Sequencing and Phylogenetic Analysis of AS-1

The sequence of the 18S rRNA-ITS1-5.8S rRNA-ITS2 operon was recovered when the partial genome of AS-1 was sequenced. The total DNA of AS-1 was extracted using a modified phenol–chloroform protocol [32 ]. The purity and quantity of the extracted DNA were assessed with a NanoDrop Spectrophotometer ND-1000 (ThermoFisher Scientific, Inc., Waltham, MA, USA. The genome sequence was determined using the New-Generation Illumina MiSeq (Illumina, Inc., San Diego, CA, USA) at the BioAnalytical Services Laboratory at the Institute of Marine and Environmental Technology. This sequencing effort was mainly to sequence the chloroplast genome of AS-1, not the chromosomal genome of AS-1. Genome data were assembled using SPAdes (Version 3.15.4). The 18S rRNA-ITS1-5.8S rRNA-ITS2 region of AS-1 was identified from the genome data using the 18S rRNA-ITS1-5.8S rRNA-ITS2 operon of A. protothecoides SAG 211/8D (Accession number: FR865686.1). The sequence of the 18S rRNA-ITS1-5.8S rRNA-ITS2 operon of AS-1 was aligned using MEGA 11 with the available data from the public NCBI nr database based on the BLAST result. This operon sequence of AS-1 was deposited in the NCBI database under accession number PP623876. Sequences were trimmed and aligned using the ClustalW method with MEGA 11. Maximum likelihood trees were constructed with 100 bootstrap values. The reference sequences used for the phylogenetic tree construction were retrieved from the NCBI nr database, and the corresponding accession numbers were included.