DOI: 10.5061/dryad.bk3j9kdnb
title: Data for Saccharina latissima RNA seq experiment

description: Sugar kelp (Saccharina latissima) is an ecologically and increasingly
 economically important kelp, distributed from temperate to Arctic rocky
 shores. However, S. latissima is presently threatened by ongoing climate
 changes. Genetic variations have previously been identified across S.
 latissima populations. However, little is known regarding the genetic
 basis for adaptation and acclimation to different environmental
 conditions. In this study, a common garden experiment was performed with
 sporophytes originating from North-Norway (NN), Mid-Norway (MN), and
 South-Norway (SN), representing areas with highly different temperatures
 and photoperiods. Transcriptomic analyses revealed significant variation
 in the gene expression of cultures from North-Norway, associated with low
 temperature and long photoperiods, compared to Mid- and South-Norway.
 Genes that were differentially expressed under different photoperiod and
 temperature conditions included genes linked to photosynthesis,
 chlorophyll biosynthesis, heat response, growth, protein synthesis, and
 translation. However, the transcriptional responses to variations in
 photoperiod and temperature differed between different populations of S.
 latissima  (NN, MN, and SN), indicating genotypic adaptations.
 Overall, our study provides deeper insight into the local adaptations
 of S. latissima populations along the Norwegian Coast with
 implications for the conservation of natural populations.
description: A common garden experiment (CGE) was carried out between 2
 February – 13 March 2018 at the University of Bergen, with material of
 <em>S. latissima </em>collected in North-Norway (69° 38’ N,
 18° 57’ E) (NN), Mid-Norway (63° 43’ N, 8° 49’ E) (MN) and South-Norway
 (60° 16’ N, 5° 13’ E) (SN). Tissue pieces carrying mature sori with
 sporangia were cut from 10-12 <em>Saccharina latissima</em>,
 collected at 1-5 m depth between 8-12 January 2018 at all three sites in
 Norway. The material from NN and MN was wrapped up in moist paper with
 cooling elements and transported to the laboratory and seeded the day of
 arrival, during two successive days. The tissue with sori was treated as
 described in Forbord et al. (2018) to prevent diatom growth in the
 cultures, and thereafter submerged in beaker glasses with cool (12°C)
 sterile sea water and stirred until spore release was observed using
 microscope. The spore solution was applied to tagged, clean and
 heat-treated granite stones (10x10 cm). After 10 min exposure to spore
 solution the stones were placed in sterile sea water. The material from SN
 was kept in moist paper in a fridge overnight to provoke spore release and
 was otherwise treated as described by Forbord et al. (2018). Additional
 plates for checking development of each culture were seeded in the same
 way. The seeded stones were transferred to a climate
 room. Each batch of gametophytic culture (NN, MN and SN) was grown in
 separate tanks with running sea water, but with similar photon fluence
 rates (around 50 µE m<sup>-2</sup>s<sup>-1</sup>,
 measured with a spherical sensor) and temperature (9-10°C) conditions. The
 running sea water of the climate rooms is from 100 m depth, filtered
 through a sand filter, and treated with UV light. The development of each
 culture batch was checked regularly, and 1 February minute sporophytes
 were observed in all three batches. The CGE was carried
 out in a climate room divided into three compartments through complete
 enclosures of black and opaque plastic, where each compartment had two
 tanks (30 cm x 50 cm x 25 cm (height)) with running seawater, representing
 replicates. The conditions of the three compartments were set to mimic
 temperature and day-length conditions of NN, MN and SN in mid-May: NN with
 4°C and 24 h light, MN with 6°C and 19 h light and SN with 9°C and 17 h
 light. The temperature conditions are within the ranges measured in
 April-May in the three regions by Forbord et al. (2020) or in mid-May
 according to Sætre (1973), which represent a period of rapid growth of
 <em>S. latissima</em>. The granite stones with growing
 sporophytes were added to the tanks with three stones in each,
 representing NN, MN and SN genotypes. Flow rate of running sea water was
 about 1 L per 20 sec in all the tanks, and photon fluence rates were kept
 at similar levels (46-50 µE
 m<sup>-2</sup>s<sup>-1</sup>). The tanks were
 rinsed regularly to prevent diatom growth, and temperatures of the tanks
 were checked every second or third day and adjusted when needed. Small
 variations normally within 1°C occurred during the experiment. On
 termination the stones were photographed, and two or three of the largest
 sporophytes per stone were sampled, put on RNA-later in a fridge
 overnight, and frozen at -80°C. <em>RNA-seq
 library / dataset</em> Total RNA was extracted
 from the flash frozen kelp tissue using 2% CTAB and 2M DTT, followed by
 separation of DNA/RNA from lipids and proteins using chloroform:isoamyl
 alcohol (24:1). The supernatant was removed, precipitated using
 isopropanol, pelleted, and washed with absolute alcohol, and finally
 resuspended in Low TE buffer. Isolation of RNA from extractions was
 achieved by removing DNA using the TURBO DNA-free™ Kit (Invitrogen).
 Extracted RNA quality and quantity was checked using a Qubit RNA HS kit
 and Agilent RNA HS kit on a 4200 TapeStation System.
 RNA libraries were constructed using the NEBNext® Ultra II RNA
 RNA Library Prep Kit for Illumina® (New England Biolabs) and quality
 checked using the Agilent 4200 TapeStation System. Paired-end sequencing
 (2x75bp) of transcriptomes was performed on an Illumina HiSeq4000, giving
 ca. 30 million reads per sample. RNA-seq data was
 available from 35 samples. Within the RNA-seq subset, sample replicates
 were categorized according to genotype and temperature/light conditions.
 Specifically, for the SN genotype there were 4 samples each at 6°C/19 h
 and 4°C/24 h, along with 3 reference samples at 9°C/17 h. For the MN
 genotype, the sample distribution was 3 samples at 9°C/17 h, 5 samples at
 4°C/24 h and 5 reference samples at 6°C/19 h. For the NN genotype there
 were 3 reference samples at 4°C/24 h, 4 samples at 6°C/19 h, and 4 samples
 at 9°C/17 h. Transcripts were aligned to a reference genome obtained
 through the France Génomique National infrastructure project Phaeoexplorer
 (ANR-10-INBS-09). <em>Bioinformatics
 analysis</em> To assess the quality of the raw
 sequencing reads, the quality control software FastQC (version 0.11.9) was
 used. The reads were trimmed using Trim Galore (version 0.6.6). Minimum
 read length before cutoff were set to 60 bp since shorter reads often have
 low qualities (Krueger, 2015). Trim Galore was run with default setting,
 i.e. a Phredscore threshold of 20 and a maximum error rate of 0.1 to
 obtain clean reads of high quality. MultiQC was used to summarize the
 results from the read quality trimming done with Trim Galore (Ewels
 <em>et al.</em>, 2016). Trimmed reads were aligned to the
 reference genome of <em>S. latissima</em> using STAR (version
 2.7.10a) (Dobin <em>et al.</em>, 2015; Denoeud <em>et
 al.</em>, 2024). Prior to alignment, the reference genome was
 indexed by running --runmode genomeGenerate. No Gene Transer Format (GTF)
 file was available and the provided General Feature Format (GFF) file was
 formatted in STAR to avoid problems when running sequence alignment. This
 was solved by replacing --sjdbGTFfile with
 --sjdbGTFtagExonParentTranscript. The setting instructs STAR to use
 “Parent” tag in the GFF-file to link exons to transcripts.  The
 featureCounts software (version 2.0.1) was used to count the number of
  reads mapping to each gene in the  <em>Saccharina
 latissimi</em> reference genome. Pairwise comparisons of
 differential gene expression were performed using DESeq2 (version 1.34.0)
 (Love et al., 2014). Reference replicates were compared against replicate
 groups with same genotype under different conditions. An absolute LFC
 (log2FoldChange) of ≥ 2 and a p-value of &lt; 0.05 was used as
 thresholds to determine the significance in gene expression between
 groups. The identifier from the GFF-file was used to find annotation from
 Interproscan. A GO enrichment analysis was carried out using topGO
 (version 2.46.0) to identify overrepresented GO-terms.
description: # Data for Saccharina latissima RNA seq experiment ### File:
 CommonGardenExperiment\_metadata.xlsx #### Description Metadata associated
 with a Common Garden Experiment involving three populations of Saccharina
 latissima (sugar kelp). The populations were obtained from North-Norway
 (i.e. Tromso), Mid-Norway (i.e. Trondheim), and South-Norway (i.e.
 Bergen). Growth experiments were conducted in the laboratory under
 conditions mimicking the in situ temperatures and photoperiod for each
 population. At the end of each growth experiments RNA was extracted using
 3-5 replicates. Sequencing of RNA was performed with Illumina paired-end
 sequencing. This resulted in 70 fastq files aviable from SRA
 (PRJNA1164979) (3 populations x 3 conditions x (3-5)replicates x 2(paired
 end) = 70). Reads were mapped to a reference genome of S. latissima
 available from the Phaeoexplorer Database (ANR-10-INBS-09). #### Variables
 * **Sample for RNAseq analyses:** This indicates the source of the
 population and the growth conditions. E.g.
 'Slatissima_Tromso_TrondheimCondition' is a sample of S.
 latissima grown under Trondheim conditions * **Organism**: Species name
 (i.e. S. latissima) * **Source of population**: The area along the
 Norwegian coast the population was collected from. * **Sporophyte age at
 time of sampling for RNAseq**: The age of the Sporophytes at the end of
 the growth experiment (at the time of sampling for RNAseq). * **Date of
 sampling for RNAseq**: Date of ampling for RNAseq * **Growth stage at the
 time of sampling for RNAseq**: Growth stage of S. latissima at the time of
 sampling for RNAseq * **Population collected by**: Name of person
 collecting each population of S. latissima. * **Geolocation of population
 sampling**: Geographical coordinates of source of the three S. latissima
 populations. * **Temperature (degC) in common garden experiment**: Growth
 condition (Temperature in degrees Celsius) in the growth experiment *
 **Photoperiod (hours of light) in Common Garden Experiment**: Growth
 condition (hours of light per day) in growth experiment. ### File:
 DescriptionOfRawdata.xlsx Raw data are available from Sequence Read
 Archive (SRA) under accession number PRJNA1164979. #### Variables *
 **Sample for RNAseq analyses**: Same as in the
 CommonGardenExperiment_metadata.xlsx file. The name indicates the source
 of the population and the growth conditions. E.g.
 'Slatissima_Tromso_TrondheimCondition' is a sample of S.
 latissima grown under Trondheim conditions * **SampleID**: ID of one RNA
 extraction (there are replicate RNA extractions from each experiment) *
 **Description**: All data is RNA-Seq of Saccharina latissima (sporohytes)
 * **Sequencer**: All sequences are derived from sequencing with Illumina
 HiSeq 4000 * **Filename**: Filename of each raw data file. Each file can
 be downloaded from SRA (PRJNA1164979) ### File: Trimming.sh Script for
 trimming paired end reads with Trim Galore. ### File: Mapping.sh Script
 for mapping reads to reference genome using STAR. ### File:
 FeatureCounts.sh Command used for gene counting ### File:
 DESeq2-DGE-analysis-MBR.qmd DESeq2 pipeline for identification and
 analyses of differentially expressed genes. ### File:
 topGO\_analyses\_R.txt R script for enrichment analysis of Gene Ontology
 (GO) terms using topGO.