"The usual suspects"- analysis of transcriptome sequences reveals deviating B gene activity in C. vulgaris bud bloomers.

BACKGROUND: The production of heather (Calluna vulgaris) in Germany is highly dependent on cultivars with mutated flower morphology, the so-called diplocalyx bud bloomers. So far, this unique flower type of C. vulgaris has not been reported in any other plant species. The flowers are characterised by an extremely extended flower attractiveness, since the flower buds remain closed throughout the complete flowering season. The flowers of C. vulgaris bud bloomers are male sterile, because the stamens are absent. Furthermore, petals are converted into sepals. Therefore the diplocalyx bud bloomer flowers consist of two whorls of sepals directly followed by the gynoecium. RESULTS: A broad comparison was undertaken to identify genes differentially expressed in the bud flowering phenotype and in the wild type of C. vulgaris. Transcriptome sequence reads were generated using 454 sequencing of two flower type specific cDNA libraries. In total, 360,000 sequence reads were obtained, assembled to 12,200 contigs, functionally mapped, and annotated. Transcript abundances were compared and 365 differentially expressed genes detected. Among these differentially expressed genes, Calluna vulgaris PISTILLATA (CvPI) which is the orthologue of the Arabidopsis B gene PISTILLATA (PI) was considered as the most promising candidate gene. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT PCR) was performed to analyse the gene expression levels of two C. vulgaris B genes CvPI and Calluna vulgaris APETALA 3 (CvAP3) in both flower types. CvAP3 which is the orthologue of the Arabidopsis B gene APETALA 3 (AP3) turned out to be ectopically expressed in sepals of wild type and bud bloomer flowers. CvPI expression was proven to be reduced in the bud blooming flowers. CONCLUSIONS: Differential expression patterns of the B-class genes CvAP3 and CvPI were identified to cause the characteristic morphology of C. vulgaris flowers leading to the following hypotheses: ectopic expression of CvAP3 is a convincing explanation for the formation of a completely petaloid perianth in both flower types. In C. vulgaris, CvPI is essential for determination of petal and stamen identity. The characteristic transition of petals into sepals potentially depends on the observed deficiency of CvPI and CvAP3 expression in bud blooming flowers.

Management-driven evolution in a domesticated ecosystem.

Millennia of human land-use have resulted in the widespread occurrence of what have been coined 'domesticated ecosystems'. The anthropogenic imprints on diversity, composition, structure and functioning of such systems are well documented. However, evolutionary consequences of human activities in these ecosystems are enigmatic. Calluna vulgaris (L.) is a keystone species of coastal heathlands in northwest Europe, an ancient semi-natural landscape of considerable conservation interest. Like many species from naturally fire-prone ecosystems, Calluna shows smoke-adapted germination, but it is unclear whether this trait arose prior to the development of these semi-natural landscapes or is an evolutionary response to the anthropogenic fire regime. We show that smoke-induced germination in Calluna is found in populations from traditionally burnt coastal heathlands but is lacking in naturally occurring populations from other habitats with infrequent natural fires. Our study thus demonstrates evolutionary imprints of human land-use in semi-natural ecosystems. Evolutionary consequences of historic anthropogenic impacts on wildlife have been understudied, but understanding these consequences is necessary for informed conservation and ecosystem management.

[Genetic and phenotypic differentiation of Calluna vulgaris (L.) Hull in Pritobolie and Europe].

Geographic variation and differentiation of the chloroplast DNA haplotypes and morpho-anatomical leaf parameters were assessed in a number of eastern European groups of Calluna vulgaris (L.)Hull populations and in the Pritobolien group of populations of this species in Western Siberia, which have long been isolated from the European populations. Sharply pronounced genetic and phenotypic distances and their gradients between the Pritobolien and European population groups were revealed. These distances were many times higher than those between the relatively homogeneous eastern European groups. The data obtained generally supported the hypothesis on the phenogenetic divergence of the Pritobolien marginal populations of C. vulgaris from the European, probably at the subspecies level.

AFLP-based genetic mapping of the "bud-flowering" trait in heather (Calluna vulgaris).

BACKGROUND: Calluna vulgaris is one of the most important landscaping plants produced in Germany. Its enormous economic success is due to the prolonged flower attractiveness of mutants in flower morphology, the so-called bud-bloomers. In this study, we present the first genetic linkage map of C. vulgaris in which we mapped a locus of the economically highly desired trait "flower type". RESULTS: The map was constructed in JoinMap 4.1. using 535 AFLP markers from a single mapping population. A large fraction (40%) of markers showed distorted segregation. To test the effect of segregation distortion on linkage estimation, these markers were sorted regarding their segregation ratio and added in groups to the data set. The plausibility of group formation was evaluated by comparison of the "two-way pseudo-testcross" and the "integrated" mapping approach. Furthermore, regression mapping was compared to the multipoint-likelihood algorithm. The majority of maps constructed by different combinations of these methods consisted of eight linkage groups corresponding to the chromosome number of C. vulgaris. CONCLUSIONS: All maps confirmed the independent inheritance of the most important horticultural traits "flower type", "flower colour", and "leaf colour". An AFLP marker for the most important breeding target "flower type" was identified. The presented genetic map of C. vulgaris can now serve as a basis for further molecular marker selection and map-based cloning of the candidate gene encoding the unique flower architecture of C. vulgaris bud-bloomers.

Development and application of high-throughput amplified fragment length polymorphism technique in Calluna vulgaris (Ericaceae)

Calluna vulgaris is an important ornamental crop of the horticultural industry in Europe. In order to improve breeding of this species, especially of the most important trait of ‘bud-flowering', the implementation of molecular techniques that allow rapid, reproducible and efficient screening of whole segregating populations e.g. for molecular marker and mapping approaches is a requirement. We therefore aimed to introduce the powerful tool of amplified fragment length polymorphisms (AFLP®), a widely and successfully applied method, into our methodological assortment. As an essential prerequisite, the isolated DNA should be of adequate quality which is a common obstacle when dealing with woody species and their interfering secondary components/metabolites. The results of screening different and modified DNA isolation protocols are described. As the outcome of our evaluations of reaction conditions during the AFLP® procedure, we circumstantiate a functional protocol ranging from DNA extraction to visualization of AFLP® banding patterns for the woody crop C. vulgaris. This method is suitable for high throughput genetic applications and may even be transferable to other species. In addition, costs are reduced by reasonable reagents and multiplexing assays.

'Who's who' in two different flower types of Calluna vulgaris (Ericaceae): morphological and molecular analyses of flower organ identity.

BACKGROUND: The ornamental crop Calluna vulgaris is of increasing importance to the horticultural industry in the northern hemisphere due to a flower organ mutation: the flowers of the 'bud-flowering' phenotype remain closed i.e. as buds throughout the total flowering period and thereby maintain more colorful flowers for a longer period of time than the wild-type. This feature is accompanied and presumably caused by the complete lack of stamens. Descriptions of this botanical particularity are inconsistent and partially conflicting. In order to clarify basic questions of flower organ identity in general and stamen loss in detail, a study of the wild-type and the 'bud-flowering' flower type of C. vulgaris was initiated. RESULTS: Flowers were examined by macro- and microscopic techniques. Organ development was investigated comparatively in both the wild-type and the 'bud-flowering' type by histological analyses. Analysis of epidermal cell surface structure of vegetative tissues and perianth organs using scanning electron microscopy revealed that in wild-type flowers the outer whorls of colored organs may be identified as sepals, while the inner ones may be identified as petals. In the 'bud-flowering' type, two whorls of sepals are directly followed by the gynoecium. Both, petals and stamens, are completely missing in this flower type. The uppermost whorl of green leaves represents bracts in both flower types. In addition, two MADS-box genes (homologs of AP3/DEF and SEP1/2) were identified in C. vulgaris using RACE-PCR. Expression analysis by qRT-PCR was conducted for both genes in leaves, bracts, sepals and petals. These experiments revealed an expression pattern supporting the organ classification based on morphological characteristics. CONCLUSIONS: Organ identity in both wild-type and 'bud-flowering' C. vulgaris was clarified using a combination of microscopic and molecular methods. Our results for bract, sepal and petal organ identity are supported by the 'ABCDE model'. However, loss of stamens in the 'bud-flowering' phenotype is an exceptional flower organ modification that cannot be explained by modified spatial expression of known organ identity genes.

Implementation of a model for identifying Essentially Derived Varieties in vegetatively propagated Calluna vulgaris varieties.

BACKGROUND: Variety protection is of high relevance for the horticultural community and juridical cases have become more frequent in a globalized economy due to essential derivation of varieties. This applies equally to Calluna vulgaris, a vegetatively propagated species from the Ericaceae family that belongs to the top-selling pot plants in Europe. We therefore analyzed the genetic diversity of 74 selected varieties and genotypes of C. vulgaris and 3 of Erica spp. by means of RAPD and iSSR fingerprinting using 168 mono- and polymorphisms. The same data set was utilized to generate a system to reliably identify Essentially Derived Varieties (EDVs) in C. vulgaris, which was adapted from a method suggested for lettuce and barley. This system was developed, validated and used for selected tests of interest in C. vulgaris. RESULTS: As expected following personal communications with breeders, a very small genetic diversity became evident within C. vulgaris when investigated using our molecular methods. Thus, a dendrogram-based assay to detect Essentially Derived Varieties in this species is not suitable, although varieties are propagated vegetatively. In contrast, the system applied in lettuce, which itself applies pairwise comparisons using appropriate reference sets, proved functional with this species. CONCLUSION: The narrow gene pool detected in C. vulgaris may be the genetic basis for juridical conflicts between breeders. We successfully tested a methodology for identification of Essentially Derived Varieties in highly identical C. vulgaris genotypes and recommend this for future proof of essential derivation in C. vulgaris and other vegetatively propagated crops.

Retrospective biomonitoring: a hundred years of environmental pollution at selected areas in Slovakia.

Tetrade analyses of Calluna vulgaris from herbarium specimens showed significant differences in the frequency of aborted pollen tetrads over the last 100 years within various parts of Slovakia. Specifically, we observed changes in the dynamics of pollution peaking in the year 1965 in heavily polluted industrial area of an aluminium factory in Ziar nad Hronom.