Read and assembly sequences were analysed to calculate GC content in reads and different genomic feature types. This figure shows that the gDNA reads and contigs/scaffolds have a skewed, almost bimodal GC content. The skew is absent from RNA sequence and gene feature sequences, indicating a difference in GC content in the non-genic regions of the genome.
Scaffolds over 250 kbp long, namely
were then analysed for the presence of small Compositionally Non-Homogenous Domains (GC-rich regions) using IsoPlotter 2.3. in Elhaik et al. This figure shows each of the scaffolds as black bars in which the white regions indicate statistically significant regions of non-homoegeneity, according to the method in in Elhaik et al.
These plots show in greater detail the changes in GC percent across these scaffolds. Many show strong drops in the GC content.
The pattern of GC poor patches observed here is reminiscent of the ‘isochore-like’ regions observed around the regions carrying effector genes in Leptosphaeria maculan’s that are believed to be involved in the plasticity of that organisms genome and may contribute to increased evolutionary potential and an ability to change hosts quickly see Raffaele and Kamoun, 2012.
As a preliminary analysis to identify wood degrading enzymes and any potentially raised pathogenicity via increased ability to enter the tree proteins predicted in
1. Chalara fraxinea TGAC annotation 1
2. Postia placenta JGI annotation
3. Fusarium graminareum Broad annotation
were used in PFAMSCAN to identify proteins containing domains from the list below at rough cutoff E <=1e-6:
(PFAM domains in carbohydrate active enzymes described in CAZY)
Counts of proteins containing each PFAM domain are shown in this figure (you may need to download to get the full version, its a big figure), Chalara proteins are represented on the outer track, Fusarium on the middle and Postia on the inner. Postia placenta is the brown rot fungus and is a capable digester of wood. The wheat pathogen Fusarium most often attacks the kernel. The carbohydrate enzyme protein domain in Chalara is much more similar to that in Postia than fusarium, indicating a potential for Chalara to be aggressively and actively entering the tree through the wood.
Kentaro Yoshida and Diane Saunders at TSL.
We used mRNAseq data from infected (AT1: data/ash_dieback/mixed_material/ashwellthorpe_AT1) and uninfected (ATU1: data/ash_dieback/fraxinus_excelsior/Ashwellthorpe_ATU1) to assess differential expression of Ash genes during infection by Chalara fraxinea.
- First, we aligned the reads from the infected sample (AT1) against the transcriptome assembly (assembled with the programme trinity) of an uninfected sample from the same location (ATU1).
- We aligned the reads from the uninfected sample against the assembly of this sample for comparison.
- Next, we counted the number of reads that mapped against unique positions in each transcript. Any reads that mapped to multiple locations were discarded.
- Finally, we ranked the counts based on their expression level (number of reads mapped). This is because the uninfected sample had many more reads than the infected sample so considering the total number of mapped reads would not be informative.
Usually we would run a normalisation step but the ranking should be sufficient to highlight any vast differences in the expression profiles. We would also like to run statistical analysis but so far we have only assessed one replicate and so this is not possible with the present data.
Considering the “rank_infected” column (data/ash_dieback/fraxinus_excelsior/annotations/gene_expression_analysis/
), within the top 50 expressed genes there are at least 11 that are related to defence (protease inhibitors, defence response proteins etc. ). 41 of of the top 50 are not included in the top 50 expressed in uninfected tissue. These would be very interesting to assess further.
Allan Downie, Anne Edwards and Kim Findlay at JIC
We used primordial fruit bodies growing on dead ash raciness collected from Ashwellthorpe Lower Wood on 10th February 2013 and incubated at room temperature on moist filter paper for 10 weeks.
The Hymenoscyphus pseudoalbidus primordial fruit bodies (shown here) were imaged by cryo-scanning electron microscopy (see below).