Based on results from the CLSM analysis, at 5 dpi there were consistent phenotypic differences in the rachis node and rachilla between AAC Tenacious and Roblin; as such, we selected this time-point to examine gene expression patterns by RNAseq. The spikelet at the POI and the rachis node below the POI, sections “iii” and “v,” were isolated at 5 dpi for the Fg and water inoculations. One spike per plant/pot and 15 spikes were used per replicate. Each treatment was replicated three times. RNA extraction was performed from 360 sectioned samples (180 Fg inoculated and 180 water inoculated), using the Trizol Reagent (Ambion), according to the manufacturer’s instructions. All samples were treated with DNase I (Invitrogen), according to the manufacturer’s instructions. The RNA yield and quality were monitored using a Qubit 3.0 Fluorometer with the Qubit BR Assay Kit (Invitrogen), and an Agilent 2100 Bioanalyzer and the Agilent RNA 6000 Nano Kit (Agilent). The average RNA Integrity Number (RIN) value for all the samples was 9.0. cDNA libraries were prepared by using the NEB rRNA-depleted stranded (plant) kit. Samples were sequenced at the McGill University and Genome Quebec Innovation Centre (Montreal, Canada) using the HiSeq2500 Illumina sequencer with 125-nucleotide paired-end reads.

Analysis of the RNAseq reads was performed according to widely established standards and protocols for wheat (IWGSC et al., 2018). Briefly, adapters were trimmed from raw sequence reads using Trimmomatic, which were then aligned to the Chinese Spring RefSeq v1.0 wheat genome assembly (IWGSC et al., 2018) using STAR (Dobin et al., 2012). Using the available RefSeq v1.0 high-confidence gene annotations, a raw count matrix was generated for each annotated gene using HTSeq-Count (Anders et al., 2015) and imported into DESeq2 (Love et al., 2014) for differential expression analyses. Pairwise comparisons between treatments were considered, and genes were declared differentially expressed if the log2 fold change was greater than 2 or less than −2. Differentially expressed gene (DEG) lists were extracted for each comparison and analyzed for gene ontology (GO) enrichment in R using the topGO package (Rahnenführer, 2009). Variants were called for each sample using Freebayes1 software. Filtering and annotation of variants that differentiate AAC Tenacious from Roblin was performed using SnpSift and SnpEff software (Cingolani et al., 2012).

We then performed a more detailed inspection of variants within three QTL regions identified from Sumai 3 (Fhb1, Fhb2, and Fhb5). QTL regions were identified in RefSeqv1.0 based BLASTn analysis of markers Gwm133 and Gwm644 on chromosome arm 6BS for Fhb2, (Cuthbert et al., 2007), and Gwm304 and Gwm415 on chromosome arm 5AS for Fhb5 (Xue et al., 2011). The Fhb1 genomic region was identified using sequences of candidate genes and gene containing contigs [Fhb1-1, GenBank accession KU304333.1 (Paudel et al., 2020); PFT and PFT containing contig, GenBank accessions AY587018.1 and KX907434.1 (Rawat et al., 2016); and TaHRC, GenBank accession MK450312.1 (Su et al., 2019)].

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