Gianpiero Marconi

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.

Development of S-SAP markers based on an LTR-like sequence from Medicago sativa L.

Abstract. The Sequence-Specific Amplification Polymorphism (S-SAP) method, recently derived from the Amplified Fragment Length Polymorphism (AFLP) technique, produces amplified fragments containing a retrotransposon LTR sequence at one end and a host restriction site at the other. We report the application of this procedure to the LTR of the Tms1 element from Medicago sativa L. Genomic dot-blot analysis indicated that Tms1 LTRs represent about 0.056% of the M. sativa genome, corresponding to 16×103 copies per haploid genome. An average of 66 markers were amplified for each primer combination. Overall 49 polymorphic fragments were reliably scored and mapped in a F1 population obtained by crossing diploid M. falcata with M. coerulea. The utility of the LTR S-SAP markers was higher than that of AFLP or SAMPL (Selective Amplification of Microsatellite Polymorphic Loci) markers. The efficiency index of the LTR S-SAP assay was 28.3, whereas the corresponding values for AFLP and SAMPL markers were 21.1 and 16.7, respectively. The marker index for S-SAP was 13.1, compared to 8.8 for AFLP and 9.5 for SAMPL. Application of the Tms1 LTR-based S-SAP to double-stranded cDNA resulted in a complex banding pattern, demonstrating the presence of Tms1 LTRs within exons. As the technique was successfully applied to other species of the genus Medicago, it should prove suitable for studying genetic diversity within, and relatedness between, alfalfa species.

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.

In planta production of two peptides of the Classical Swine Fever Virus (CSFV) E2 glycoprotein fused to the coat protein of potato virus X

BackgroundClassical Swine Fever (CSFV) is one of the most important viral infectious diseases affecting wild boars and domestic pigs. The etiological agent of the disease is the CSF virus, a single stranded RNA virus belonging to the family Flaviviridae.All preventive measures in domestic pigs have been focused in interrupting the chain of infection and in avoiding the spread of CSFV within wild boars as well as interrupting transmission from wild boars to domestic pigs. The use of plant based vaccine against CSFV would be advantageous as plant organs can be distributed without the need of particular treatments such as refrigeration and therefore large areas, populated by wild animals, could be easily covered.ResultsWe report the in planta production of peptides of the classical swine fever (CSF) E2 glycoprotein fused to the coat protein of potato virus X. RT-PCR studies demonstrated that the peptide encoding sequences are correctly retained in the PVX construct after three sequential passage in Nicotiana benthamiana plants. Sequence analysis of RT-PCR products confirmed that the epitope coding sequences are replicated with high fidelity during PVX infection. Partially purified virions were able to induce an immune response in rabbits.ConclusionPrevious reports have demonstrated that E2 synthetic peptides can efficiently induce an immunoprotective response in immunogenized animals. In this work we have showed that E2 peptides can be expressed in planta by using a modified PVX vector. These results are particularly promising for designing strategies for disease containment in areas inhabited by wild boars.

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.

Structure of genetic diversity in Olea europaea L. cultivars from central Italy

The olive is considered one of the most important fruit crops of the Mediterranean basin where it shows a wide range of variability, with about 2,000 cultivars. Italy, with about 500 cultivars, plays a fundamental role. The ability to discriminate olive cultivars and estimate genetic variability are important factors in better management of genetic resources and in helping to understand how genetic diversity is partitioned among cultivars. The two main objectives of the present investigation were to evaluate the identity of cultivars grown in Abruzzo region, central Italy, and to study their genetic structure. We applied amplified fragment length polymorphism (AFLP) methodology on 84 genotypes belonging to the most relevant and oldest varieties cultivated in Abruzzo and on six unknown genotypes. The information content of data was evaluated using the Marker Ratio index and the Polymorphic Index Content. Moreover, STRUCTURE software was used to investigate the genetic population structure. The analysis enabled us to clearly distinguish eight cultivars within seven clusters. Additionally, one cluster was found to have various minor cultivars and showed a relatively high level of diversity. The partitioning of genetic diversity showed that the largest amount of molecular variance was within groups. Our data suggest that both sexual and clonal propagation have played an important role in the evolution of olive cultivars. In our hypothesis, some ancestral population spread in central Italy with a relevant role of seed propagation, followed by a selection of superior clones from which more traditional varieties originated. In a few cases, hybridization should be taken into consideration to explain the diffusion of recently developed cultivars.

Exposure to cadmium-contaminated soils increases allergenicity of Poa annua L. pollen.

To cite this article: Aina R, Asero R, Ghiani A, Marconi G, Albertini E, Citterio S. Exposure to cadmium‐contaminated soils increases allergenicity of Poa annua L. pollen. Allergy 2010; 65: 1313–1321.

Alfalfa Mob1-like Genes are Expressed in Reproductive Organs during Meiosis and Gametogenesis

Mps-one-binder (Mob) proteins play an important role in chromosome separation and cell plate formation in yeast. We cloned two Mob1-like genes from alfalfa (Medicago sativa L.) and show that one gene is constitutively expressed while the other is expressed only in flower buds during sporogenesis and gametogenesis. For the analysis of gene expression during reproduction in alfalfa wild-types and apomeiotic mutants, a specific antisense riboprobe was designed for MsMob1 transcripts and a polyclonal antibody was raised against MsMob1 proteins. In situ mRNA localization as well as protein immunolocalization proved that MsMob1-like genes are specifically expressed in degenerating megaspores of normal ovules and in enlarged megaspore mother cells and embryo sacs of apomeiotic ovules. Gene products were also found in microspore tetrads at the beginning of pollen development as well as in tapetum cells of anthers undergoing programmed cell death to allow pollen dispersal at maturity. Overall results suggest that MsMob1-like genes can play a key role during the reproductive pathway in plants.

Agronomic, chemical and genetic variability of saffron (Crocus sativus L.) of different origin by LC-UV–vis-DAD and AFLP analyses

The identification of a bi-univocal correspondence between geographical origin of saffron (Crocus sativus L.) and the composition of its stigmas has recently been the subject of many research papers, which have focused on the analysis of the differences among the so called “minor components”, such as flavonoids and volatiles, in the secondary metabolic pattern of this spice. Saffron pigments (crocetin esters), on the other hand, constitute the majority of the metabolites found in its stigmas, and their spectrophotometric measurement is still used as an official method to determine the quality of the spice in terms of coloring power. To our knowledge, no attempts have been made to find a correspondence between the geographical origin of different saffron samples and their morphological traits and pigments pattern. In this paper, we have demonstrated that saffron corms of different origins, grown in the same experimental field, produce daughter corms with different dimensions and still produce stigma samples with different pigment profiles. Furthermore, daughter corm dimensions and pigment profile even more so, may be related to the origin of the sample, and therefore pigments can be used as chemotaxonomic markers. Compositional analyses results were corroborated by genetic data obtained using AFLP molecular markers.

Understanding Genetic Diversity and Population Structure of a Poa pratensis Worldwide Collection through Morphological, Nuclear and Chloroplast Diversity Analysis

Poa pratensis L. is a forage and turf grass species well adapted to a wide range of mesic to moist habitats. Due to its genome complexity little is known regarding evolution, genome composition and intraspecific phylogenetic relationships of this species. In the present study we investigated the morphological and genetic diversity of 33 P. pratensis accessions from 23 different countries using both nuclear and chloroplast molecular markers as well as flow cytometry of somatic tissues. This with the aim of shedding light on the genetic diversity and phylogenetic relationships of the collection that includes both cultivated and wild materials. Morphological characterization showed that the most relevant traits able to distinguish cultivated from wild forms were spring growth habit and leaf colour. The genome size analysis revealed high variability both within and between accessions in both wild and cultivated materials. The sequence analysis of the trnL-F chloroplast region revealed a low polymorphism level that could be the result of the complex mode of reproduction of this species. In addition, a strong reduction of chloroplast SSR variability was detected in cultivated materials, where only two alleles were conserved out of the four present in wild accessions. Contrarily, at nuclear level, high variability exist in the collection where the analysis of 11 SSR loci allowed the detection of a total of 91 different alleles. A Bayesian analysis performed on nuclear SSR data revealed that studied materials belong to two main clusters. While wild materials are equally represented in both clusters, the domesticated forms are mostly belonging to cluster P2 which is characterized by lower genetic diversity compared to the cluster P1. In the Neighbour Joining tree no clear distinction was found between accessions with the exception of those from China and Mongolia that were clearly separated from all the others.