De novo transcriptome assembly and annotation of the Antarctic hair grass Deschampsia antarctica

Antarctic hair grass (Deschampsia antarctica), one of only two native flowering plants in Antarctica, exhibits remarkable cold tolerance. To elucidate the molecular mechanisms underlying this exceptional resistance of D. antarctica to low temperatures, we constructed the de novo transcriptome reference using sequencing data from RNA samples during cold acclimation and subsequent deacclimation. Total RNA was isolated from leaves of D. antarctica using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). A total of 3 μg RNA (1000 ng/μL) from each sample was used to construct libraries with the TruSeq mRNA Library Kit (Illumina, San Diego, CA, USA). Sequencing was performed on a HiSeq 2000 system (PE, 2 × 101) (Illumina). Raw reads have been deposited and linked to NCBI BioProject PRJNA1098583. For pre-processing of raw data, Trimmomatic v0.3.9 was used to remove low-quality reads and adaptor sequences from the raw data. An additional decontamination process was performed using the BBduk v38.87 with viral, rRNA, human and bacteria RefSeq databases. For de novo assembly, rnaSPAdes v3.15.0 was used with the `--hard_filtered_transcripts` option to generate hard-filtered transcripts. The resulting dataset was then clustered using CD-HIT-EST v4.8.1 with 90% identity and a length cutoff of > 300 bp. For selecting expressed genes through NGS mapping, we set thresholds of TPM ≥ 0.2 and mapping counts > 0. To assess the completeness of our transcriptome assembly, we conducted a BUSCO v5.0 analysis using the embryophyte_odb10 database. To infer the functions of the generated unigene set, similarity analysis was performed using the DIAMOND program, which executed BLASTX analysis on the nucleotide sequences of the unigenes against the NCBI non-redundant, Araport11 and Oryza sativa (Os_IRGSP_1.0_pep_all) protein databases with an e-value cutoff of 1e-5. Protein domain analysis was conducted using InterProScan to investigate conserved domains with protein sequences. KEGG pathway analysis was carried out using the KAAS web-tool, with unigene sequences as input and employing the SBH method.