title: RNA-Seq of high salt treatment
design: Total RNA was extracted using the Plant RNA Extraction Kit (Tiangen Biotech, Beijing, China) as per the manufacturers instructions. RNA quality and concentration were assessed using a Nanodrop photometer (IMPLEN, CA, USA). Only high-quality RNA was used for constructing stranded mRNA libraries, which were then sequenced on the Illumina HiSeq platform (paired-end 150 bp reads). The sequencing procedure followed protocols previously described (Zhuo et al., 2022). The reference genome of P. yezoensis (NCBI Assembly GCA_009829735.1) was used. Three sample groups, each with three biological replicates, were analyzed: control (G, gametophyte), high-salinity stress (HS), and recovery (Re). RNA sequencing was performed on nine samples (three replications each group) using the reference genome. Clean data were obtained, with the effective data volume for each sample ranging from 6.57 to 7.18 Gb and Q30 base percentages between 92.33% and 92.66%. After alignment to the reference genome, expression levels of protein-coding genes were quantified using FPKM values. In total, 1,891 DEGs were identified in HS vs. G, 2,950 in Re vs. HS, and 3,758 in Re vs. G comparisons. Transcript abundance was quantified as FPKM. DEGs were identified using DESeq2 (Love et al., 2014) with a threshold of padj < 0.05 and |logFoldChange| > 1. GO and KEGG enrichment analyses were subsequently performed to annotate the DEGs and characterize their associated biological processes and pathways.