Nicola La Porta

The geographical and environmental determinants of genetic diversity for four alpine conifers of the European Alps

Climate is one of the most important drivers of local adaptation in forest tree species. Standing levels of genetic diversity and structure within and among natural populations of forest trees are determined by the interplay between climatic heterogeneity and the balance between selection and gene flow. To investigate this interplay, single nucleotide polymorphisms (SNPs) were genotyped in 24 to 37 populations from four subalpine conifers, Abies alba Mill., Larix decidua Mill., Pinus cembra L. and Pinus mugo Turra, across their natural ranges in the Italian Alps and Apennines. Patterns of population structure were apparent using a Bayesian clustering program, STRUCTURE, which identified three to five genetic groups per species. Geographical correlates with these patterns, however, were only apparent for P. cembra. Multivariate environmental variables [i.e. principal components (PCs)] were subsequently tested for association with SNPs using a Bayesian generalized linear mixed model. The majority of the SNPs, ranging from six in L. decidua to 18 in P. mugo, were associated with PC1, corresponding to winter precipitation and seasonal minimum temperature. In A. alba, four SNPs were associated with PC2, corresponding to the seasonal minimum temperature. Functional annotation of those genes with the orthologs in Arabidopsis revealed several genes involved in abiotic stress response. This study provides a detailed assessment of population structure and its association with environment and geography in four coniferous species in the Italian mountains.

Suppression Substractive Hybridization and NGS Reveal Differential Transcriptome Expression Profiles in Wayfaring Tree (Viburnum lantana L.) Treated with Ozone.

Tropospheric ozone (O3) is a global air pollutant that causes high economic damages by decreasing plant productivity. It enters the leaves through the stomata, generates reactive oxygen species, which subsequent decrease in photosynthesis, plant growth, and biomass accumulation. In order to identify genes that are important for conferring O3 tolerance or sensitivity to plants, a suppression subtractive hybridization analysis was performed on the very sensitive woody shrub, Viburnum lantana, exposed to chronic O3 treatment (60 ppb, 5 h d−1 for 45 consecutive days). Transcript profiling and relative expression assessment were carried out in asymptomatic leaves, after 15 days of O3 exposure. At the end of the experiment symptoms were observed on all treated leaves and plants, with an injured leaf area per plant accounting for 16.7% of the total surface. Cloned genes were sequenced by 454-pyrosequencing and transcript profiling and relative expression assessment were carried out on sequenced reads. A total of 38,800 and 12,495 high quality reads obtained in control and O3-treated libraries, respectively (average length of 319 ± 156.7 and 255 ± 107.4 bp). The Ensembl transcriptome yielded a total of 1241 unigenes with a total sequence length of 389,126 bp and an average length size of 389 bp (guanine-cytosine content = 49.9%). mRNA abundance was measured by reads per kilobase per million and 41 and 37 ensembl unigenes showed up- and down-regulation respectively. Unigenes functionally associated to photosynthesis and carbon utilization were repressed, demonstrating the deleterious effect of O3 exposure. Unigenes functionally associated to heat-shock proteins and glutathione were concurrently induced, suggesting the role of thylakoid-localized proteins and antioxidant-detoxification pathways as an effective strategy for responding to O3. Gene Ontology analysis documented a differential expression of co-regulated transcripts for several functional categories, including specific transcription factors (MYB and WRKY). This study demonstrates that a complex sequence of events takes place in the cells at intracellular and membrane level following O3 exposure and elucidates the effects of this oxidative stress on the transcriptional machinery of the non-model plant species V. lantana, with the final aim to provide the molecular supportive knowledge for the use of this plant as O3-bioindicator.

ChloroMitoSSRDB: Open Source Repository of Perfect and Imperfect Repeats in Organelle Genomes for Evolutionary Genomics

Microsatellites or simple sequence repeats (SSRs) are repetitive stretches of nucleotides (A, T, G, C) that are distributed either as single base pair stretches or as a combination of two- to six-nucleotides units that are non-randomly distributed within coding and in non-coding regions of the genome. ChloroMitoSSRDB is a complete curated web-oriented relational database of perfect and imperfect repeats in organelle genomes. The present version of the database contains perfect and imperfect SSRs of 2161 organelle genomes (1982 mitochondrial and 179 chloroplast genomes). We detected a total of 5838 chloroplast perfect SSRs, 37 297 chloroplast imperfect SSRs, 5898 mitochondrial perfect SSRs and 50 355 mitochondrial imperfect SSRs across these genomes. The repeats have been further hyperlinked to the annotated gene regions (coding or non-coding) and a link to the corresponding gene record in National Center for Biotechnology Information(www.ncbi.nlm.nih.gov/) to identify and understand the positional relationship of the repetitive tracts. ChloroMitoSSRDB is connected to a user-friendly web interface that provides useful information associated with the location of the repeats (coding and non-coding), size of repeat, motif and length polymorphism, etc. ChloroMitoSSRDB will serve as a repository for developing functional markers for molecular phylogenetics, estimating molecular variation across species. Database URL: ChloroMitoSSRDB can be accessed as an open source repository at www.mcr.org.in/chloromitossrdb.

Micro- and macro-geographic scale effect on the molecular imprint of selection and adaptation in Norway spruce

Forest tree species of temperate and boreal regions have undergone a long history of demographic changes and evolutionary adaptations. The main objective of this study was to detect signals of selection in Norway spruce (Picea abies [L.] Karst), at different sampling-scales and to investigate, accounting for population structure, the effect of environment on species genetic diversity. A total of 384 single nucleotide polymorphisms (SNPs) representing 290 genes were genotyped at two geographic scales: across 12 populations distributed along two altitudinal-transects in the Alps (micro-geographic scale), and across 27 populations belonging to the range of Norway spruce in central and south-east Europe (macro-geographic scale). At the macrogeographic scale, principal component analysis combined with Bayesian clustering revealed three major clusters, corresponding to the main areas of southern spruce occurrence, i.e. the Alps, Carpathians, and Hercynia. The populations along the altitudinal transects were not differentiated. To assess the role of selection in structuring genetic variation, we applied a Bayesian and coalescent-based F ST-outlier method and tested for correlations between allele frequencies and climatic variables using regression analyses. At the macro-geographic scale, the F ST-outlier methods detected together 11 F ST-outliers. Six outliers were detected when the same analyses were carried out taking into account the genetic structure. Regression analyses with population structure correction resulted in the identification of two (micro-geographic scale) and 38 SNPs (macro-geographic scale) significantly correlated with temperature and/or precipitation. Six of these loci overlapped with F ST-outliers, among them two loci encoding an enzyme involved in riboflavin biosynthesis and a sucrose synthase. The results of this study indicate a strong relationship between genetic and environmental variation at both geographic scales. It also suggests that an integrative approach combining different outlier detection methods and population sampling at different geographic scales is useful to identify loci potentially involved in adaptation.

Leaf plasticity to light intensity in Italian cypress (Cupressus sempervirens L.): adaptability of a Mediterranean conifer cultivated in the Alps.

Italian cypress (Cupressus sempervirens L.) is native to the eastern Mediterranean, an area characterised by hot, dry summers and mild winters. Over the centuries, however, the species has been introduced into more northerly regions, a long way from its native range. The current, generally warmer climatic conditions brought about by global warming have favoured its cultivation in even more northerly areas in the Alps and other European alpine regions. Given that not only temperature, but also light availability are limiting factors for the spread of cypress in these environments, it is important to ascertain how this species copes with low light conditions. The photosynthetic characteristics of cypress leaves collected from different portions of the crown with contrasting light availability were evaluated by several methods. Chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoid (Car) content was found to be higher in shade leaves than in sun leaves when measured on a fresh mass basis, although enzymatic activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) and nitrate reductase were lower in shade leaves. When the efficiency of PSII was measured by chlorophyll fluorescence, a marked reduction in F(m) was found in shade leaves, while F(o) remained unchanged. The use of exogenous electron donors diphenyl carbazide (DPC) and NH(2)OH actually improved the photosynthetic efficiency of shade leaves, and the same effect was found when PSII electron transport activity was measured as O(2) evolution. Altogether, these results seem to indicate lesser photosynthetic efficiency in shade leaves, probably an impairment on the donor side of the PSII. At the same time, analysis by SDS-PAGE revealed differences in the polypeptide composition of the thylakoid membranes of sun and shade leaves: the bands corresponding to 23 kDa, 28-25 kDa and 33 kDa polypeptides were less intense in the thylakoid membranes extracted from shade leaves. These results were further confirmed by an immunological study showing that the content of the 33 kDa protein, corresponding to the extrinsic PSII protein PsbO, was significantly diminished in shade leaves. The high plasticity of cypress leaves appears to be an advantageous trait in the plants response to variations in environmental conditions, including global change. Implications for the management of this Mediterranean species at the northern edge of its distribution are discussed.

Climate-related adaptive genetic variation and population structure in natural stands of Norway spruce in the South-Eastern Alps

Forest trees dominate many Alpine landscapes that are currently exposed to changing climate. Norway spruce is one of the most important conifer species of the Italian Alps, and natural populations are found across steep environmental gradients with large differences in temperature and moisture availability. This study seeks to determine and quantify patterns of genetic diversity in natural populations toward understanding adaptive responses to changing climate. Across the Italian species range, 24 natural stands were sampled with a major focus on the Eastern Italian Alps. Sampled trees were genotyped for 384 selected single nucleotide polymorphisms (SNPs) from 285 genes. A wide array of potential candidate genes was tested for correlation with climatic parameters. To minimize false-positive association between genotype and climate, population structure was investigated. Pairwise FST estimates between sampled populations ranged between 0.000 and 0.075, with the highest values involving the two disjoint populations, Valdieri, on the western Italian Alps, and Campolino, the most southern population on the Apennines. Despite considerable genetic admixture among populations, both Bayesian and multivariate approach identified four genetic clusters. Selection scans revealed five FST outliers, and the environmental association analysis detected ten SNPs associated to one or more climatic variables. Overall, 13 potentially adaptive loci were identified, three of which have been reported in a previous study on the same species conducted on a broader geographical scale. In our study, precipitation, more than temperature, was often associated with genotype; therefore, it appears as the most important environmental variable associated with the high sensitivity of Norway spruce to soil water supply. These findings provide relevant information for understanding and quantifying climate change effects on this species and its ability to genetically adapt.

ChloroMitoSSRDB 2.00: more genomes, more repeats, unifying SSRs search patterns and on-the-fly repeat detection

Organelle genomes evolve rapidly as compared with nuclear genomes and have been widely used for developing microsatellites or simple sequence repeats (SSRs) markers for delineating phylogenomics. In our previous reports, we have established the largest repository of organelle SSRs, ChloroMitoSSRDB, which provides access to 2161 organelle genomes (1982 mitochondrial and 179 chloroplast genomes) with a total of 5838 perfect chloroplast SSRs, 37 297 imperfect chloroplast SSRs, 5898 perfect mitochondrial SSRs and 50 355 imperfect mitochondrial SSRs across organelle genomes. In the present research, we have updated ChloroMitoSSRDB by systematically analyzing and adding additional 191 chloroplast and 2102 mitochondrial genomes. With the recent update, ChloroMitoSSRDB 2.00 provides access to a total of 4454 organelle genomes displaying a total of 40 653 IMEx Perfect SSRs (11 802 Chloroplast Perfect SSRs and 28 851 Mitochondria Perfect SSRs), 275 981 IMEx Imperfect SSRs (78 972 Chloroplast Imperfect SSRs and 197 009 Mitochondria Imperfect SSRs), 35 250 MISA (MIcroSAtellite identification tool) Perfect SSRs and 3211 MISA Compound SSRs and associated information such as location of the repeats (coding and non-coding), size of repeat, motif and length polymorphism, and primer pairs. Additionally, we have integrated and made available several in silico SSRs mining tools through a unified web-portal for in silico repeat mining for assembled organelle genomes and from next generation sequencing reads. ChloroMitoSSRDB 2.00 allows the end user to perform multiple SSRs searches and easy browsing through the SSRs using two repeat algorithms and provide primer pair information for identified SSRs for evolutionary genomics. Database URL: http://www.mcr.org.in/chloromitossrdb

Cold tolerance in cypress (Cupressus sempervirens L.): a physiological and molecular study

Twenty cypress accessions were tested for freezing tolerance. After freezing to −15°C, differences among cypress accessions were tested by measuring electrolyte leakage and chlorophyll fluorescence. Based on these data, cypress accessions showing contrasting freezing tolerance were subjected to transcript profiling of candidate genes upon the development of cold hardening, with the ultimate goal of providing a scientific basis for selecting/breeding cypress genotypes with higher tolerance to low temperature. Nine different cypress genes were selected: a heat shock protein, a putative chaperonin, a chlorophyll-binding protein, a serine/threonine protein kinase, a putative exonuclease, a dehydrin, and three senescence-associated proteins. Transcript levels of these genes were profiled during cold hardening under controlled conditions using real-time reverse-transcription-polymerase chain reaction. While the genes showed regulation patterns common to both cypress accessions, in the case of chaperonin, exonuclease, and some senescence-associated proteins, clonal differences in gene regulation were found. The potential relationship of these differences with cold tolerance is discussed.

A multi-temporal approach in MaxEnt modelling: A new frontier for land use/land cover change detection

Abstract Land-cover change, a major driver of the distribution and functioning of ecosystems, is characterized by a high diversity of patterns of change across space and time. Thus, a large amount of information is necessary to analyse change and develop plans for proper management of natural resources. In this work we tested MaxEnt algorithm in a completely remote land-cover classification and change analysis. In order to provide an empirical example, we selected south-eastern Italian Alps, manly Trentino-South Tyrol, as test region. We classified two Landsat images (1976 and 2001) in order to forecast probability of occurrence for unsampled locations and to determine the best subset of predictors (spectral bands). A difference map for each land cover class, representing the difference between 1976 and 2001 probability of occurrence values, was built. In order to better address the patterns of change analysis, we put together difference maps and topographic variables. The latter are considered, at least in the study area, as the main environmental drivers of land-use change, in connection with climate change. Our results indicate that the selected algorithm, applied to land cover classes, can provide reliable data, especially when referring to classes with homogeneous texture properties and surface reflectance. The performed models had satisfactory predictive performance, showing relatively clear patterns of difference between the two considered time steps. The development of a methodology that, in the absence of field data, allow to obtain data on land use change dynamics, is of extreme importance for land planning and management.

Xylella fastidiosa: Host Range and Advance in Molecular Identification Techniques

In the never ending struggle against plant pathogenic bacteria, a major goal is the early identification and classification of infecting microorganisms. Xylella fastidiosa, a Gram-negative bacterium belonging to the family Xanthmonadaceae, is no exception as this pathogen showed a broad range of vectors and host plants, many of which may carry the pathogen for a long time without showing any symptom. Till the last years, most of the diseases caused by X. fastidiosa have been reported from North and South America, but recently a widespread infection of olive quick decline syndrome caused by this fastidious pathogen appeared in Apulia (south-eastern Italy), and several cases of X. fastidiosa infection have been reported in other European Countries. At least five different subspecies of X. fastidiosa have been reported and classified: fastidiosa, multiplex, pauca, sandyi, and tashke. A sixth subspecies (morus) has been recently proposed. Therefore, it is vital to develop fast and reliable methods that allow the pathogen detection during the very early stages of infection, in order to prevent further spreading of this dangerous bacterium. To this purpose, the classical immunological methods such as ELISA and immunofluorescence are not always sensitive enough. However, PCR-based methods exploiting specific primers for the amplification of target regions of genomic DNA have been developed and are becoming a powerful tool for the detection and identification of many species of bacteria. The aim of this review is to illustrate the application of the most commonly used PCR approaches to X. fastidiosa study, ranging from classical PCR, to several PCR-based detection methods: random amplified polymorphic DNA (RAPD), quantitative real-time PCR (qRT-PCR), nested-PCR (N-PCR), immunocapture PCR (IC-PCR), short sequence repeats (SSRs, also called VNTR), single nucleotide polymorphisms (SNPs) and multilocus sequence typing (MLST). Amplification and sequence analysis of specific targets is also mentioned. The fast progresses achieved during the last years in the DNA-based classification of this pathogen are described and discussed and specific primers designed for the different methods are listed, in order to provide a concise and useful tool to all the researchers working in the field.