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The present study aimed to characterize the genetic structure of Artemisia eriantha Ten. and the diversity of the rhizosphere microbiota. Plant leaves and rhizosphere soils were sampled from three areas of Central Italy, namely Monte Corvo, Monte Portella (both from the Gran Sasso massif), and Monte Focalone (Majella massif). The plant samples were subjected to genetic structure analysis by amplified fragment length polymorphism (AFLP) markers. The microbiota from the rhizosphere soils was investigated by 16S rRNA gene metabarcoding. The within and among population variability was typical of outbreeding species. The AFLP polymorphisms revealed a marked closeness among plant populations collected in Monte Focalone and Monte Corvo, despite the geographical proximity of the latter with Monte Portella, a result confirmed by cluster, STRUCTURE, and discriminant analyses. 16S rRNA gene metabarcoding showed higher values of diversity for Monte Corvo (H, 5.7; Chao1, 445) and Monte Focalone (H′, 5.57; Chao1, 446) than Monte Portella (H′, 5.3; Chao1, 275). At the phylum level, the communities were mainly represented by Proteobacteria, Actinobacteria, and Acidobacteria (>10%). At the genus level, the Monte Focalone and Monte Corvo microbiotas were closer than Monte Portella, thus confirming the results from the plant communities. The findings provided evidence for the first time of an association between the Artemisia eriantha plant and microbiota communities. The relevance of the results in terms of biodiversity and the conservation strategies of plant and microbiota communities in the Central Apennines are discussed. [ABSTRACT FROM AUTHOR]

The article focuses on the advances in the cytogenetics of Annonaceae, the case of Annona cherimola. Topics inlcude the chromosome structure of A. cherimola has obtained by the self-GISH procedure, the repeated DNA sequences has localized in the centromeric regions of all chromosomes, and the s-GISH signals with DAPI banding suggests that these sequences are the principal component of the centromeric heterochromatin of this species.

In the last 50 years, intensive farming systems have been boosted by modern agricultural techniques and newly bred cultivars. The massive use of few and related cultivars has dramatically reduced the apple genetic diversity of local varieties, confined to marginal areas. In Central Italy a limited spread of intensive fruit orchards has made it possible to preserve much of the local genetic diversity, but at the same time the coexistence of both modern and ancient varieties has generated some confusion. The characterization and clarification of possible synonyms, homonyms, and/or labeling errors in old local genetic resources is an issue in the conservation and management of living collections. 175 accessions provided by 10 apple collections, mainly local varieties, some of unknown origin, and well-known modern and ancient varieties, were studied by using 19 SSRs, analyzed by STRUCTURE, Ward's clustering and parentage analysis. We were able to identify 25 duplicates, 9 synonyms, and 9 homonyms. As many as 37 unknown accession were assigned to well known local or commercial varieties. Polyploids made up 20%. Some markers were found to be significantly correlated with morphological traits and the loci associated with the fruit over color were related to QTLs for resistance to biotic stresses, aroma compounds, stiffness, and acidity. In conclusion the gene pool of Central Italy seems to be rather consistent and highly differentiated compared with other European studies (F ST = 0.147). The importance of safeguarding this diversity and the impact on the management of the germplasm living collection is discussed. [ABSTRACT FROM AUTHOR]

Saffron is one of the most highly priced agricultural products in the world. Its prize can reach 20,000 €/kg retail and this is due both to the high demand and to the great cultivation and production costs. Several studies have demonstrated that differences in saffron quality are mainly due to the methodology followed in the processing of stigmas, and environmental conditions independent of the origin. Some authors found phenotypic variations within cultivated saffron, but very limited genetic diversity. The reason for the very limited genetic diversity in cultivated saffron is explained by its asexual mode of reproduction (propagation). The aim of this study was to assess the antimicrobial activity of stigma saffron, which were tested against different bacteria strains. The results obtained from the antimicrobial activity study indicate that stigmas of C. sativus have some antimicrobial effect. [ABSTRACT FROM AUTHOR]

The molecular mechanisms of transferred DNA (T-DNA) integration into the plant genome are still not completely understood. A large number of integration events have been analyzed in different species, shedding light on the molecular mechanisms involved, and on the frequent transfer of vector sequences outside the T-DNA borders, the so-called vector backbone (VB) sequences. In this work, we characterized 46 transgenic alfalfa (Medicago sativa L.) plants (events), generated in previous works, for the presence of VB tracts, and sequenced several T-DNA/genomic DNA (gDNA) junctions. We observed that about 29% of the transgenic events contained VB sequences, within the range reported in other species. Sequence analysis of the T-DNA/gDNA junctions evidenced larger deletions at LBs compared to RBs and insertions probably originated by different integration mechanisms. Overall, our findings in alfalfa are consistent with those in other plant species. This work extends the knowledge on the molecular events of T-DNA integration and can help to design better transformation protocols for alfalfa. [ABSTRACT FROM AUTHOR]

Pear is one of the oldest fruit tree crops and the third most important temperate fruit species. Its domestication took place independently in the Far East (China) and in the Caucasus region. While the origin of Eastern Asian cultivars is clear, that of European cultivars is still in doubt. Italy has a wealth of local varieties and genetic resources safeguarded by several public and private collections to face the erosion caused by the introduction of improved varieties in specialized orchards. The objectives of the present study were: (i) to characterize the existing germplasm through nuclear (SSR) and (ii) to clarify the genetic divergence between local and cultivated populations through chloroplast DNA (cpDNA) markers in order to provide insights into phylogenetic relationships of Pyrus spp. For this reason, 95 entries from five different germplasm collections, including nine European, Mediterranean and Eastern Asian species, were analyzed, and the intergenic accD-psaI sequences were compared to the worldwide distributed dataset encompassing a total of 298 sequences from 26 different Pyrus species. The nine nuclear SSRs were able to identify a total of 179 alleles, with a loci polymorphism P = 0.89. Most of the variation (97%) was found within groups. Five accessions from different sources were confirmed to be the same. Eight out of 20 accessions of unknown origin were identified, and six synonyms were detected. Locus NH030a was found to be monomorphic in all the cultivated accessions and in reference species interfertile with P. communis, leading to hypothesize selection pressures for adaptation to cultivation. The cpDNA sequences of the 95 accessions were represented by 14 haplotypes, six of which (derived from P. communis, P. cossonii and P. ussuriensis) are recorded here for the first time and may suggest the ancient origin of some local varieties. The network analysis of the 298 cpDNA sequences allowed two different haplogroups, Eastern and Western Eurasia, to be defined, supporting recent views of a clear division between Occidental and Oriental species. By combining the results from nuclear and uniparental markers, it was possible to better define many unknown accessions. [ABSTRACT FROM AUTHOR]

Key message: A mutant glutamate 1-semialdehyde aminotransferase gene from the Synechococcus , inserted into tobacco plastid DNA by means of particle bombardment and antibiotic selection, conferred gabaculine resistance allowing to attain homoplasmy. Abstract: Many plant species are recalcitrant to plastid genome transformation. New selections systems may help to overcome this limitation and to extend the application of this technology. A mutant hemL gene from the photosynthetic cyanobacterium Synechococcus, encoding a gabaculine-insensitive glutamate 1-semialdehyde aminotransferase (GSA), is an efficient selectable marker gene for nuclear transformation of tobacco, alfalfa and durum wheat. Since GSA functions in the plastid, we introduced the mutant hemL gene into the tobacco plastid genome along with the conventional antibiotic resistance aadA gene, in the attempt to develop a new selection system for plastome transformation. Although we were unable to directly regenerate gabaculine resistant transplastomic plants, we demonstrated the functionality of hemL in tobacco plastids by using gabaculine selection in the second and third rounds of in vitro selection that permitted to obtain the homoplasmic state in transgenic plants. Thus, the mutant hemL gene functions as a secondary selection marker in tobacco plastids. Our results encourage further attempts to test gabaculine resistant GSA for plastome transformation of crop plants in which gabaculine has stronger regeneration-inhibiting effects with respect to tobacco. [ABSTRACT FROM AUTHOR]

Heat shock proteins (HSPs) are molecular chaperones involved in many cellular functions. It has been shown that mammalian cytosolic HSP70 binds antigenic peptides mediating the activation of the immune system, and that it plays a determining role in tumour immunogenicity. This suggests that HSP70 may be used for the production of conjugated vaccines. Human and plant HSPs share high sequence similarity and some important biological functions in vitro. In addition, plant HSPs have no endotoxic side effects. Extraction of HSP70 from plants for use as vaccine adjuvant requires enhancing its concentration in plant tissues. In this work, we explored the possibility to produce HSP70 in both transgenic and non-transgenic plants, using alfalfa as a model species. First, a transcriptional analysis of a constitutive and an inducible HSP70 genes was conducted in Arabidopsis thaliana. Then the coding sequence of the inducible form was cloned and introduced into alfalfa by Agrobacterium-mediated transformation, and the accumulation of the protein in leaf tissue of transgenic plants was demonstrated. We also tested diverse alfalfa varieties for heat-inducible expression of endogenous HSP70, revealing variety-specific responses to heat shock. [ABSTRACT FROM AUTHOR]

Transferring a transgenic trait from a lab genotype into a cultivated plant variety requires selection of single-locus T0 plants, a backcrossing breeding program with transgenic progeny selection at each cycle and, generally, attaining the homozygous state for the transgene locus. Polyploidy and outcrossing complicate this process, as in the case of alfalfa, an autotetraploid, cross-fertilizing forage legume. Recently, a new, efficient selectable marker gene, MsGSAgr, derived from the alfalfa glutamate 1-semialdehyde aminotransferase (GSA) gene was described. Since MsGSAgr differs from the wild-type GSA by one nucleotide, high resolution melting (HRM) analysis could be used to screen transgenic plants for MsGSAgr, and linked transgene(s), copy number. An HRM assay was developed by simulating different copy numbers with mixes of plasmids containing mutated and wild-type GSA sequences. The assay was validated by analyzing transgenic alfalfa plants containing one or multiple MsGSAgr loci. HRM enabled us to clearly discriminate transgenic from non-transgenic plants, and the single copy from the multicopy state, thus providing a tool to streamline molecular plant breeding. [ABSTRACT FROM AUTHOR]

Landraces are heterogeneous plant varieties that are reproduced by farmers as populations that are subject to both artificial and natural selection. Landraces are distinguished by farmers due to their specific traits, and different farmers often grow different populations of the same landrace. We used simple sequence repeats (SSRs) to analyse 12 barley landrace populations from Sardinia from two collections spanning 10 years. We analysed the population structure, and compared the population diversity of the landraces that were collected at field level (population). We used a representative pool of barley varieties for diversity comparisons and to analyse the effects of gene flow from modern varieties. We found that the Sardinian landraces are a distinct gene pool from those of both two-row and six-row barley varieties. There is also a low, but significant, mean level and population-dependent level of introgression from the modern varieties into the Sardinian landraces. Moreover, we show that the Sardinian landraces have the same level of gene diversity as the representative sample of modern commercial varieties grown in Italy in the last decades, even within population level. Thus, these populations represent crucial sources of germplasm that will be useful for crop improvement and for population genomics studies and association mapping, to identify genes, loci and genome regions responsible for adaptive variations. Our data also suggest that landraces are a source of valuable germplasm for sustainable agriculture in the context of future climate change, and that in-situ conservation strategies based on farmer use can preserve the genetic identity of landraces while allowing adaptation to local environments. [ABSTRACT FROM AUTHOR]

In the world lentil is grown on more than 3 million hectares and is one of the most important, low-cost, food source of protein. In Italy lentil has been cultivated since ancient times, but in the last decades its cultivation has been confined to marginal areas, small islands and hilly, mountainous areas of central and southern Italy. Local varieties are still common and are often greatly appreciated for their taste and cooking qualities. Several accessions from the Santo Stefano di Sessanio area, Abruzzo Region, were collected and phenotypically and genotypically characterized in order to look for the existing variability within and between populations. Image analysis of seeds was also used. Populations grown in Santo Stefano di Sessanio and in the neighbouring area basically share most of their characteristics. However, some of the accessions anonymously gathered from the local market were shown to be different from those collected from farmers. The paper reports and discusses how this local product needs be characterized and promoted in order to avoid fraud that could negatively affect the local economy and put valuable, adapted, genetic resources at risk of erosion. [ABSTRACT FROM AUTHOR]

Methods to avoid the presence of selectable marker genes (SMG) in transgenic plants are available but not implemented in many crop species. We assessed the efficiency of simple marker-free Agrobacterium-mediated transformation techniques in alfalfa: regeneration without selection, or marker-less, and co-transformation with two vectors, one containing the SMG and one containing a non-selected gene. To easily estimate the efficiency of marker-less transformation, the nptII and the GUS markers were used as non-selected genes. After Agrobacterium treatment, somatic embryos were regenerated without selection. The percentage of transgenic embryos was determined by a second cycle of regeneration using the embryos as starting material, in the presence of kanamycin, by PCR screening of T1 progenies, and by the GUS test. In two experiments, from 0 to 1.7% of the somatic embryos were transgenic. Co-transformation was performed with two vectors, one with the hemL SMG and one with the unselected nptII gene, each carried by a different culture of Agrobacterium. Only 15 putative co-transformed plants were regenerated from two experiments, with an average co-transformation percentage of 3.7. Southern blot hybridizations and/or T progeny segregation were used to confirm transgene integration, and qPCR was also used to estimate the T-DNA copy number. In the T progenies obtained by crossing with a non-transgenic pollinator, marker-free segregants were obtained. Both marker-free approaches showed very low efficiency. [ABSTRACT FROM AUTHOR]

Parenteral and oral administration of autoantigens can induce immune tolerance in autoimmune diseases. Prophylactic therapy based on oral administration of human autoantigens is not, however, feasible when sufficient quantities of candidate autoantigens are not available. Transgenic plants that express high levels of recombinant proteins would allow large quantities of autoantigens to be produced at relatively low costs. In addition, transgenic food would provide a simple and direct method of delivering autoantigens. The production and the characterization of transgenic tobacco and carrot plants expressing human GAD65, a major autoantigen in human insulin-dependent diabetes mellitus (IDDM), is reported. Immunogold labeling and electron microscopy of transgenic tobacco tissue shows the selective targeting of human GAD65 to chloroplast tylacoids and mitochondria. In planta expressed GAD65 has a correct immunoreactivity with IDDM-associated autoantibodies and retains enzymatic activity, a finding that suggests a correct protein folding. In transgenic tobacco and carrot the expression levels of human GAD65 varies between 0.01% and 0.04% of total soluble proteins. Transgenic edible plant organs are now available to study the feasibility of inducing immune tolerance in IDDM animals by oral administration of GAD65. [ABSTRACT FROM AUTHOR]

Fennel (Foeniculum vulgare) is a species belonging to the Apiaceae family, well known for its nutritional and pharmacological properties. Despite the economic and agricultural relevance, its genomic and transcriptomic data remain poor. Microsatellites—also known as simple sequence repeats (SSRs)—are codominant markers widely used to perform cross-amplification tests starting from markers developed in related species. SSRs represent a powerful tool, especially for those species lacking genomic information. In this study, a set of primers previously designed in Daucus carota for polymorphic SSR loci was tested in commercial varieties and breeding lines of fennel in order to: (i) test their cross-genera transferability, (ii) look at their efficiency in assessing genetic diversity, and (iii) identify their usefulness for marker-assisted selection (MAS) in breeding programs. Thirty-nine SSR markers from carrot were selected and tested for their transferability score, and only 23% of them resulted suitable for fennel. The low rate of SSR transferability between the two species evidences the difficulties of the use of genomic SSR in cross-genera transferability. [ABSTRACT FROM AUTHOR]

"Roveja di Civita di Cascia" is a landrace of Pisum sativum grown in marginal land habitats of the Apennines, Central Italy, and is one of the eleven herbaceous crop landraces listed in the Regional Register of local varieties. The objective of the present paper was to assess its genetic structure using 62 morphological traits and five microsatellites. As many as 55 traits showed significant differences with the control entries (P. sativum subsp. sativum var. arvense and var. sativum). We tested P. sativum Simple Sequence Repeats (SSRs) for their transferability to "Roveja", and found that only 12 out of 35 performed well. Of these, we demonstrated that five were sufficient to assess the genetic structure of this landrace, characterized by several private alleles, differentiating it from Paladio and Bluemoon, which were used as controls. Phenotypic and genotypic data evidenced a genetic structure based on a blend of several pure-bred lines. The sustainability of on-farm landrace conservation is discussed. [ABSTRACT FROM AUTHOR]