Use of MSAP markers to analyse the effects of salt stress on DNA methylation in rapeseed (Brassica napus var. oleifera).

Excessive soil salinity is a major ecological and agronomical problem, the adverse effects of which are becoming a serious issue in regions where saline water is used for irrigation. Plants can employ regulatory strategies, such as DNA methylation, to enable relatively rapid adaptation to new conditions. In this regard, cytosine methylation might play an integral role in the regulation of gene expression at both the transcriptional and post-transcriptional levels. Rapeseed, which is the most important oilseed crop in Europe, is classified as being tolerant of salinity, although cultivars can vary substantially in their levels of tolerance. In this study, the Methylation Sensitive Amplified Polymorphism (MSAP) approach was used to assess the extent of cytosine methylation under salinity stress in salinity-tolerant (Exagone) and salinity-sensitive (Toccata) rapeseed cultivars. Our data show that salinity affected the level of DNA methylation. In particular methylation decreased in Exagone and increased in Toccata. Nineteen DNA fragments showing polymorphisms related to differences in methylation were sequenced. In particular, two of these were highly similar to genes involved in stress responses (Lacerata and trehalose-6-phosphatase synthase S4) and were chosen to further characterization. Bisulfite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied. In particular, our data show that salinity stress influences the expression of the two stress-related genes. Moreover, we quantified the level of trehalose in Exagone shoots and found that it was correlated to TPS4 expression and, therefore, to DNA methylation. In conclusion, we found that salinity could induce genome-wide changes in DNA methylation status, and that these changes, when averaged across different genotypes and developmental stages, accounted for 16.8% of the total site-specific methylation differences in the rapeseed genome, as detected by MSAP analysis.

Comparative cytogenetic study on Trifolium subterraneum (2n = 16) and Trifolium israeliticum (2n = 12).

Changes in chromosome number have played an important role in the evolution of the genus Trifolium. Along with a few species of polyploid origin there are several cases of dysploidy as evidenced by the presence of four basic chromosome numbers (x = 8, 7, 6, 5). Trifolium subterraneum and Trifolium israeliticum are related species with chromosome complements 2n = 16 and 2n = 12, respectively. Although they represent an interesting case of speciation based on chromosome number reduction, no attempts to demonstrate their cytogenetic affinity have been carried out to date. With this study we performed a comparative cytogenetic study with the purpose of clarifying the evolutionary relationship between these species and to verify whether genomic rearrangements, other than modification of the chromosome number, are associated with the speciation process. Although karyomorphological analysis supports the hypothesis that chromosome rearrangements had a role in the reduction of the chromosome number, the physical mapping of the rDNA sequences revealed a significant remodelling of the 45S and 5S rDNA sites that greatly contributed to the differentiation of the 2n = 16 and 2n = 12 karyotypes. The nucleotide analysis of 5S rDNA repeats confirmed that the two species are related but constitute distinct entities. The observed genomic changes lead to the hypothesis that the 2n = 12 species is the result of an evolutionary pathway that passed through intermediate forms. It cannot be excluded that the most direct ancestor of T. israeliticum is a species with 2n = 14.

In planta expression of a mature Der p 1 allergen isolated from an Italian strain of Dermatophagoides pteronyssinus.

European (Dermatophagoides pteronyssinus) and American (Dermatophagoides farinae) house dust mite species are considered the most common causes of asthma and allergic symptoms worldwide. Der p 1 protein, one of the main allergens of D. pteronyssinus, is found in high concentration in mites faecal pellets, which can became easily airborne and, when inhaled, can cause perennial rhinitis and bronchial asthma. Here we report the isolation of the Der p 1 gene from an Italian strain of D. pteronyssinus and the PVX-mediated expression of its mature form (I-rDer p 1) in Nicotiana benthamiana plants. Human sera from characterized allergic patients were used for IgE binding inhibition assays to test the immunological reactivity of I-rDer p 1 produced in N. benthamiana plants. The binding properties of in planta produced I-rDer p 1 versus the IgE of patients sera were comparable to those obtained on Der p 1 preparation immobilized on a microarray. In this paper we provide a proof of concept for the production of an immunologically active form of Der p 1 using a plant viral vector. These results pave the way for the development of diagnostic allergy tests based on in planta produced allergens.

Ectomycorrhizal communities in a productive Tuber aestivum Vittad. orchard: composition, host influence and species replacement.

Truffles (Tuber spp.) and other ectomycorrhizal species form species-rich assemblages in the wild as well as in cultivated ecosystems. We aimed to investigate the ectomycorrhizal communities of hazels and hornbeams that are growing in a 24-year-old Tuber aestivum orchard. We demonstrated that the ectomycorrhizal communities included numerous species and were phylogenetically diverse. Twenty-nine ectomycorrhizal taxa were identified. Tuber aestivum ectomycorrhizae were abundant (9.3%), only those of Tricholoma scalpturatum were more so (21.4%), and were detected in both plant symbionts with a variation in distribution and abundance between the two different hosts. The Thelephoraceae family was the most diverse, being represented by 12 taxa. The overall observed diversity represented 85% of the potential one as determined by a jackknife estimation of richness and was significantly higher in hazel than in hornbeam. The ectomycorrhizal communities of hornbeam trees were closely related phylogenetically, whereas no clear distribution pattern was observed for the communities in hazel. Uniform site characteristics indicated that ectomycorrhizal relationships were host mediated, but not host specific. Despite the fact that different plant species hosted diverse ectomycorrhizal communities and that the abundance of T. aestivum differed among sites, no difference was detected in the production of fruiting bodies.

Tracing the biological origin of animal glues used in paintings through mitochondrial DNA analysis.

We report the development of a suitable protocol for the identification of the biological origin of binding media on tiny samples from ancient paintings, by exploitation of the high specificity and high sensitivity offered by the state-of-the art DNA analysis. In particular, our aim was to molecularly characterize mitochondrial regions of the animal species traditionally employed for obtaining glues. The model has been developed using aged painting models and then tested to analyze the organic components in samples from the polychrome terracotta Madonna of Citerna by Donatello (1415-1420), where, by GC-MS and FTIR spectroscopy, animal glues and siccative oils were identified. The results obtained are good in terms of both sensibility and specificity of the method. First of all, it was possible to confirm that Donatello used animal glue for the preparation of the painted layers of the Madonna of Citerna and, specifically, glue derived from Bos taurus. Data obtained from sequencing confirm that each sample contains animal glue, revealing that it was mostly prepared from two common European taurine lineages called T2 and T3. There is one remarkable exception represented by one sample which falls into a surviving lineage of the now extinct European aurochs.

SERK and APOSTART. Candidate genes for apomixis in Poa pratensis.

Seed production generally requires the mating of opposite sex gametes. Apomixis, an asexual mode of reproduction, avoids both meiotic reduction and egg fertilization. The essential feature of apomixis is that an embryo is formed autonomously by parthenogenesis from an unreduced egg of an embryo sac generated through apomeiosis. If apomixis were well understood and harnessed, it could be exploited to indefinitely propagate superior hybrids or specific genotypes bearing complex gene sets. A more profound knowledge of the mechanisms that regulate reproductive events would contribute fundamentally to understanding the genetic control of the apomictic pathway. In Poa pratensis, we isolated and characterized two genes, PpSERK (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE) and APOSTART. These full-length genes were recovered by rapid amplification of cDNA ends and their temporal and spatial expression patterns were assessed by reverse transcription-polymerase chain reaction and in situ hybridization, respectively. The expression of PpSERK and APOSTART differed in apomictic and sexual genotypes. Their putative role in cell-signaling transduction cascades and trafficking events required during sporogenesis, gametogenesis, and embryogenesis in plants is reported and discussed. We propose that, in nucellar cells of apomictic genotypes, PpSERK is the switch that channels embryo sac development and that it may also redirect signaling gene products to compartments other than their typical ones. The involvement of APOSTART in meiosis and programmed cell death is also discussed.

Isolation of candidate genes for apomixis in Poa pratensis L.

The essential feature of apomixis is that an embryo is formed autonomously by parthenogenesis from an unreduced egg of an embryo sac generated through apomeiosis. The genetic constitution of the offspring is, therefore, usually identical to the maternal parent, a trait of great interest to plant breeders. If apomixis were well understood and harnessed, it could be exploited to indefinitely propagate superior hybrids or specific genotypes bearing complex gene sets. A fundamental contribution to the understanding of the genetic control of the apomictic pathway could be provided by a deep knowledge of molecular mechanisms that regulate the reproductive events. In Poa pratensis the cDNA-AFLP method of mRNA profiling allowed us to visualize a total of 2248 transcript-derived fragments and to isolate 179 sequences that differed qualitatively or quantitatively between apomictic and sexual genotypes at the time of flowering when the primary stages of apomixis occur. Three ESTs were chosen for further molecular characterization because of their cDNA-AFLP expression pattern and BLAST information retrieval. The full-lengths of the newly isolated genes were recovered by RACE and their temporal expression patterns were assessed by RT-PCR. Their putative role in cell signaling transduction cascades and trafficking events required during sporogenesis, gametogenesis and embryogenesis in plants is reported and discussed.

Microsatellite-AFLP for genetic mapping of complex polyploids.

In spite of the economical relevance of polyploid crops, genetic mapping of these species has been relatively overlooked. This is because of intrinsic difficulties such as the uncertainty of the chromosome behavior at meiosis I and the need for very large segregating populations. An important, yet underestimated issue, in mapping polyploids is the choice of the molecular marker system. An ideal molecular marker system for polyploid mapping should maximize the percentage of single dose markers (SDMs) detected and the possibility of recognizing allelic markers. In the present work, the marker index for genetic mapping (MIgm) of M-AFLP is compared with that of AFLP and SAMPL. M-AFLPs have the highest MIgm values (22 vs. 18.5 of SAMPL and 9.83 of AFLP) mostly because of their high power to detect polymorphism. Owing to their prevalent codominant inheritance, it is proposed that M-AFLP can be used for the preliminary identification of hom(e)ologous groups.