Daphniphyllum macropodum transcriptome assembly

Plant material was obtained from plants growing in the UK either at Ray Wood, Castle Howard or at The University of York greenhouses. Plant tissues were collected from 7 individual plants at 6 different visits and harvested onto dry ice (Table S1). The harvested plant tissues were leaf buds, male and female flowers and immature leaves (Table S2). These tissues were collected due to their accessibility and ease of harvest from the available trees/plants.

DNA-depleted RNA was isolated using a Direct-Zol RNA miniprep kit (Zymo Research, Irvine, CA, USA). RNA quantity and quality were assessed using the Agilent Tapestation system with RNA nano screentape. For short read sequencing, library preparation was performed from 200 ng high quality total RNA input using the NEBNext Ultra II Directional Library prep kit for Illumina in conjunction with the NEBNext Poly(A) mRNA Magnetic Isolation Module and unique dual index set (New England Biolabs, Ipswich, MA, USA), according to the manufacturer's instructions. Libraries were pooled at equimolar ratios and sent for paired end 150 base sequencing at Novogene (UK) Company Ltd. on a NovaSeq 6000 instrument (Illumina, San Diego, CA, USA), with 10 million reads per sample on average.

For full length cDNA sequencing, libraries were prepared (and sequenced by the Bioscience Technology Facility at the University of York) from 100 ng total RNA per species using the Oxford Nanopore Technologies (ONT) PCR cDNA barcoding library prep kit SQK-PCB109, including a 12 cycle PCR amplification of full length cDNA, with unique barcodes for each sample. Following cDNA amplification, equal quantities of cDNA from each species were pooled into final 100 fmol cDNA pool for adapter ligation and loading onto Oxford Nanopore R9.4.1 flow cells and sequencing on an ONT promethION instrument running MinKNOW version 20.06.9. Basecalling and barcode demultiplexing were performed using ONT's guppy basecaller software version 4.0.11 and quality-checked with PycoQC. Reads from all samples were then combined for a de novo transcriptome assembly using Rattle. The resulting assembly was polished with Medaka using the ONT reads, followed by 3 rounds of Pilon polishing using all Illumina short reads combined. The Illumina reads were quality checked with FastQC, trimmed with Cutadapt, and any ribosomal RNA removed with BBSplit prior to use in polishing. Transcripts were then filtered using the Perl script tr2aacds.pl from Evigene to produce a final transcriptome.