Nucleic acid isolation and sequencing

A single genotypic isolate (V1) of C. lentillifera, which was an offspring of strains originated from Nha Trang, Vietnam, was collected from our marine culture base in Changjiang, Hainan province and then cultured in sterilized seawater. To minimize the contamination of environmental microbes, the thallus of V1 was treated with a combination of various antibiotics following Brawley et al. [58] for 2 weeks.

For genome sequencing, DNA of the C. lentillifera isolate V1 was extracted using a Plant Genomic DNA Kit (DP305, Tiangen Inc., Beijing, China) following the manufacturer’s instructions. A 20-kb insert SMRTbell library was prepared and sequenced by Novogene (Beijing, China) using the PacBio Sequel platform (Pacific Biosciences, CA, USA).

For PacBio full-length cDNA isoform sequencing (Iso-Seq), C. lentillifera samples were treated under multiple conditions, such as high temperature (30 °C), low temperature (18 °C), high salinity (50 PSU), low salinity (15 PSU), high light (260 μmol photons·m^− 2·s^− 1), shading, desiccation and normal conditions (25 °C, 32PSU and 10 μmol photons·m^− 2·s^− 1). Total RNA of each treatment was isolated with the RNAprep pure plant kit (Tiangen Inc., Beijing, China) and treated with RNase-free DNaseI (RT411, Tiangen Inc., Beijing, China) following the manufacturer’s instructions. The quality of RNA was checked on a Bioanalyzer 2100 system (Agilent, Palo Alto, CA, USA). Then extracted RNAs were pooled evenly, and Iso-Seq libraries were constructed with SMARTer™ PCR cDNA Synthesis Kit (Clontech, CA, USA) according to the manufacturer’s instructions, which uses a modified oligo (dT) primer (CDS Primer II A) for reverse transcription of polyA+ tail transcripts. cDNA size was selected by BluePippin™ Size-Selection System (Sage Science), and then sequenced on the PacBio Sequel platform.