Novaseq platform

M. xanthus DK1622 and ∆msuB mutants were grown in a CTT medium for 24 h at 30 °C. Total RNA was isolated and then the mRNA was purified using probes to remove rRNA for mRNA-seq library construction. The libraries were sequenced on an Illumina Novaseq platform to generate paired-end reads of 150 bp in length (Novogene Bioinformatics Technology Co., Ltd.). Clean reads were trimmed to remove adapter sequences and mapped to the DK1622 genome using Bowtie2-2.2.3. Differentially expressed genes (DEGs) were calculated with the cut-off of a fold change of >0 or a fold change of <0, and P_adj < 0.05 using DESeq2. For the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, DEGs with a fold change of >0 or a fold change of <0 (P_adj < 0.01) were included and the statistical enrichment of differentially expressed genes in the KEGG pathways were analyzed using KOBAS software.

Library construction, quality control, and sequencing were performed at Novogene (Beijing, China). PCR amplification of ITS2 regions was performed using ITS3/ITS4 primers,⁵⁶ using Novogene’s pipeline. The PCR products were subjected to 2% agarose gel electrophoresis. PCR products from each sample were pooled, end-repaired, A-tailed, and ligated using Illumina adapters. The library was quantified using Qubit and real-time PCR, and size distribution was estimated using a bioanalyzer. Quantified libraries were pooled and sequenced on an Illumina NovaSeq platform (Novogene, Beijing, China) according to the effective library concentration and data amount required. Paired-end reads were assigned to the samples based on their unique barcodes and truncated by cutting off the barcode and primer sequences. Paired-end reads were merged using FLASH^{57 (link)} based on the overlap of the reads.

RNA was purified from RPE1 cells and then reverse-transcribed into cDNA. The RNA-seq libraries were prepared with the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina according to the manufacturer’s instructions. Index-coded samples were clustered on the cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, library preparations were sequenced on the Illumina Novaseq platform and 150 bp paired-end reads were generated by Novogene (Novogene, Tianjin, China). RNA-seq data were aligned to the Ensembl human reference transcriptome (GRCh38, version 94) by HiSat2 and summarized by StringTie as fragments per kilobase transcript per million mapped reads (FPKM). Heatmaps to visualize the data were generated using Excel.

For RNA-seq sequencing, the wild-type PH-1 and BP1^ΔIDR2-C strain was incubated in YEPD liquid medium with agitation (180 rpm) for 24 h at 25 °C. The fresh mycelial samples were harvested from liquid medium and ground in liquid nitrogen. Total RNA was isolated from three independent biological replicates from the wild-type PH-1 and BP1^ΔIDR2-C. DNA-free total RNA was generated by the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s recommendations. The library was sequenced on an Illumina Novaseq platform with paired end reads (150 bp) by Novogene Corporation (Beijing, China). The sequencing reads were mapped to the F. graminearum PH-1 genome sequence using Hisat2. Feature Counts v1.5.0-p3 was used to count the reads mapped to each gene. Genes with log2 Fold change ≥ 1 or ≤  − 1 and P-value ≤ 0.05 were divided into differentially expressed.

We constructed a comprehensive Fagaceae dataset consisting of 122 individuals from 91 species representing all eight currently recognized genera^{58 ,120} . Complete taxon sampling was achieved for three small genera: Chrysolepis (2 species), Notholithocarpus (2 species) and Trigonobalanus (3 species). For the remaining genera, representative samples for all major lineages were included: Fagus (2), Castanea (5), Castanopsis (12) and Lithocarpus (10). For the well-studied genus Quercus, extensive sampling (54 species) was conducted to represent all eight recognized sections^{42 ,48} . Several species were represented by multiple accessions collected from different natural populations or cultivated plants. Betula pendula was selected as an outgroup due to its close relationship to Fagaceae and the availability of an assembled genome^{121 (link)}. Accession information is provided in Supplementary Data 1.
Total genomic DNA was extracted from silica-dried leaf tissue using BioTeke Genomic DNA Extraction Kit (Beijing, China). High-quality DNA was used to constructed paired-end sequencing libraries with an insert size of 500–600 bp according to the Illumina library preparation protocol. Sequencing (150 bp pair-end) was carried out on the NovaSeq platform at Novogene (Beijing, China) to a coverage of 25–40× for all samples.