Characterization and phylogenetic analysis of the complete chloroplast genome of Asterothamnus centraliasiaticus Novopokr. (Asteraceae: Asterothamnus)

Abstract Asterothamnus centraliasiaticus Novopokr., a species of perennial deciduous semi-shrub within the family Asteraceae, has excellent medical, economic, ecological and genetic value. In this study, the chloroplast genome of A. centraliasiaticus was first assembled using Illumina HiSeq2500 sequences. The results indicate that the complete cp genome of A. centraliasiaticus is 152,205 bp in length, and comprises a pair of inverted repeat (IR) regions of 25,031 bp each, a large single-copy (LSC) region of 83,956 bp and a small single-copy (SSC) region of 18,187 bp. The GC content of A. centraliasiaticus is 37.3%. A total of 130 genes were successfully annotated containing 85 protein-coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes. The maximum likelihood (ML) phylogenetic analysis based on the complete chloroplast genome data highly supported that A. centraliasiaticus was close to Aster lavandulifolius. These results will provide significant genetic information for the germplasm protection and reasonable development.


Introduction
Asterothamnus centraliasiaticus Novopokr 1950 (Novopokrovsky 1950), is a species of perennial deciduous semi-shrub within the family Asteraceae, and is natively distributed in arid and semi-arid areas of the Qinghai-Tibetan Plateau in China and Northwest Mongolia, naturally occurring at elevations of 1300-3900 m (Figure 1). A. centraliasiaticus is an ecologically important species within its range especially by functioning in soil stabilization and as a food resource for livestock and wildlife (Chai et al. 2010).Furthermore, A. centraliasiaticus possesses bright-colored flowers, and can also be exploited as ornamental plant.However, this valuable wild germplasm has been seriously threatened by human activities and climate changes during the past decades, and need urgent conservation (Chai et al. 2011).A good insight into its population genetics would be fundamental to formulating efficient strategies for its conservation, management and exploitation (Yang et al. 2018).Unfortunately, to date there are no reports of complete chloroplast (cp) genomes for this species or even for plants of the same genus.To contribute to such efforts, we reconstructed its complete chloroplast genome from high-throughput illumina sequencing data and analyzed its phylogenetic position within Asteraceae based on 20 cp genome sequences.This study will provide significant genetic information for the germplasm protection and reasonable development.

Materials and methods
The fresh leaves of a single individual were collected from Guide County, Hainan Tibetan Autonomous Prefecture, Qinghai Province, China (101 � 53 0 47" E, 36 � 09 0 23" N, 2050 m a.s.l.), in August 2022.After on-site collection, the samples were immediately flash-frozen in −80 liquid nitrogen and then directly mailed to the sequencing company (Biotechnologies Inc.).The specimens (voucher no.QHGN-YLW24A5) have been deposited in the Key Laboratory of Superior Forage Germplasm in the Qinghai-Tibetan Plateau.College of Qinghai Academy of Animal and Veterinary Sciences, Qinghai University, Xining, China (URL: https://mky.qhu.edu.cn/index.htm,contact person: Ying Liu and email is liuying_yanhong@sina.com).
The total genomic DNA was extracted from leaf tissues using the TruSeq DNA sample Preparation kit (Vanzyme, China), and then sequenced using the Illumina Hiseq 2500 platform (Illumina, SanDiego, CA) with paired-end reads of 150 bp by Genesky Biotechnologies Inc, Shanghai, China.In total, 32,019,330 raw reads (2 � 150 bp) were obtained, which were trimmed using FastQC v 0.11.8.After trimming, the high-quality paired-end reads were assembled through metaSPAdes software (version 3.13.0).The sequence was matched to the genome, and all positions read mapping depth of assembled genome was statistically obtained, CONTACT Ying Liu liuying_yanhong@sina.com Qinghai University, Qinghai Academy of Animal and Veterinary Sciences, Qinghai Provincial Key Laboratory of Adaptive Management on Alpine Grassland, Key Laboratory of Superior Forage Germplasm in the Qinghai-Tibetan Plateau, Xining 810016, Qinghai, China Supplemental data for this article can be accessed online at https://doi.org/10.1080/23802359.2024.2306207.
respectively.In the process, cp genome of Aster indicus (GenBank: NC040126) was used as reference genome, and the genome annotation was performed with the program by CPGAVAS2 software (version N/A) (Shi et al. 2019) comparing the sequences with the cp genome of A. indicus.The map of cis/trans-splicing gene and plastome using the online program CPGView (http://www.1kmpg.cn/cpgview)(Liu et al. 2018), and then the annotated cp genome sequence was submitted to the GenBank with the accession number OP909739.
The complete chloroplast sequences of A. centraliasiaticus, and other thirteen species in Aster were used in the phylogenetic analysis.Two Medicago species were used as outgroups.All of the 16 cp genome sequences were obtained from GenBank, and were aligned using MEGA7 with default parameter.The maximum likelihood (ML) tree was built using MEGA7 with bootstrap set to 1,000.
The complete chloroplast sequences of A. centraliasiaticus, and other thirteen species in Aster were used in the phylogenetic analysis.Two Medicago species were used as outgroups.Phylogenetic analysis indicated that A. centraliasiaticus was close to Aster lavandulifolius (Figure 3).
and genetic information that can be used to enhance agronomic traits in plants or to produce high value agricultural or biomedical products (Daniell et al. 2016).In this study, the chloroplast genome of A. centraliasiaticus was first assembled using Illumina HiSeq2500 sequences.The results indicate that the complete cp genome of A. centraliasiaticus is 152,205 bp in length.Similar to previous studies (Kwon et al. 2022;Liu et al. 2018), it is also a standard quadripartite structure, comprises a pair of inverted repeat (IR) regions of 25,031 bp each, a large single-copy (LSC) region of 83,956 bp and a small single-copy (SSC) region of 18,187 bp.A total of 130 genes were successfully annotated containing 85 protein-coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes.In addition, the sequence of the genome has been widely used to determine evolutionary relationships in plants (Liu et al. 2022;Yang et al. 2018).In this study, the maximum likelihood (ML) phylogenetic analysis based on the complete chloroplast genome data highly supported that A. centraliasiaticus was close to Aster lavandulifolius.The obtained results will yield substantial genetic data pertaining to germplasm preservation and rational advancement.Furthermore, our findings indicate that certain species within the Aster genus exhibit a greater degree of dissimilarity at the chloroplast genome level compared to A. centraliasiaticus.This discovery necessitates a reassessment of the botanical species classification of A. centraliasiaticus, which in turn will furnish novel perspectives and theoretical substantiation for subsequent plant taxonomy.

Figure 2 .
Figure 2. Chloroplast genome map of Asterothamnus centraliasiaticus.The species name is shown in the left top corner.From the center outward, the first track shows the dispersed repeats.The dispersed repeats consist of direct (D) and palindromic (P) repeats, connected with red and green arcs.The second track shows the long tandem repeats as short blue bars.The third track shows the short tandem repeats or microsatellite sequences as short bars with different colors.The colors, the type of repeat they represent, and the descriptions of the repeat types are as follows.Black: c (complex repeat); green: p1 (repeat unit size ¼ 1); yellow: p2 (repeat unit size ¼ 2); purple: p3 (repeat unit size ¼ 3); blue: p4 (repeat unit size ¼ 4); orange: p5 (repeat unit size ¼ 5); red: p6 (repeat unit size ¼ 6).The small single-copy (SSC), inverted repeat (IRa and IRb), and large single-copy (LSC) regions are shown on the fourth track.The GC content along the genome is plotted on the fifth track.