Transcriptomics and metabarcoding approaches for the study of lichen symbionts

This research has focused on two main aspects of lichen symbioses: the characterization of stress responses in lichen photobionts and the fungal diversity present inside the lichen thallus. The study of the molecular mechanisms that underlie the stress tolerance considered a member of the most common genus of lichenized green algae, Trebouxia gelatinosa. Lichens can cope with extreme environmental conditions and this capacity is linked to the desiccation tolerance, in particular of their associated photobionts. In nature, lichen thalli continuously undergo cycles of dehydration and rehydration and in desiccated states they are extremely resistant. A transcriptome analysis on T. gelatinosa was performed under control conditions, in desiccated state and after rehydration. Desiccation and rehydration affected mainly the gene expression of components of the photosynthetic apparatus, the ROS-scavenging system, heat shock proteins (HSPs) and desiccation related proteins (DRPs). The obtained data were used to investigate in more detail the relationship between the expression of genes of interest and physiological parameters, important to understand the global response of T. gelatinosa under environmental stresses. Oxidative stress and desiccation were considered. The antioxidant activity of the photobiont was studied by quantifying the gene expression of ROS scavenging and stress-related enzymes on samples treated with hydrogen peroxide (H2O2). The data produced have been evaluated in relation to the chlorophyll a fluorescence (ChlaF) activity. H2O2 treatments produced dose and time dependent oxidative effects. The gene expression in dependence to the cell water content during desiccation was also evaluated. From the hydrated to the desiccated state, both relative water content (RWC) and water potential (\u3a8) were monitored. The turgor loss point was found as a key moment that triggered changes in gene transcription. Lichens are regarded as ecological microniches as their thalli harbor complex fungal communities (mycobiota) consisting of species with divergent trophic and ecological strategies. Complexity and diversity of lichen mycobiomes are still largely unknown, despite surveys combining culture-based and high throughput sequencing (HTS) have been applied. This research assessed the taxonomic composition (species diversity) of a well-characterized, alpine rock lichen community which included both thalli symptomatically infected by lichenicolous fungi and asymptomatic thalli. Taxonomic composition was assessed by HTS of the ITS2 barcode. Chaetothyriales was the major component of the observed lichen mycobiomes; Basidiomycota were recorded in the least amount. Sequences representative of morphologically characterized lichenicolous fungi were predicted and were used to assess whether there was an asymptomatic presence of these taxa in other thalli

New features of desiccation tolerance in the lichen photobiont Trebouxia gelatinosa are revealed by a transcriptomic approach.

Trebouxia is the most common lichen-forming genus of aero-terrestrial green algae and all its species are desiccation tolerant (DT). The molecular bases of this remarkable adaptation are, however, still largely unknown. We applied a transcriptomic approach to a common member of the genus, T. gelatinosa, to investigate the alteration of gene expression occurring after dehydration and subsequent rehydration in comparison to cells kept constantly hydrated. We sequenced, de novo assembled and annotated the transcriptome of axenically cultured T. gelatinosa by using Illumina sequencing technology. We tracked the expression profiles of over 13,000 protein-coding transcripts. During the dehydration/rehydration cycle c. 92 % of the total protein-coding transcripts displayed a stable expression, suggesting that the desiccation tolerance of T. gelatinosa mostly relies on constitutive mechanisms. Dehydration and rehydration affected mainly the gene expression for components of the photosynthetic apparatus, the ROS-scavenging system, Heat Shock Proteins, aquaporins, expansins, and desiccation related proteins (DRPs), which are highly diversified in T. gelatinosa, whereas Late Embryogenesis Abundant Proteins were not affected. Only some of these phenomena were previously observed in other DT green algae, bryophytes and resurrection plants, other traits being distinctive of T. gelatinosa, and perhaps related to its symbiotic lifestyle. Finally, the phylogenetic inference extended to DRPs of other chlorophytes, embryophytes and bacteria clearly pointed out that DRPs of chlorophytes are not orthologous to those of embryophytes: some of them were likely acquired through horizontal gene transfer from extremophile bacteria which live in symbiosis within the lichen thallus

ITS2 metabarcoding analysis complements lichen mycobiome diversity data

Lichen thalli harbor complex fungal communities (mycobiomes) of species with divergent trophic and ecological strategies. The complexity and diversity of lichen mycobiomes are still largely unknown, despite surveys combining culture-based methods and high-throughput sequencing (HTS). The results of such surveys are strongly influenced by the barcode locus chosen, its sensitivity in discriminating taxa, and the depth to which public sequence repositories cover the phylogenetic spectrum of fungi. Here, we use HTS of the internal transcribed spacer 2 (ITS2) to assess the taxonomic composition and diversity of a wellcharacterized, alpine rock lichen community that includes thalli symptomatically infected by lichenicolous fungi as well as asymptomatic thalli. Taxa belonging to the order Chaetothyriales are the major components of the observed lichen mycobiomes. We predict sequences representative of lichenicolous fungi characterized morphologically and assess their asymptomatic presence in lichen thalli. We demonstrated the limitations of metabarcoding in fungi and show how the estimation of species diversity widely differs when ITS1 or ITS2 are used as barcode, and particularly biases the detection of Basidiomycota. The complementary analysis of both ITS1 and ITS2 loci is therefore required to reliably estimate the diversity of lichen mycobiomes

Whole-Genome Analysis of Diversity and SNP-Major Gene Association in Peach Germplasm

Peach was domesticated in China more than four millennia ago and from there it spread world-wide. Since the middle of the last century, peach breeding programs have been very dynamic generating hundreds of new commercial varieties, however, in most cases such varieties derive from a limited collection of parental lines (founders). This is one reason for the observed low levels of variability of the commercial gene pool, implying that knowledge of the extent and distribution of genetic variability in peach is critical to allow the choice of adequate parents to confer enhanced productivity, adaptation and quality to improved varieties. With this aim we genotyped 1,580 peach accessions (including a few closely related Prunus species) maintained and phenotyped in five germplasm collections (four European and one Chinese) with the International Peach SNP Consortium 9K SNP peach array. The study of population structure revealed the subdivision of the panel in three main populations, one mainly made up of Occidental varieties from breeding programs (POP1OCB), one of Occidental landraces (POP2OCT) and the third of Oriental accessions (POP3OR). Analysis of linkage disequilibrium (LD) identified differential patterns of genome-wide LD blocks in each of the populations. Phenotypic data for seven monogenic traits were integrated in a genome-wide association study (GWAS). The significantly associated SNPs were always in the regions predicted by linkage analysis, forming haplotypes of markers. These diagnostic haplotypes could be used for marker-assisted selection (MAS) in modern breeding programs

Intrathalline diversity of lichen-inhabiting fungi assessed by metabarcoding analysis

Lichen-inhabiting fungi are a large and diversified, but still relatively unknown, ecological group. They can live on host thalli symptomatically, showing characteristic phenotypes and different degrees of specificity (lichenicolous fungi), or asymptomatically (endolichenic fungi), detectable only through culture and molecular techniques. Here we aim at studying the whole diversity of lichen-associated fungi using metabarcoding analysis of the internal transcribed-spacer region 2, as parts of the fungal standard barcode. We analyzed thalli with and without symptoms of fungal infection, collected from Alpine lichen communities on the Koralpe Massif (Austria)

Nuova segnalazione per l\u2019Italia Nord-Orientale(Foresta del Cansiglio, Veneto, NE-Italia) di Campanula bertolae tramite la tecnica del DNA barcoding

Dall\u2019analisi morfologica degli esemplari raccolti nella Foresta del Cansiglio risulta che essi hanno la base del fusto glabra e la papillosit\ue0 dell\u2019ovario assente. I 2 campioni provenienti dal Cansiglio-Alpago e analizzati per il DNA barcoding hanno mostrato una similarit\ue0 del 100% per entrambi i geni di Campanula bertolae. E\u2019 quindi una segnalazione nuova per questa specie nell\u2019Italia Nordorientale e nel Veneto. Nella\u201cFlora d\u2019Italia\u201d(Pignatti, 1982) viene data come endemismo delle Alpi Occidentali, presente su pendii aridi e sassosi (serpentino) in Piemonte. In\u201cFlora alpina\u201d(Aeschimann et al., 2004) la presenza viene data per certa nelle province di Cuneo, Torino, Bergamo e Brescia e ne viene confermato l\u2019endemismo; risulta presente su una vasta gamma di substrati: preferibilmente serpentino, pietre verdi, granito, gneiss, scisti silicei, e, anche se in misura minore, calcare e dolomia. In Cansiglio si sviluppa su scaglia grigia (calcare marnoso)

Lichens and the omics

The massive development of tools, such as the omics technologies, which allow scientists to look at biological processes on large scales, is a fundamental component of the so-called Systems Biology. This subject studies biological systems in order to achieve a comprehensive and integrated view of their biology (Ideker et al., 2001). For these aims, the complementation of different disciplines such as molecular biology, biochemistry, and bioinformatics is of key importance (Graves & Haystead, 2002). The main purpose of omics is the quantification and characterization of the complete set of the biological molecules which shape structures, functions and dynamics of cells and organisms (Sim\uf2 et al., 2014). The -ome suffix refers to the whole composition of groups of molecules like nucleic acids, proteins and metabolites at a given time or place: genomics is the study of the genome, transcriptomics is the study of the total RNA, proteomics is the study of the entire set of proteins and metabolomics is the study of the set of low molecular weight metabolites (Smith et al., 2005). When the analysis is referred to genes, transcripts, proteins or metabolites recovered from environmental samples, which may contain many different organisms, the recently introduced term meta-omics is used (Valles-Colomer et al., 2016). For instance, metagenomics is defined as the analysis of all the genomes contained in an environmental sample (Thomas et al., 2012). Here we offer an overview of these up-to date tools and present what has already been done in the study of the lichen symbiosis

Integrated eDNA metabarcoding and morphological analyses assess spatio-temporal patterns of airborne fungal spores

Fungi represent relevant allergens and plant pathogens that can disperse on long ranges, potentially producing severe consequences on public health and agriculture. Up to 11% of the bioaerosol particles are fungal spores and mycelium fragments. Estimation of fungal species diversity in time and space is decisive but may be biased by abiotic conditions and sampling methods. Traditional morphological analyses of fungal spores have been widely applied in aerobiology in the past, while recently eDNA metabarcoding can complement these studies. Here, we used both morphological analysis (spore count and taxon identification) and high-throughput sequencing to disentangle spatio-temporal variation of fungi across Northern and Central Italy and to evaluate the detection efficiency of the two approaches. Our results showed that eDNA metabarcoding detects about three times more genera and has a higher detection efficiency than the morphological analyses. However, the efficiency is high in both spore count and eDNA metabarcoding methods when the most abundant or the rarest genera are considered but it can substantially vary between the two approaches when moderately abundant genera are analyzed. Furthermore, morphological spore determination resulted in higher variance explained by PERMANOVA analysis with respect to eDNA metabarcoding (26% and 13%, respectively), which leads to a better spatio-temporal characterization of the fungal genera. As both morphological analyses and eDNA metabarcoding methods capture significant interactions between seasons and sites, they could be preferably used as complementing approaches to reliably study airborne fungal diversity and variation

Relation between water status and desiccation-affected genes in the lichen photobiont Trebouxia gelatinosa

9siThe relation between water status and expression profiles of desiccation -related genes has been studied in the desiccation tolerant (DT) aeroterrestrial green microalga Trebouxia gelatinosa, a common lichen photobiont. Algal colonies were desiccated in controlled conditions and during desiccation water content (WC) and water potential (Ψ) were measured to find the turgor loss point (Ψtlp). Quantitative real-time PCR was performed to measure the expression of ten genes related to photosynthesis, antioxidant defense, expansins, heat shock proteins (HSPs), and desiccation related proteins in algal colonies collected during desiccation when still at full turgor (WC > 6 g H2O g−1 dry weight), immediately before and after Ψtlp (−4 MPa; WC∼1 g H2O g−1 dry weight) and before and after complete desiccation (WC < 0.01 g H2O g−1 dry weight), quantifying the HSP70 protein levels by immunodetection. Our analysis showed that the expression of eight out of ten genes changed immediately before and after Ψtlp. Interestingly, the expression of five out of ten genes changed also before complete desiccation, i.e. between 0.2 and 0.01 g H2O g−1 dry weight. However, the HSP70 protein levels were not affected by changes in water status. The study provides new evidences of the link between the loss of turgor and the expression of genes related to the desiccation tolerance of T. gelatinosa, suggesting the former as a signal triggering inducible mechanisms.partially_openembargoed_20200901_20210901Banchi, Elisa*; Candotto Carniel, Fabio; Montagner, Alice; Petruzzellis, Francesco; Pichler, Gregor; Giarola, Valentino; Bartels, Dorothea; Pallavicini, Alberto; Tretiach, MauroBanchi, Elisa; Candotto Carniel, Fabio; Montagner, Alice; Petruzzellis, Francesco; Pichler, Gregor; Giarola, Valentino; Bartels, Dorothea; Pallavicini, Alberto; Tretiach, Maur