soft selection case. However, multiple peaks are present when the costs become higher, and the course of evolution is then determined by initial conditions, and the region of attraction of each peak. This implies that the evolution of specialization and phenotypic plasticity may not only depend on selection regimes within habitats, but also on contingent, historical events (migration, mutation). Furthermore, the evolutionary dynamics in changing environments can be widely different for populations under hard and soft selection. Approaches to measure costs in natural and experimental populations are discussed.