Characterization of the chloroplast genome of the marine microalga Tetraselmis marina (Cienkowski) R.E.Norris, Hori & Chihara 1980

Abstract Tetraselmis marina (Cienkowski) R.E.Norris, Hori & Chihara 1980, a costal green microalga, is considered as a promising animal feed in aquaculture due to the high content of fatty acids and carotenoid. Furthermore, T. marina plays important roles in bioremediation. In this study, we assembled the complete chloroplast genome of T. marina. Results showed that the full length of the complete chloroplast genome was 96,151 bp, containing a large single-copy region of 62,574 bp, a small single-copy region of 1261 bp, and a pair of inverted repeat regions of 16,158 bp. The GC content of the genome was 36.6%. A total of 125 genes were annotated, including 81 protein coding genes, 38 tRNA genes, and six rRNA genes. Phylogenetic analysis based on 22 chloroplast genomes suggested that T. marina was closely related to Tetraselmis sp. CCMP 881.


Introduction
Tetraselmis marina (Cienkowski) R.E.Norris, Hori & Chihara 1980, a common coastal microalga, belongs to genus Tetraselmis (Chlorodendraceae).Tetraselmis sp. is commonly considered as a promising potential source of antioxidants or animal feed due to the high contents of fatty acid and carotenoid profile (Moussa et al. 2017;Oliveira Moser et al. 2022).Additionally, several species of Tetraselmis sp. are commonly potential candidates for radioactive Sr bioremediation (Fukuda et al. 2014).Interestingly, several species of Tetraselmis sp.possess biomineralization capacity, and can produce intracellular inclusions of amorphous calcium carbonate (i.e.micropearls, Martignier et al. 2018Martignier et al. , 2020)).It implies that Tetraselmis sp. may play a role in the ocean carbon cycle.However, the information about the chloroplast genome of T. marina has been not reported.In the present study, we sequenced and assembled the chloroplast genome of T. marina, and analyzed its phylogenetic position.

Methods
Whole-genome DNA was extracted according to a modified CTAB protocol (Doyle 1987).The lysis incubation was changed from 30 min to 60 min and then 2 lL RNase A was added at 37 � C for 30 min.The purified genomic DNA was sheared into 350 bp fragments to construct a paired-end (PE) library according to the Nextera XT sample preparation procedures (Illumina, San Diego, CA).The PE reads of 150 bp were generated using a Novaseq 6000 sequencer (Illumina, San Diego, CA) (Fig. S1).A total of 4.15 G of raw data was obtained for further analysis.The GC content, Q20 value and Q30 value of the clean data were 54.94%, 96.53%, and 91.09%, respectively.High-quality reads were assembled into the chloroplast genome using the de novo assembler SPAdes v.3.14.1 software.Finally, the PGA program was used to annotate the chloroplast genome (Qu et al. 2019), using the chloroplast genome of Tetraselmis sp.CCMP 881 (GenBank accession number KU167097.1)as the reference.In order to identify the phylogenetic relationship of T. marina, the 12 common protein-coding genes in each complete mitochondrial genome of 22  chloroplast genomes of related marine microalga species were aligned using the MAFFT version 7 software with the FFT-NS-2 strategy (Katoh and Standley 2016).Then, a phylogenetic tree was conducted based on the maximum-likelihood method using 1000 bootstrap replicates by IQ-TREE 2.0 (Nguyen et al. 2015).Oltmannsiellopsis viridis (GenBank accession number DQ291132.1)was used as an outgroup species.

Results
The complete chloroplast genome sequence of T. marina was submitted to GenBank under accession number ON645926.The length of chloroplast genome sequence of T. marina was 96,151 bp, consisting of two inverted repeat regions of 16,158 bp, separated by a large single-copy region of 62,574 bp, and a small single-copy region of 1261 bp (Figure 2).The overall GC content was 36.6%.There is a cis-splicing gene called atpB (Figure S2).A total of 125 genes were annotated, consisting of 81 protein-coding genes, six rRNA, and 38 tRNA genes.Phylogenetic analysis results showed that T. marina was closely related to Tetraselmis sp.CCMP 881 (Figure 3).

Discussion and conclusions
In this study, the complete chloroplast genome of T. marina was assembled and annotated for the first time.It was 96,151 bp, containing a large single-copy region of 62,574 bp, a small single-copy region of 1,261 bp, and a pair of inverted repeat regions of 16,158 bp.Compared with the chloroplast genome of the marine microalga previously published data, this result indicated that the chloroplast genome of T. marina showed a high level of gene synteny with one publicly available Tetraselmis sp.CCMP 881 (Turmel et al. 2020).Phylogenetic trees analysis provided new insight into the genetic relationship of T. marina.Further investigations are necessary to understand and document the evolution of the genus Tetraselmis.

Figure 1 .
Figure 1.The morphology of T. marina (A) in flasks and (B) under a microscope.Photograph was taken by Fangfang Yang.It is a unicellular with a size range between 10 and 20 lm.

Figure 2 .
Figure 2. Gene map of the complete chloroplast genome of T. marina using OGDRAW (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html).Genes shown on the outside of the circle are transcribed clockwise, while those inside are transcribed counterclockwise.Arrangement of 125 genes represented in the map, including 81 protein-coding genes, six rRNA, and 38 tRNA genes.