Transcriptomic dataset
For each seedling, up to three fascicles from the current year’s growth
were sampled and flash frozen in liquid nitrogen for RNA extraction.
Tissue sampling was performed across two days with consistent weather
conditions between the hours of 12:00 and 17:00. A maximum of 100 mg of
needle tissue per seedling was ground into a fine powder using liquid
nitrogen with a mortar and pestle with 40 mg of polyvinylpyrrolidone
(PVP-40) added. Total RNA was extracted using Spectrum Plant Total RNA
Kits (Sigma-Aldrich). Following polyA tail selection to enrich for mRNA,
libraries were prepared following standard protocols for the NEBNext
Ultra II RNA Library Prep (Illumina) and sequenced on the Novaseq6000
platform at Novogene Corporation (Sacramento, CA).
Raw data were assessed for quality using fastqc and trimmed to remove
adapters and low-quality reads using TrimGalore v.0.6.4 (Krueger, 2015).
Due to lack of a well-annotated genome from a closely related species,
we built a de novo transcriptome assembly using Trinity v.2.8.5
(Grabherr et al ., 2011; Haas et al ., 2013) with ak -mer length of 24 and a minimum contig size of 300bp. Further
details about the assembly pipeline, filtering steps, quality assessment
and annotations using EnTAP (Hart et al ., 2020) are detailed in
the Supporting Information (Methods S1).