DNA metabarcoding uncovers fungal diversity of mixed airborne samples in Italy - Fig 5

<p>A) Bar charts showing the taxonomic composition at genus level in the four sampling sites. Abundances of taxa are reported with the percentage values of reads. Taxa accounting for <0.1% of reads are grouped as “Other”. B) Venn diagram shows the number of unique and shared taxa identified at the genus level among sites (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194489#pone.0194489.s006" target="_blank">S2 Table</a>).</p

DNA metabarcoding uncovers fungal diversity of mixed airborne samples in Italy

<div><p>Fungal spores and mycelium fragments are particles which become and remain airborne and have been subjects of aerobiological studies. The presence and the abundance of taxa in aerobiological samples can be very variable and impaired by changeable climatic conditions. Because many fungi produce mycotoxins and both their mycelium fragments and spores are potential allergens, monitoring the presence of these taxa is of key importance. So far data on exposure and sensitization to fungal allergens are mainly based on the assessment of few, easily identifiable taxa and focused only on certain environments. The microscopic method used to analyze aerobiological samples and the inconspicuous fungal characters do not allow a in depth taxonomical identification. Here, we present a first assessment of fungal diversity from airborne samples using a DNA metabarcoding analysis. The nuclear ITS2 region was selected as barcode to catch fungal diversity in mixed airborne samples gathered during two weeks in four sites of North-Eastern and Central Italy. We assessed the taxonomic composition and diversity within and among the sampled sites and compared the molecular data with those obtained by traditional microscopy. The molecular analyses provide a tenfold more comprehensive determination of the taxa than the traditional morphological inspections. Our results prove that the metabarcoding analysis is a promising approach to increases quality and sensitivity of the aerobiological monitoring. The laboratory and bioinformatic workflow implemented here is now suitable for routine, high-throughput, regional analyses of airborne fungi.</p></div

Summary of manual annotation of mapped probes genotyped in a <i>Malus</i> germplasm collection.

<p>The number of probes displaying either robust, reliable genotypes; ambiguous but scoreable genotypes; and unscoreable genotypes following genotyping of a collection of 192 <i>Malus</i> cultivars.</p

Jackknifed principal coordinate analysis (PCoA) plot of Bray-Curtis distances between the samples of the four sites.

<p>Ellipsoids show the statistical confidence of the analysis.</p

The number of SNP markers derived from both <i>Malus</i> and <i>Pyrus</i> genomic sequence mapped in this investigation displaying evidence for probe sequence variants, null alleles and non-specific probe binding.

<p>The number of SNP markers derived from both <i>Malus</i> and <i>Pyrus</i> genomic sequence mapped in this investigation displaying evidence for probe sequence variants, null alleles and non-specific probe binding.</p

Geographical location of the sampling sites in the North-Eastern and Central Italy.

<p>Region and city names are reported (FVG: Friuli Venezia Giulia). The map has been retrieved and modified from <a href="http://www.d-maps.com" target="_blank">http://www.d-maps.com</a>.</p

A comparison of genetic and physical positions of probes mapped in M432.

<p>A comparison of the percentage of probes mapped to each linkage group on the M432 linkage map with their physical positions on the ‘Golden Delicious’ genome sequence.</p

Doughnut charts showing fungal taxonomic composition at division (A) and class (B) level in the four sampling sites.

<p>Relative abundances of taxa are reported in percentage. “Unidentified fungi” comprehends Fungi sp. and fungi with no blast hit. Taxa accounting for <0.1% of reads are grouped as “other”.</p

Comparison of probe annealing numbers, behaviour and probe GenTrain scores.

<p>(a) The number of probes mapped in the M432 progeny showing evidence of annealing to single loci, multiple paralogous loci and probes showing evidence of probe annealing site divergence. (b) Box and whisker plots comparing the GenTrain scores of probes associated with single loci, and with multiple loci in the ‘Golden Delicious’ genome following BLAST analysis, and probes for which divergent annealing sites contained additional segregating SNPs or created null alleles.</p

Cluster plots of IRSC probes.

<p>Mean normalised Theta and normalised R values from markers genotyped in the M432 progeny using the IRSC array plotted for (a) probes to which a single significant genome match was returned and (b) probes returning multiple hits to the ‘Golden Delicious’ genome sequence.</p