Inter-annual and spatial climatic variability have led to a balance between local fluctuating selection and wide-range directional selection in a perennial grass species.

BACKGROUND AND AIMS: The persistence of a plant population under a specific local climatic regime requires phenotypic adaptation with underlying particular combinations of alleles at adaptive loci. The level of allele diversity at adaptive loci within a natural plant population conditions its potential to evolve, notably towards adaptation to a change in climate. Investigating the environmental factors that contribute to the maintenance of adaptive diversity in populations is thus worthwhile. Within-population allele diversity at adaptive loci can be partly driven by the mean climate at the population site but also by its temporal variability. METHODS: The effects of climate temporal mean and variability on within-population allele diversity at putatively adaptive quantitative trait loci (QTLs) were evaluated using 385 natural populations of Lolium perenne (perennial ryegrass) collected right across Europe. For seven adaptive traits related to reproductive phenology and vegetative potential growth seasonality, the average within-population allele diversity at major QTLs (HeA) was computed. KEY RESULTS: Significant relationships were found between HeA of these traits and the temporal mean and variability of the local climate. These relationships were consistent with functional ecology theory. CONCLUSIONS: Results indicated that temporal variability of local climate has likely led to fluctuating directional selection, which has contributed to the maintenance of allele diversity at adaptive loci and thus potential for further adaptation.

Cold storage to overcome dormancy affects the carbon balance of azalea.

Flower bud dormancy in azalea (Rhododendron simsii) is broken by artificial cold treatment and this will have its consequences on carbon reserves and photosynthesis. The effect of cold storage at 7 °C on carbohydrate and starch content in leaves and flower buds of an early ('Nordlicht') and semi-early ('M. Marie) flowering cultivar was quantified. Carbon loss due to respiration was lowest for 'M. Marie'. Photosynthetic measurements on 'Nordlicht' showed that photosynthesis 3 days after cold treatment (plants ready to flower) was improved compared to before cold treatment (plants with dormant flower buds).

Postglacial recolonization history of the European crabapple (Malus sylvestris Mill.), a wild contributor to the domesticated apple.

Understanding the way in which the climatic oscillations of the Quaternary Period have shaped the distribution and genetic structure of extant tree species provides insight into the processes driving species diversification, distribution and survival. Deciphering the genetic consequences of past climatic change is also critical for the conservation and sustainable management of forest and tree genetic resources, a timely endeavour as the Earth heads into a period of fast climate change. We used a combination of genetic data and ecological niche models to investigate the historical patterns of biogeographic range expansion of a wild fruit tree, the European crabapple (Malus sylvestris), a wild contributor to the domesticated apple. Both climatic predictions for the last glacial maximum and analyses of microsatellite variation indicated that M. sylvestris experienced range contraction and fragmentation. Bayesian clustering analyses revealed a clear pattern of genetic structure, with one genetic cluster spanning a large area in Western Europe and two other genetic clusters with a more limited distribution range in Eastern Europe, one around the Carpathian Mountains and the other restricted to the Balkan Peninsula. Approximate Bayesian computation appeared to be a powerful technique for inferring the history of these clusters, supporting a scenario of simultaneous differentiation of three separate glacial refugia. Admixture between these three populations was found in their suture zones. A weak isolation by distance pattern was detected within each population, indicating a high extent of historical gene flow for the European crabapple.

Genetic relationships in an international collection of Puccinia horiana isolates based on newly identified molecular markers and demonstration of recombination.

The obligate biotrophic pathogen Puccinia horiana is the causal agent of chrysanthemum white rust. Although P. horiana is a quarantine organism, it has been able to spread to most chrysanthemum-producing regions in the world since the 1960s; however, the transfer routes are largely obscure. An extremely low level of allelic diversity was observed in a geographically diverse set of eight isolates using complexity reduction of polymorphic sequences (CRoPS) technology. Only 184 of the 16,196 contigs (1.1%) showed one or more single-nucleotide polymorphisms (SNPs). Thirty-two SNPs and one simple-sequence repeat were translated into molecular markers and used to genotype 45 isolates originating from North and South America, Asia, and Europe. In most cases, phylogenetic clustering was related to geographic origin, indicating local establishment. The European isolates mostly grouped in two major populations that may relate to the two historic introductions previously reported. However, evidence of recent geographic transfer was also observed, including transfer events between Europe and South America and between Southeast Asia and Europe. In contrast with the presumed clonal propagation of this microcyclic rust, strong indications of marker recombination were observed, presumably as a result of anastomosis, karyogamy, and somatic meiosis. Recombination and transfer also explain the geographic dispersal of specific markers. A near-to-significant correlation between the genotypic data and previously obtained pathotype data was observed and one marker was associated with the most virulent pathotype group. In combination with a fast SNP detection method, the markers presented here will be helpful tools to further elucidate the transfer pathways and local survival of this pathogen.

Recolonization after habitat restoration leads to decreased genetic variation in populations of a terrestrial orchid.

Colonization is crucial to habitat restoration projects that rely on the spontaneous regeneration of the original vegetation. However, as a previously declining plant species spreads again, the likelihood of founder effects increases through recurrent population founding and associated serial bottlenecks. We related Amplified Fragment Length Polymorphism markers genetic variation and fitness to colonization history for all extant populations of the outcrossing terrestrial orchid Dactylorhiza incarnata in an isolated coastal dune complex. Around 1970, D. incarnata suffered a severe bottleneck yet ultimately persisted and gradually spread throughout the spatially segregated dune slacks, aided by the restoration of an open vegetation. Genetic assignment demonstrated dispersal to vacant sites from few nearby extant populations and very limited inflow from outside the spatially isolated reserve. Results further indicated that recurrent founding from few local sources resulted in the loss of genetic diversity and promoted genetic divergence (F(ST) = 0.35) among populations, but did not influence population fitness. The few source populations initially available and the limited inflow of genes from outside the study reserve, as a consequence of habitat degradation and spatial isolation, may have magnified the genetic effects of recurrent population founding.

The consequences of mating over a range of parental genetic similarity in a selfing allopolyploid plant species.

In diploids, F(1) offspring performance is expected to increase with increasing genetic dissimilarity between the parents until an optimum is reached because outbreeding mitigates inbreeding depression and maximizes heterosis. However, many flowering plant species are derived through allopolyploidization, i.e. interspecific hybridization with genome doubling. This mode of plant speciation can be expected to considerably alter the consequences of inbreeding and outbreeding. We investigated the F1 fitness consequences of mating over a range of (genetic) distances in the allohexaploid plant species Geum urbanum. Offspring was raised under controlled conditions (632 plants). The performance of outcrossed progeny was not significantly better than that of their selfed half-siblings and did not increase with parental genetic dissimilarity (0-0.83). Our findings support low, if any, inbreeding depression and heterosis. We attribute this to the peculiar state of quasi-permanent heterozygosity in allopolyploids and frequent selfing.

A direct assessment of realized seed and pollen flow within and between two isolated populations of the food-deceptive orchid Orchis mascula.

Gene flow can counteract the loss of genetic diversity caused by genetic drift in small populations. For this reason, clearly understanding gene flow patterns is of the highest importance across fragmented landscapes. However, gene flow patterns are not only dependent upon the degree of spatial isolation of fragmented populations, but are also dependent upon the life-history traits of the species. Indeed, habitat fragmentation effects appear especially unpredictable for food-deceptive orchid species, because of their highly specialised seed and pollen dispersal mechanisms. In this study we used amplified fragment length polymorphism markers and subsequent parentage and spatial autocorrelation analysis to quantify the extent and the patterns of realized gene flow within and between two adjacent fragmented populations of the food-deceptive Orchis mascula. We observed considerable gene flow between both populations, occurring mainly through pollen dispersal. Seed dispersal, on the other hand, was mainly limited to the first few meters from the mother plant in both populations, although at least one among-population seed dispersal event was observed. This, in turn, resulted in a significant spatial genetic structure for both populations. Although genetic diversity was high in both populations and mainly outcrossing occurred, reproductive output was strongly skewed toward a limited number of successful adult plants. These observed patterns are likely due to the different pollinator behaviour associated with food-deceptive plants. We conclude that these populations can be considered viable under their current fragmented state.

Clade classification of monolignol biosynthesis gene family members reveals target genes to decrease lignin in Lolium perenne.

In monocots, lignin content has a strong impact on the digestibility of the cell wall fraction. Engineering lignin biosynthesis requires a profound knowledge of the role of paralogues in the multigene families that constitute the monolignol biosynthesis pathway. We applied a bioinformatics approach for genome-wide identification of candidate genes in Lolium perenne that are likely to be involved in the biosynthesis of monolignols. More specifically, we performed functional subtyping of phylogenetic clades in four multigene families: 4CL, COMT, CAD and CCR. Essential residues were considered for functional clade delineation within these families. This classification was complemented with previously published experimental evidence on gene expression, gene function and enzymatic activity in closely related crops and model species. This allowed us to assign functions to novel identified L. perenne genes, and to assess functional redundancy among paralogues. We found that two 4CL paralogues, two COMT paralogues, three CCR paralogues and one CAD gene are prime targets for genetic studies to engineer developmentally regulated lignin in this species. Based on the delineation of sequence conservation between paralogues and a first analysis of allelic diversity, we discuss possibilities to further study the roles of these paralogues in lignin biosynthesis, including expression analysis, reverse genetics and forward genetics, such as association mapping. We propose criteria to prioritise paralogues within multigene families and certain SNPs within these genes for developing genotyping assays or increasing power in association mapping studies. Although L. perenne was the target of the analyses presented here, this functional subtyping of phylogenetic clades represents a valuable tool for studies investigating monolignol biosynthesis genes in other monocot species.

Cold storage to overcome dormancy affects the carbohydrate status and photosynthetic capacity of Rhododendron simsii.

Global warming leads to increasing irregular and unexpected warm spells during autumn, and therefore natural chilling requirements to break dormancy are at risk. Controlled cold treatment can provide an answer to this problem. Nevertheless, artificial cold treatment will have consequences for carbon reserves and photosynthesis. In this paper, the effect of dark cold storage at 7 °C to break flower bud dormancy in the evergreen Rhododendron simsii was quantified. Carbohydrate and starch content in leaves and flower buds of an early ('Nordlicht'), semi-early ('M. Marie') and late ('Mw. G. Kint') flowering cultivar showed that carbon loss due to respiration was lowest in 'M. Marie', while 'Mw. G. Kint' was completely depleted of starch reserves at the end of cold treatment. Gene isolation resulted in a candidate gene for sucrose synthase (SUS) RsSus, which appears to be homologous to AtSus3 and had a clear increase in expression in leaves during cold treatment. Photosynthesis measurements on 'Nordlicht' and the late-flowering cultivar 'Thesla' showed that during cold treatment, dark respiration decreased 58% and 63%, respectively. Immediately after cold treatment, dark respiration increased and stabilised after 3 days. The light compensation point followed the same trend as dark respiration. Quantum efficiency showed no significant changes during the first days after cold treatment, but was significantly higher than in plants with dormant flower buds at the start of cold treatment. In conclusion, photosynthesis stabilised 3 days after cold treatment and was improved compared to the level before cold treatment.

AFLP markers demonstrate local genetic differentiation between two indigenous oak species [ Quercus robur L. and Quercus petraea (Matt.) Liebl.] in Flemish populations.

The nuclear genetic variation within and between four sessile ( Q. petraea) and six pedunculate ( Q. robur) autochthonous Flemish oak populations was investigated with AFLP markers. One sessile and one pedunculate oak population were additionally screened for detailed leaf characteristics using an image analysis system. Principal coordinate analysis on the AFLP data classified the oaks in two main groups, according to their taxonomic status. No species-specific AFLP markers were found using four primer combinations, but marker frequency differences up to 71% were recorded between both species. Analysis of the genetic structure showed that the divergence between species, as observed by ordination, was significant. Both species revealed similar diversity levels. A smaller though significant differentiation was also revealed for both species among populations within species. Molecular and morphology based approaches showed a high degree of consistency. Screening of 60 AFLP primer combinations using a bulking strategy did not allow identifying species-specific markers, which supports the conclusions reached in previous studies. The distribution of genetic variability at the species and at the population level is discussed.

SNP discovery using Paired-End RAD-tag sequencing on pooled genomic DNA of Sisymbrium austriacum (Brassicaceae).

Single nucleotide polymorphisms SNPs are rapidly replacing anonymous markers in population genomic studies, but their use in non model organisms is hampered by the scarcity of cost-effective approaches to uncover genome-wide variation in a comprehensive subset of individuals. The screening of one or only a few individuals induces ascertainment bias. To discover SNPs for a population genomic study of the Pyrenean rocket (Sisymbrium austriacum subsp. chrysanthum), we undertook a pooled RAD-PE (Restriction site Associated DNA Paired-End sequencing) approach. RAD tags were generated from the PstI-digested pooled genomic DNA of 12 individuals sampled across the species distribution range and paired-end sequenced using Illumina technology to produce ~24.5 Mb of sequences, covering ~7% of the specie's genome. Sequences were assembled into ~76 000 contigs with a mean length of 323 bp (N(50)  = 357 bp, sequencing depth = 24x). In all, >15 000 SNPs were called, of which 47% were annotated in putative genic regions based on homology with the Arabidopsis thaliana genome. Gene ontology (GO) slim categorization demonstrated that the identified SNPs covered extant genic variation well. The validation of 300 SNPs on a larger set of individuals using a KASPar assay underpinned the utility of pooled RAD-PE as an inexpensive genome-wide SNP discovery technique (success rate: 87%). In addition to SNPs, we discovered >600 putative SSR markers.

Landscape genetics of the self-compatible forest herb Geum urbanum: effects of habitat age, fragmentation and local environment.

To investigate the role of habitat fragmentation, fragment age and local environment in shaping the genetics of plant populations, we examined the genetic structure of the self-compatible forest herb Geum urbanum using microsatellite markers. A historical land-use reconstruction assigned the studied populations to two age classes: populations in primary forest fragments, and populations in secondary fragments. Local environmental conditions were quantified on the basis of the herb-layer community composition. A stepwise general linear model revealed that levels of within-population genetic diversity were best explained by population size, landscape connectivity and the interaction between both. Connectivity was positively correlated with the genetic diversity of small populations, but did not significantly affect the diversity of large populations. Contrary to what we expected, secondary-forest populations showed lower divergence relative to populations located in primary patches. Small populations were genetically more diverged compared to large populations. Mantel tests showed no significant isolation by distance and no significant correlation between habitat similarity and genetic differentiation. We conclude that gene flow has probably prevented founder events from being reflected in the present genetic structure of G. urbanum. Gene flow towards low-connectivity populations, however, seemed to be insufficient to counteract the effects of drift in small populations.

Chloroplast diversity in the genus Malus: new insights into the relationship between the European wild apple (Malus sylvestris (L.) Mill.) and the domesticated apple (Malus domestica Borkh.).

To unravel the relationship between the European wild apple, Malus sylvestris (L.) Mill., and its domesticated relative M. domestica Borkh., we studied chloroplast DNA variation in 634 wild and 422 domesticated accessions originating from different regions. Hybridization between M. sylvestris and M. domestica was checked using 10 nuclear microsatellites and a Bayesian assignment approach. This allowed us to identify hybrids and feral plants escaped from cultivation. Sixty-eight genotypes belonging to 12 other wild Malus species, including 20 M. sieversii (Ledeb.) Roem. accessions were also included in the analysis of chloroplast diversity. Marker techniques were developed to type a formerly described duplication and a newly detected transversion in the matK gene. Chloroplast DNA variation was further investigated using PCR-RFLP (Polymerase Chain Reaction-Random Fragment Length Polymorphism), and haplotypes were constructed based on all mutational combinations. A closer relationship than presently accepted between M. sylvestris and M. domestica was established at the cytoplasmic level, with the detection of eight chloroplast haplotypes shared by both species. Hybridization between M. sylvestris and M. domestica was also apparent at the local level with sharing of rare haplotypes among local cultivars and sympatric wild trees. Indications of the use of wild Malus genotypes in the (local) cultivation process of M. domestica and cytoplasmic introgression of chloroplast haplotypes into M. sylvestris from the domesticated apple were found. Only one of the M. sieversii trees studied displayed one of the three main chloroplast haplotypes shared by M. sylvestris and M. domestica. This is surprising as M. sieversii has formerly been described as the main maternal progenitor of the domesticated apple. This study hereby reopens the exciting discussion on the origin of M. domestica.

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

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

Four QTLs determine crown rust (Puccinia coronata f. sp. lolii) resistance in a perennial ryegrass (Lolium perenne) population.

Crown rust resistance is an important selection criterion in ryegrass breeding. The disease, caused by the biotrophic fungus Puccinia coronata, causes yield losses and reduced quality. In this study, we used linkage mapping and QTL analysis to unravel the genomic organization of crown rust resistance in a Lolium perenne population. The progeny of a pair cross between a susceptible and a resistant plant were analysed for crown rust resistance. A linkage map, consisting of 227 loci (AFLP, SSR, RFLP and STS) and spanning 744 cM, was generated using the two-way pseudo-testcross approach from 252 individuals. QTL analysis revealed four genomic regions involved in crown rust resistance. Two QTLs were located on LG1 (LpPc4 and LpPc2) and two on LG2 (LpPc3 and LpPc1). They explain 12.5, 24.9, 5.5 and 2.6% of phenotypic variance, respectively. An STS marker, showing homology to R genes, maps in the proximity of LpPc2. Further research is, however, necessary to check the presence of functional R genes in this region. Synteny at the QTL level between homologous groups of chromosomes within the Gramineae was observed. LG1 and LG2 show homology with group A and B chromosomes of oat on which crown rust-resistance genes have been identified, and with the group 1 chromosomes of the Triticeae, on which leaf rust-resistance genes have been mapped. These results are of major importance for understanding the molecular background of crown rust resistance in ryegrasses. The identified markers linked to crown rust resistance have the potential for use in marker-assisted breeding.

Data from amplified fragment length polymorphism (AFLP) markers show indication of size homoplasy and of a relationship between degree of homoplasy and fragment size.

We investigate the distribution of sizes of fragments obtained from the amplified fragment length polymorphism (AFLP) marker technique. We find that empirical distributions obtained in two plant species, Phaseolus lunatus and Lolium perenne, are consistent with the expected distributions obtained from analytical theory and from numerical simulations. Our results indicate that the size distribution is strongly asymmetrical, with a much higher proportion of small than large fragments, that it is not influenced by the number of selective nucleotides nor by genome size but that it may vary with genome-wide GC-content, with a higher proportion of small fragments in cases of lower GC-content when considering the standard AFLP protocol with the enzyme MseI. Results from population samples of the two plant species show that there is a negative relationship between AFLP fragment size and fragment population frequency. Monte Carlo simulations reveal that size homoplasy, arising from pulling together nonhomologous fragments of the same size, generates patterns similar to those observed in P. lunatus and L. perenne because of the asymmetry of the size distribution. We discuss the implications of these results in the context of estimating genetic diversity with AFLP markers.