Fig. 4

Genome-wide identification and characterization of B. hordei lncRNAs. A Using the total RNA-seq data mapped to the genome of B. hordei isolate DH14 [10] with HISAT2 [45], we assembled transcripts with StringTie [43]. Next, we filtered out coding genes from the transcriptome by Gffcompare [46], transcripts shorter than 200 bp using Gffread [46], transcripts with coding potential using CPC2 [47], and transcripts accounting for ribosomal RNA, transfer RNA, small nuclear RNA, and small nucleolar RNA via CMscan search against the Rfam database [48]. Then, we used FEELnc [49] for lncRNA annotation and classification, resulting in 17,226 putative lncRNAs in the reference genome of B. hordei. Lastly, we manually inspected the predicted lncRNA models by our Web Apollo [44] instance, yielding in total 5,127 unique lncRNA loci in B. hordei. B Histogram for the transcript length in base pairs (bp; x-axis) against the number of coding genes (mRNAs, purple) and lncRNAs (orange; y-axis). The violin plot above shows the overall distribution of gene lengths; data points represent individual transcripts. C Bar graph of the exon number per transcript (x-axis) against the gene number (y-axis). Purple, mRNAs; orange, lncRNAs. D The violin plot shows the expression in Log₂(transcripts per million (TPM)) for mRNAs (purple), lncRNAs in sense orientation of associated genes (blue), lncRNAs in antisense orientation of TEs (orange), intronic lncRNAs (grey), and intergenic lncRNAs (green). The number of transcripts (n) contributing to the respective subset are given on the top. E The box plot shows the transcript levels in Log₂(TPM) for lncRNAs (orange) and mRNAs (purple) depending on the transcript exon number (x-axis). F The histogram shows the number of transcripts (x-axis) encoded by lncRNA genes (y-axis). G The stacked bar graph displays the occurrence of alternatively spliced lncRNA transcripts (AS, alternative splicing; orange bar) and the number and type of alternative splicing events in B. hordei. The types of events are illustrated by the drawings, where the red portion of the exon indicates the alternative event and black lines connecting exons splice events, colored in shades of red and orange according to event. From dark red to dark orange, events are shown in this order: retained intron, alternative 3’ splice site, alternative 5’ splice site, skipped exon, alternative last exon, and alternative first exon. H The dot plots show the transcript levels in TPM for alternatively spliced isoforms of the lncRNA BLGHnc_000769 (y-axis) at six time points of host infection (x-axis). Note that isoform BLGHnc_000769-RA was not expressed above background levels and thus omitted from this Figure. The black bar indicates the median of three independent replicates (n = 3)