Sample collection and treatment
S. japonica juvenile sporophytes (“Zhongke No. 1”) were cultivated and collected from the Gaolv Aquacultural Ltd. Co. in Lidao, Rongcheng, Shandong, China. After eliminating the epiphytes via sterilized seawater washing, kelps were kept in cold conditions during shipping. The collected kelps were pre-cultured in the dark at 10 °C for 24 h and were exposed to the dark, white light (70 µmol photons m− 2 s− 1) and blue light (25 µmol photons m− 2 s− 1) for 3 h, respectively. White fluorescent lamps (Philips, Shanghai, China) and blue light-emitting diodes (460 ~ 475 nm wavelengths; Ichia, Japan) were applied as light sources. After exposure, the juvenile sporophytes were immediately frozen with liquid nitrogen and stored at -80 °C for subsequent RNA extraction. The kelp (“Zhongke No. 1”) was originally cultivated after nine generations of hybridization with long and wide S. japonica phenotypes. One voucher specimen of S. japonica was deposited in the Resource-sharing Platform of Specimens Marine Biological Museum Chinese Academy of Sciences (No. MBM436715).
RNA isolation and library preparation
Total RNA was isolated using TRIzol reagent (Invitrogen, CA, USA). The extracted RNA was qualitatively examined using 1% agarose gel electrophoresis, and the concentration was determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, USA). After extraction, RNAs ranging from 18 to 30 nt were separated by 15% polyacrylamide gel electrophoresis (PAGE). Next, 3’ adapters were added, and the 36–44 nt RNAs were enriched. Subsequently, 5’ adapters were ligated to the RNAs. The ligation products were reverse transcribed and the 140–160 bp PCR products were enriched to generate a cDNA library. Finally, sequencing was conducted on an Illumina HiSeqTM 2500 at Gene Denovo Biotechnology Co. (Guangzhou, China). The RNA integrity and concentration were further measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). The small RNAs and mRNAs were reverse transcribed to cDNA and then sequenced with a HiSeq 2500 and HiSeq 4000 (Illumina, San Diego, CA, USA), respectively.
Identification of miRNAs and target gene prediction
After removing reads containing adapters or low-quality bases, all clean tags were aligned with small RNAs in the Rfam and GenBank databases (Release 209.0) with our previous S. japonica genome data registered in the NCBI database (accession: MEHQ00000000). The rRNA, scRNA, snoRNA, snRNA and tRNA sequences were filtered out. All clean tags were validated using the miRBase database with known miRNAs. We selected the following prediction criteria for novel miRNAs: length, 18–25 nt; maximal free energy allowed for a miRNA precursor, 18 kcal/mol; space between miRNA and miRNA*, 14–35 nt; maximal asymmetry of the miRNA/miRNA* duplex, 5 nt; and flank sequence length of miRNA precursor, 10 nt. Finally, the identified miRNAs were predicted by Patmatch (v1.2) software. The minimum free energy of the miRNA/target duplex was set at ≥ 74% and there were no more than two adjacent mismatches in the miRNA/target duplex and no mismatches at positions 10–11 of the miRNA/target duplex.
Target gene function enrichment analysis
Blast2Go was employed for exploring the Gene Ontology (GO) annotation terms. Database for Annotation, Visualization and Integrated Discovery (DAVID) was adopted for pathway analysis with the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database [30, 31]. P values less than 0.05 indicated enriched gene sets.
Analysis of miRNAs and target genes by qRT-PCR
Total RNAs were reverse transcribed using the PrimeScript RT reagent Kit (TaKaRa, Shiga, Japan). qRT-PCR was conducted with the SYBR® PrimeScript™ miRNA RT-PCR Kit (RR716, Takara, China). Poly(A) tail addition and reverse transcription reactions were conducted in a total volume of 20 µΛ. The miRNA PrimeScript RT enzyme mix from the SYBR® PrimeScript™ miRNA RT-PCR Kit was used in the reverse transcription reaction mixtures, and the qRT-PCR primers used are listed in Table S2. Actin and U6 were adopted as internal control markers, and the relative expression of the miRNAs was calculated by the 2−ΔΔCt method. All qRT-PCR tests were performed with three biological replicates.
Degradome library construction and data analysis
Three degradome libraries (DR, BL and WL) were constructed using the juvenile sporophytes of S. japonica. Following the enrichment of mRNA, the obtained poly(A)-enriched RNA was ligated to oligonucleotide adaptors harbouring an MmeI recognition site. First-strand cDNA was generated from the ligated sequence via reverse transcription. After PCR amplification, the additional DNA products were yielded. After purification, digestion and ligation, the cDNA library was subjected for sequencing with Illumina Hiseq 2500.
Raw data obtained from HiSeq sequencing were processed to filter out the low-quality tags. The 5′ adapters, 3′ adapter contaminants, insert tags, and reads shorter than 18 nts were removed to obtain clean data, which were further mapped to the reference genome of S. japonica. By performing Blastn searches against the Rfam and National Centre for Biotechnology Information (NCBI) databases with an E-value cutoff of 10− 2, the full-length sRNA tags were annotated to non-coding RNAs, and all of which were discarded. Additionally, t-plots were constructed according to the category of sites to analyse the miRNA targets and RNA degradation patterns.
Statistical analysis
Statistical differences were examined using one-way analysis of variance (ANOVA) by SPSS 22.0. P-values less than 0.05 were considered to be significant.