Evolution of floral scent in relation to self-incompatibility and capacity for autonomous self-pollination in the perennial herb Arabis alpina

Background and AimsThe transition from outcrossing to selfing is a frequent evolutionary shift in flowering plants and is predicted to result in reduced allocation to pollinator attraction if plants can self-pollinate autonomously. The evolution of selfing is associated with reduced visual floral signalling in many systems, but effects on floral scent have received less attention. We compared multiple populations of the arctic–alpine herb Arabis alpina (Brassicaceae), and asked whether the transition from self-incompatibility to self-compatibility has been associated with reduced visual and chemical floral signalling. We further examined whether floral signalling differ between self-compatible populations with low and high capacity for autonomous self-pollination, as would be expected if benefits of signalling decrease with reduced dependence on pollinators for pollen transfer.MethodsIn a common garden we documented flower size and floral scent emission rate and composition in eight self-compatible and nine self-incompatible A. alpina populations. These included self-compatible Scandinavian populations with high capacity for autonomous self-pollination, self-compatible populations with low capacity for autonomous self-pollination from France and Spain, and self-incompatible populations from Italy and Greece.Key ResultsThe self-compatible populations produced smaller and less scented flowers than the self-incompatible populations. However, flower size and scent emission rate did not differ between self-compatible populations with high and low capacity for autonomous self-pollination. Floral scent composition differed between self-compatible and self-incompatible populations, but also varied substantially among populations within the two categories.ConclusionsOur study demonstrates extensive variation in floral scent among populations of a geographically widespread species. Contrary to expectation, floral signalling did not differ between self-compatible populations with high and low capacity for autonomous self-pollination, indicating that dependence on pollinator attraction can only partly explain variation in floral signalling. Additional variation may reflect adaptation to other aspects of local environments, genetic drift, or a combination of these processes.     

Seed traits are pleiotropically regulated by the flowering time gene PERPETUAL FLOWERING 1 (PEP1) in the perennial Arabis alpina

The life cycles of plants are characterized by two major life history transitions—germination and the initiation of flowering—the timing of which are important determinants of fitness. Unlike annuals, which make the transition from the vegetative to reproductive phase only once, perennials iterate reproduction in successive years. The floral repressor PERPETUAL FLOWERING 1 (PEP1), an ortholog of FLOWERING LOCUS C, in the alpine perennial Arabis alpina ensures the continuation of vegetative growth after flowering and thereby restricts the duration of the flowering episode. We performed greenhouse and garden experiments to compare flowering phenology, fecundity and seed traits between A. alpina accessions that have a functional PEP1 allele and flower seasonally and pep1 mutants and accessions that carry lesions in PEP1 and flower perpetually. In the garden, perpetual genotypes flower asynchronously and show higher winter mortality than seasonal ones. PEP1 also pleiotropically regulates seed dormancy and longevity in a way that is functionally divergent from FLC. Seeds from perpetual genotypes have shallow dormancy and reduced longevity regardless of whether they after‐ripened in plants grown in the greenhouse or in the experimental garden. These results suggest that perpetual genotypes have higher mortality during winter but compensate by showing higher seedling establishment. Differences in seed traits between seasonal and perpetual genotypes are also coupled with differences in hormone sensitivity and expression of genes involved in hormonal pathways. Our study highlights the existence of pleiotropic regulation of seed traits by hub developmental regulators such as PEP1, suggesting that seed and flowering traits in perennial plants might be optimized in a coordinated fashion.     

Development of an Arabis alpina genomic contig sequence data set and application to single nucleotide polymorphisms discovery

The alpine plant Arabis alpina is an emerging model in the ecological genomic field which is well suited to identifying the genes involved in local adaptation in contrasted environmental conditions, a subject which remains poorly understood at molecular level. This study presents the assembly of a pool of A. alpina genomic fragments using next‐generation sequencing technologies. These contigs cover 172 Mb of the A. alpina genome (i.e. 50% of the genome) and were shown to contain sequences giving positive hits against 96% of the 458 CEGMA core genes (Core Eukaryotic Genes Mapping Approach), a set of highly conserved eukaryotic genes. Regions presenting high nucleic sequence identity with 77% of the close relative Arabidopsis thaliana's genes were found with an unbiased distribution across the different functional categories of A. thaliana genes. This new resource was tested using a resequencing assay to identify polymorphic sites. Sixteen samples were successfully analysed and 127 041 single‐nucleotide polymorphisms identified. This contig data set will contribute to improving our understanding of the ecology of Arabis alpina, thus constituting an important resource for future ecological genomic studies.     

QTL mapping of vernalization response in perennial ryegrass (Lolium perenne L.) reveals co-location with an orthologue of wheat VRN1

The objective of this study was to map quantitative trait loci (QTL) for the vernalization response in perennial ryegrass (Lolium perenne L.). The mapping population consisted of 184 F₂ genotypes produced from a cross between one genotype of a synthetic perennial ryegrass variety Veyo and one genotype from the perennial ryegrass ecotype Falster. Veyo and Falster were chosen among four different populations because of their contrasting vernalization requirements. In total, five QTL for the vernalization response, measured as days to heading, were identified and mapped to linkage groups (LG) LG2, LG4, LG6 and LG7. Individually, these QTL explained between 5.4 and 28.0% of the total phenotypic variation. The overall contribution of these five QTL was 80% of the total phenotypic variation. A putative orthologue of Triticum monococcum VRN1 was amplified from genomic DNA from perennial ryegrass. PCR fragments covering the proximal part of the promoter and the 5 end of the orthologue were subsequently PCR-amplified from both parents of the mapping population and shown to possess 95% DNA sequence identity to VRN1. Several polymorphisms were identified between Veyo and Falster in this fragment of the putative VRN1 orthologue. A CAPS marker, vrn-1, was developed and found to co-segregate with a major QTL on LG4 for the vernalization response. This indicates that the CAPS marker vrn-1 could be located in an orthologous gene of the wheat VRN1.     

Evolution of the Selfing Syndrome in Arabis alpina (Brassicaceae)

Introduction

The transition from cross-fertilisation (outcrossing) to self-fertilisation (selfing) frequently coincides with changes towards a floral morphology that optimises self-pollination, the selfing syndrome. Population genetic studies have reported the existence of both outcrossing and selfing populations in Arabis alpina (Brassicaceae), which is an emerging model species for studying the molecular basis of perenniality and local adaptation. It is unknown whether its selfing populations have evolved a selfing syndrome.

Methods

Using macro-photography, microscopy and automated cell counting, we compared floral syndromes (size, herkogamy, pollen and ovule numbers) between three outcrossing populations from the Apuan Alps and three selfing populations from the Western and Central Alps (Maritime Alps and Dolomites). In addition, we genotyped the plants for 12 microsatellite loci to confirm previous measures of diversity and inbreeding coefficients based on allozymes, and performed Bayesian clustering.

Results and Discussion

Plants from the three selfing populations had markedly smaller flowers, less herkogamy and lower pollen production than plants from the three outcrossing populations, whereas pistil length and ovule number have remained constant. Compared to allozymes, microsatellite variation was higher, but revealed similar patterns of low diversity and high Fis in selfing populations. Bayesian clustering revealed two clusters. The first cluster contained the three outcrossing populations from the Apuan Alps, the second contained the three selfing populations from the Maritime Alps and Dolomites.

Conclusion

We conclude that in comparison to three outcrossing populations, three populations with high selfing rates are characterised by a flower morphology that is closer to the selfing syndrome. The presence of outcrossing and selfing floral syndromes within a single species will facilitate unravelling the genetic basis of the selfing syndrome, and addressing which selective forces drive its evolution.     

Genomic Signatures of Sexual Selection on Pollen-Expressed Genes in Arabis alpina

Fertilization in angiosperms involves the germination of pollen on the stigma, followed by the extrusion of a pollen tube that elongates through the style and delivers two sperm cells to the embryo sac. Sexual selection could occur throughout this process when male gametophytes compete for fertilization. The strength of sexual selection during pollen competition should be affected by the number of genotypes deposited on the stigma. As increased self-fertilization reduces the number of mating partners, and the genetic diversity and heterozygosity of populations, it should thereby reduce the intensity of sexual selection during pollen competition. Despite the prevalence of mating system shifts, few studies have directly compared the molecular signatures of sexual selection during pollen competition in populations with different mating systems. Here we analyzed whole-genome sequences from natural populations of Arabis alpina, a species showing mating system variation across its distribution, to test whether shifts from cross- to self-fertilization result in molecular signatures consistent with sexual selection on genes involved in pollen competition. We found evidence for efficient purifying selection on genes expressed in vegetative pollen, and overall weaker selection on sperm-expressed genes. This pattern was robust when controlling for gene expression level and specificity. In agreement with the expectation that sexual selection intensifies under cross-fertilization, we found that the efficacy of purifying selection on male gametophyte-expressed genes was significantly stronger in genetically more diverse and outbred populations. Our results show that intra-sexual competition shapes the evolution of pollen-expressed genes, and that its strength fades with increasing self-fertilization rates.     

The quantitative response of wheat vernalization to environmental variables indicates that vernalization is not a response to cold temperature

The initiation of flowering is a crucial trait that allows temperate plants to flower in the favourable conditions of spring. The timing of flowering initiation is governed by two main mechanisms: vernalization that defines a plant's requirement for a prolonged exposure to cold temperatures; and photoperiod sensitivity defining the need for long days to initiate floral transition. Genetic variability in both vernalization and photoperiod sensitivity largely explains the adaptability of cultivated crop plants such as bread wheat (Triticum aestivum L.) to a wide range of climatic conditions. The major genes controlling wheat vernalization (VRN1, VRN2, and VRN3) and photoperiod sensitivity (PPD1) have been identified, and knowledge of their interactions at the molecular level is growing. However, the quantitative effects of temperature and photoperiod on these genes remain poorly understood. Here it is shown that the distinction between the temperature effects on organ appearance rate and on vernalization sensu stricto is crucial for understanding the quantitative effects of the environmental signal on wheat flowering. By submitting near isogenic lines of wheat differing in their allelic composition at the VRN1 locus to various temperature and photoperiod treatments, it is shown that, at the whole-plant level, the vernalization process has a positive response to temperature with complex interactions with photoperiod. In addition, the phenotypic variation associated with the presence of different spring homoeoalleles of VRN1 is not induced by a residual vernalization requirement. The results demonstrate that a precise definition of vernalization is necessary to understand and model temperature and photoperiod effects on wheat flowering. It is suggested that this definition should be used as the basis for gene expression studies and assessment of functioning of the wheat flowering gene network, including an explicit account of the quantitative effect of environmental variables.     

Contemporary gene flow and mating system of Arabis alpina in a Central European alpine landscape

Background and Aims

Gene flow is important in counteracting the divergence of populations but also in spreading genes among populations. However, contemporary gene flow is not well understood across alpine landscapes. The aim of this study was to estimate contemporary gene flow through pollen and to examine the realized mating system in the alpine perennial plant, Arabis alpina (Brassicaceae).

Methods

An entire sub-alpine to alpine landscape of 2 km² was exhaustively sampled in the Swiss Alps. Eighteen nuclear microsatellite loci were used to genotype 595 individuals and 499 offspring from 49 maternal plants. Contemporary gene flow by pollen was estimated from paternity analysis, matching the genotypes of maternal plants and offspring to the pool of likely father plants. Realized mating patterns and genetic structure were also estimated.

Key Results

Paternity analysis revealed several long-distance gene flow events (≤1 km). However, most outcrossing pollen was dispersed close to the mother plants, and 84 % of all offspring were selfed. Individuals that were spatially close were more related than by chance and were also more likely to be connected by pollen dispersal.

Conclusions

In the alpine landscape studied, genetic structure occurred on small spatial scales as expected for alpine plants. However, gene flow also covered large distances. This makes it plausible for alpine plants to spread beneficial alleles at least via pollen across landscapes at a short time scale. Thus, gene flow potentially facilitates rapid adaptation in A. alpina likely to be required under ongoing climate change.     

Validation of outlier loci through replication in independent data sets: a test on Arabis alpina

Outlier detection and environmental association analysis are common methods to search for loci or genomic regions exhibiting signals of adaptation to environmental factors. However, a validation of outlier loci and corresponding allele distribution models through functional molecular biology or transplant/common garden experiments is rarely carried out. Here, we employ another method for validation, namely testing outlier loci in specifically designed, independent data sets. Previously, an outlier locus associated with three different habitat types had been detected in Arabis alpina. For the independent validation data set, we sampled 30 populations occurring in these three habitat types across five biogeographic regions of the Swiss Alps. The allele distribution model found in the original study could not be validated in the independent test data set: The outlier locus was no longer indicative of habitat‐mediated selection. We propose several potential causes of this failure of validation, of which unaccounted genetic structure and technical issues in the original data set used to detect the outlier locus were most probable. Thus, our study shows that validating outlier loci and allele distribution models in independent data sets is a helpful tool in ecological genomics which, in the case of positive validation, adds confidence to outlier loci and their association with environmental factors or, in the case of failure of validation, helps to explain inconsistencies.     

BROTHER OF FT AND TFL1 (BFT) has TFL1‐like activity and functions redundantly with TFL1 in inflorescence meristem development in Arabidopsis

The FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family is a small gene family that encodes important regulators that control flower development in Arabidopsis. Here, we investigated the biological role of the product of BROTHER OF FT AND TFL1 (BFT), a member of this family, whose function remains unknown. Comparison of the critical residues that play a role in distinguishing FT‐ or TFL1‐like activity revealed that BFT is more similar to FT. Similar to FT expression, BFT expression showed a diurnal oscillation pattern, peaking in the evening. In situ hybridization revealed BFT expression in the shoot apical meristem, young leaf and axillary inflorescence meristem. Transgenic plants over‐expressing BFT exhibited delayed flowering and severe floral defects (floral indeterminacy and compact inflorescences surrounded by serrate leaves), similar to 35S::TFL1 plants. LEAFY (LFY) and APETALA1 (AP1) expression was significantly reduced in 35S::BFT plants. BFT over‐expression failed to rescue the terminal flower phenotype of tfl1 mutants; however, it delayed both terminal flower formation in the primary inflorescence and axillary inflorescence development in the tfl1 mutant background. Consistent with this, the loss‐of‐function BFT alleles, bft‐2 and an BFT RNAi line, accelerated termination of the primary inflorescence and formation of axillary inflorescences in the tfl1 mutant background. Taken together, our results suggest that, despite similarities in the critical residues of BFT and FT, BFT possesses a TFL1‐like activity and functions redundantly with TFL1 in inflorescence meristem development, and possibly contributes to the regulation of plant architecture.     

Rpa1 mediates an immune response to avrRpm1Psa and confers resistance against Pseudomonas syringae pv. actinidiae

The type three effector AvrRpm1_Pma from Pseudomonas syringae pv. maculicola (Pma) triggers an RPM1‐mediated immune response linked to phosphorylation of RIN4 (RPM1‐interacting protein 4) in Arabidopsis. However, the effector–resistance (R) gene interaction is not well established with different AvrRpm1 effectors from other pathovars. We investigated the AvrRpm1‐triggered immune responses in Nicotiana species and isolated Rpa1 (R esistance to P seudomonas syringae pv. a ctinidiae 1) via a reverse genetic screen in Nicotiana tabacum. Transient expression and gene silencing were performed in combination with co‐immunoprecipitation and growth assays to investigate the specificity of interactions that lead to inhibition of pathogen growth. Two closely related AvrRpm1 effectors derived from Pseudomonas syringae pv. actinidiae biovar 3 (AvrRpm1_Psa) and Pseudomonas syringae pv. syringae strain B728a (AvrRpm1_Psy) trigger immune responses mediated by RPA1, a nucleotide‐binding leucine‐rich repeat protein with an N‐terminal coiled‐coil domain. In a display of contrasting specificities, RPA1 does not respond to AvrRpm1_Pma, and correspondingly AvrRpm1_Psa and AvrRpm1_Psy do not trigger the RPM1‐mediated response, demonstrating that separate R genes mediate specific immune responses to different AvrRpm1 effectors. AvrRpm1_Psa co‐immunoprecipitates with RPA1, and both proteins co‐immunoprecipitate with RIN4. In contrast with RPM1, however, RPA1 was not activated by the phosphomimic RIN4^T166D and silencing of RIN4 did not affect the RPA1 activity. Delivery of AvrRpm1_Psa by Pseudomonas syringae pv. tomato (Pto) in combination with transient expression of Rpa1 resulted in inhibition of the pathogen growth in N. benthamiana. Psa growth was also inhibited by RPA1 in N. tabacum.     

Overexpression of ubiquitin‐like LpHUB1 gene confers drought tolerance in perennial ryegrass

HUB1, also known as Ubl5, is a member of the subfamily of ubiquitin‐like post‐translational modifiers. HUB1 exerts its role by conjugating with protein targets. The function of this protein has not been studied in plants. A HUB1 gene, LpHUB1, was identified from serial analysis of gene expression data and cloned from perennial ryegrass. The expression of this gene was reported previously to be elevated in pastures during the summer and by drought stress in climate‐controlled growth chambers. Here, pasture‐type and turf‐type transgenic perennial ryegrass plants overexpressing LpHUB1 showed improved drought tolerance, as evidenced by improved turf quality, maintenance of turgor and increased growth. Additional analyses revealed that the transgenic plants generally displayed higher relative water content, leaf water potential, and chlorophyll content and increased photosynthetic rate when subjected to drought stress. These results suggest HUB1 may play an important role in the tolerance of perennial ryegrass to abiotic stresses.     

Genome‐wide variation in nucleotides and retrotransposons in alpine populations of Arabis alpina (Brassicaceae)

Advances in high‐throughput sequencing have promoted the collection of reference genomes and genome‐wide diversity. However, the assessment of genomic variation among populations has hitherto mainly been surveyed through single‐nucleotide polymorphisms (SNPs) and largely ignored the often major fraction of genomes represented by transposable elements (TEs). Despite accumulating evidence supporting the evolutionary significance of TEs, comprehensive surveys remain scarce. Here, we sequenced the full genomes of 304 individuals of Arabis alpina sampled from four nearby natural populations to genotype SNPs as well as polymorphic long terminal repeat retrotransposons (polymorphic TEs; i.e., presence/absence of TE insertions at specific loci). We identified 291,396 SNPs and 20,548 polymorphic TEs, comparing their contributions to genomic diversity and divergence across populations. Few SNPs were shared among populations and overall showed high population‐specific variation, whereas most polymorphic TEs segregated among populations. The genomic context of these two classes of variants further highlighted candidate adaptive loci having a putative impact on functional genes. In particular, 4.96% of the SNPs were identified as nonsynonymous or affecting start/stop codons. In contrast, 43% of the polymorphic TEs were present next to Arabis genes enriched in functional categories related to the regulation of reproduction and responses to biotic as well as abiotic stresses. This unprecedented data set, mapping variation gained from SNPs and complementary polymorphic TEs within and among populations, will serve as a rich resource for addressing microevolutionary processes shaping genome variation.