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Characterization of the complete chloroplast genome of Zanthoxylum esquirolii Levl. (Rutaceae)

Abstract Zanthoxylum esquirolii Léveillé 1914 is mainly distributed in southwest China, and its wild germplasm resources are scarce and in urgent need of conservation. In this study, we report the first complete chloroplast genome sequence of Z. esquirolii using next-generation sequencing. The circular genome is 158,390 bp in length, containing two inverted repeat (IR) regions of 27,622 bp separated by a large single copy (LSC) region of 85,580 bp and a small single copy (SSC) region of 17,566 bp. The chloroplast genome contains a total of 132 genes, including 87 protein-coding genes, 37 tRNA genes, and eight rRNA genes. The overall GC content of the chloroplast genome was 38.46%, with corresponding values in the LSC, SSC, and IR regions of 36.84%, 33.55%, and 42.51%, respectively. The phylogenetic tree revealed that Z. esquirolii Levl. formed a clade with Z. piperitum DC., Z. bungeanum Maxim., Z. simulans Hance and Z. sp. NH-2018, and had a strongly supported sister relationship with Z. bungeanum.

In 1914, L eveill e H. first described of Zanthoxylum esquirolii Levl. (L eveill e 1914). Z. esquirolii is an important economic forest tree species with good development and utilization value and is an excellent medicinal tree species mainly distributed in the provinces of Guizhou, Sichuan, Tibet and Yunnan in China (Huang, 1997). Z. esquirolii has the functions of warming the middle and dispelling cold, promoting blood circulation and relieving pain, and treating cold-related aches and pains, bruises, blood stasis, swelling and pain (He et al., 2011). This plant has great potential for use as a feedstock and medicine. However, the natural habitat of the species's fragmented, and wild resources of Z. esquirolii have been dramatically depleted and need urgent conservation. A large body of knowledge regarding its genetic information would contribute to the formulation of a protection strategy. No genomic information of Z. esquirolii has been reported to date. In this study, we present the first complete chloroplast genome sequence of Z. esquirolii and construct its phylogenetic relationships with related species based on Illumina paired-end sequencing data. These data will provide a reference for the development and protection of germplasm resources in the future.
The fresh leaves of a single individual of Z. esquirolii were collected from Xishui County, Guizhou,China (28.6712 N,106.4561 E), and a voucher specimen was deposited at the Chongqing University of Arts and Sciences Herbarium (GZLX1) under accession number CUAS-GZ20180720 (Xia Liu, liuxiavip8@163.com). Genomic DNA was extracted using a modified CTAB method (Doyle 1987). The DNA library was sequenced by Hefei Bio&Data Biotechnologies Inc. (Hefei, China) on the BGISEQ-500 platform with PE150 read lengths. The clean reads were used for the de novo assembly of the chloroplast genome using SPAdes Assembler v3.9.0 (Bankevich et al. 2012). With Z. bungeanum Maxim. (NC_031386) as the reference. The annotation of the complete genome was performed using CpGAVAS (Liu et al. 2012) and GeSeq software (Michael et al. 2017). After a manual check and adjustment, the annotated chloroplast genome sequence of Z. esquirolii was submitted to GenBank (MZ676709).
The chloroplast genome of Z. esquirolii is a double stranded, circular DNA 158,390 bp in length that contains two inverted repeat (IR) regions of 27,622 bp separated by a large single-copy (LSC) region and a small single-copy (SSC) region of 85,580 bp and 17,566 bp, respectively. The chloroplast genome encodes a total of 132 genes (87 proteincoding, 37 tRNA, and 8 rRNA genes), with 18 duplicated genes (7 protein-coding, 7 tRNA, and 4 rRNA genes). Nineteen genes contain two exons and four protein-coding genes (ycf3, clpP, and two rps12) contain three exons. The overall GC content of Z. esquirolii is 38.46% and the values in the LSC, SSC and IR regions are 36.84%, 33.55%, and 42.51%, respectively.
The phylogenetic analysis was performed using 12 complete plastid genomes, with Phellodendron amurense Rupr. and Phellodendron chinense Schneid. as the outgroup (Figure 1). The 12 complete chloroplast genome sequences were subjected to multiple sequence alignment using MAFFT software (Katoh and Standley 2013). The best models of the complete chloroplast genomes for the ML and BI phylogenetic analyses were determined by jModelTest 2.1.1 (Posada 2008) with an Akaike Information Criterion (AICc). The best fitting evolutionary model for the combined complete chloroplast genome dataset was TVM þ I þ G in the ML and Bayesian analyses. A maximum likelihood (ML) phylogenetic tree was built using the RAxML version 8 program (Alexandros 2014) with 1000 bootstrap replicates. For BI in MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003), two independent Markov chain Monte Carlo (MCMC) runs were performed and contained four MCMC chains that were run for 1000,000 generations and sampled every 1000 generations; all other parameters were set to default. The first 25% of the sampled trees were abandoned as burn-in to check the stability of each run, and the posterior probabilities (PP) were calculated from the remaining trees. All phylogenetic trees were viewed using the Figtree v1.4.2 program (Rambaut 2014). The phylogenetic trees generated by the ML and Bayesian methods were most similar to each other but with different branch support values in some clades (Figure 1). Phylogenetic analysis showed that the Zanthoxylum species formed a monophyletic group. Z. esquirolii is most closely related to Z. bungeanum and is sister to Z. sp. NH-2018 and Z. simulans Hance, with 100% bootstrap support.
The limited number of polymorphic loci produced by lowresolution markers hinders the phylogenetic research on Zanthoxylum species in previous studies (Medhi et al. 2014;Feng et al. 2015;Kim et al. 2017;Appelhans et al. 2018). The emergence of the chloroplast genomes of a large number of Zanthoxylum species can provide important insights into the evolution of Zanthoxylum in eastern Asia. This complete chloroplast genome can be used for phylogenetic, population, and chloroplast genetic engineering studies of Z. esquirolii and is fundamental for the creation of new conservation and management strategies for this important medicinal plant species.

Ethical approval
The present study was approved by the authors' institution (the Chongqing University of Arts and Sciences) and national. The research does not involve a threatened/endangered species. All the research meets ethical guidelines and adheres to the legal requirements of the study country. The collection of plant material has been carried out in accordance with the International Union for Conservation of Nature (IUCN) policies research involving species at risk of extinction, the Convention on Biological Diversity and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.

Author contributions
Xia Liu, Qinqin Huang and Zexiong Chen designed the study, writing and revised the manuscript; Chong Sun and Han Liu involved in the process of sequences editing and phylogenetic analyses; Can He, Fengting Huang and Haowen Liu participated in the collection and identification of plant material. All authors read and approved the final manuscript, and agreed to be accountable for all aspects of the work.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Data availability statement
The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at (https://www.ncbi.nlm.nih.gov/) under the accession no. MZ676709. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA680256, SRR17163937, and SAMN23766595, respectively.