Characterization and phylogenetic analysis of the complete chloroplast genome of Saussurea sagittifolia (Asteraceae, Cardueae)

Abstract The species of Saussurea sagittifolia Y. S. Chen & S. R. Yi belongs to the family Asteraceae (Cardueae). The complete chloroplast genome of S. sagittifolia was assembled and annotated for the first time in this study. The complete chloroplast genome of S. sagittifolia was 152,535 bp, including a large single-copy (LSC) region of 83,511 bp, a small single-copy (SSC) region of 18,632 bp, and a pair of inverted repeats (IRs) of 25,196 bp. The overall GC content of the chloroplast genome was 37.7%. The chloroplast genome encoded 131 genes, including 87 protein-coding genes, 36 tRNA genes, and eight rRNA genes. Phylogenetic analysis based on complete chloroplast sequences revealed that it related closely to Saussurea medusa.


Introduction
The Saussurea is one of the largest genera in the tribe Cardueae of family Asteraceae, with highly medicinal and ornamental value in China (Shi and Raab-Straube 2011;Li et al. 2022).It is mainly distributed in Asia, Europe, and North America (Shi and Raab-Straube 2011).Saussurea sagittifolia Y. S. Chen & S. R. Yi is a new species from the Bashan Mountains region in North Sichuan province, China in 2020 (Xu et al. 2020).The species of S. sagittifolia is close to S. oligocephala in morphological characteristics.However, it is different with the basal leaves triangular-ovate, a margin of small sharp teeth, apex acuminate, base with an auricular protrusion, and cauline leaves of more than 10, triangular, lanceolate, linear to subulate (Xu et al. 2020).In this study, we collected this species in Guangwu Shan Mountain, Bazhong City, Sichuan Province, China.We sequenced and reported the complete chloroplast genome of S. sagittifolia using next-generation sequencing technology for the first time.It will be helpful for well understanding the species delimitation studies and phylogenetic position of the genus Saussurea and S. sagittifolia in the Asteraceae.

Sampling, extraction, and genome sequencing
The fresh leaves and specimen of S. sagittifolia (Figure 1) were collected from Guangwu Shan in Nanjiang County, Sichuan Province, China (106 � 48 0 3.16 00 E, 32 � 39 0 45.8 00 N).The voucher specimen was deposited at the Herbarium of Sichuan Normal University (SCNU) (Zhi-Xi Fu, fuzx2017@sicnu.edu.cn) under the voucher number: Ya Deng, DY158.Total genomic DNA was extracted using a modified CTAB method (Doyle and Doyle 1987).A paired-end library with an insert size of 150 bp was constructed, and the library was sequenced using Illumina NovaSeq 6000 platform at Beijing Genomics Institute (BGI, Shenzhen, China).

Genome assembly and annotation
The high-quality reads were assembled with SPAdes v3.10.1 (Bankevich et al. 2012) and annotated by PGA (Qu et al. 2019) with default settings.To assess the accuracy of the assembly, we calculated the depth of coverage by mapping the reads to the chloroplast genome sequence using a specific protocol (https://doi.org/10.17504/protocols.io.4r3l27jkxg1y/v1).The  annotation result was drawn using the CPGview program (http://www.1kmpg.cn/cpgview/)(Liu et al. 2023).The complete chloroplast sequence of S. sagittifolia deposited in GenBank of the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov,accession number ON094066).

Phylogenetic analysis
In order to reveal the phylogenetic position of S. sagittifolia with other members of Asteraceae, a phylogenetic analysis was performed based on 24 complete chloroplast genomes of Asteraceae, and Anthriscus cerefolium (Apiaceae), Kalopanax septemlobus (Araliaceae) as outgroups.Sequence alignment was achieved using the MAFFT (Katoh and Standley 2013).A maximum-likelihood (ML) analysis was carried out with RaxML v7.2.8 based on the GTRGAMMA model on the CIPRES (https://www.phylo.org/)using 1000 bootstrap replicates (Stamatakis 2014).

Phylogenetic analysis
The phylogenetic analysis revealed that S. sagittifolia was belonging to the subfamily Carduoideae tribe Cardueae.Furthermore, S. sagittifolia was closely related to S. medusa.The genus Saussurea was recovered as a monophyletic group (Figure 3).The chloroplast genome sequence of S. sagittifolia in this study might provide vital information for phylogenetic and evolutionary studies in Asteraceae.

Figure 1 .
Figure 1.The morphology of leaf of S. sagittifolia.(a) The basal leaves of S. sagittifolia.(b) The leaves abaxially of S. sagittifolia.Leaf blades in middle and upper stem triangular, lanceolate, linear to subulate, apex long-caudate, base attenuate, margin with a few small sharp teeth to subentire.Leaves abaxially usually dark purple, capitula fewer, and involucres narrowly campanulate.The photos of S. sagittifolia were taken by the Zhi-Xi Fu in Guangwushan Mountain, Nanjiang county, Bazhong city, Sichuan province, China (the voucher number: Ya Deng, DY158).

Figure 2 .
Figure 2. The chloroplast genome map of S. sagittifolia generated using CPGview.Boxes of different sizes and colors in the outermost circle represent genes and their lengths.Genes inside the circle are transcribed clockwise, and those on the outside are transcribed counter-clockwise.The grey area in the middle circle represents the variation of GC content at different positions, and the regions and lengths represented by the tetrameric structures (LSC, SSC, IRa, and IRb) are plotted in different colors on the inner circle.