Comparative Chloroplast Genomes and Phylogenetic Analyses of Zanthoxylum L. Provide Insights into Species Delimitation and Phylogenetic Relationships

doi:10.21203/rs.3.rs-2188002/v1

Background: Zanthoxylum L. (Rutaceae), including a large number of economically, ecologically and medicinally important species, is widely distributed all over the world. However, there are few studies about its chloroplast genome information to date.

Results: Our results showed that the chloroplast genomes have a typical quadripartite structure with one large single copy, one small single copy, and two inverted repeat regions. The size of the genomes ranged from 157,231 bp to 158,728 bp in length, and 132 genes were identified in four species, which including 87 protein-coding genes, 37 transfer RNA genes and 8 ribosomal RNAs, while contained 133 genes (88 protein-coding genes) in Z. piasezkii chloroplast genome. The IR-SC boundary regions exhibited great variation among these five chloroplast genomes.

Conclusions: Our findings suggest that the rpl22 gene was truncated in Z. piasezkii, which was was different from other Zanthoxylum species and could be used as a marker for accurate identify Z. piasezkii. Our plastid tree indicates the possibility that the earlier intrageneric classifications, especially subgenus, need to be further refined. These findings will provide insights into the genetic diversity, evolutionary history, and species identification of Zanthoxylum.

Zanthoxylum

Complete chloroplast genome

Phylogeny

Molecular evolution

Ancestral populations of Chinese peppers inhabited the mountain regions in Shanxi, Henan, Hunan, Gansu, Hubei and Sichuan Provinces and are highly adapted to a cold and dry environment, which may indicate the presence of an important gene pool. However, wild populations are largely absent from marginal regions or have been replaced by cultivated populations [1, 2]. Therefore, the identification and evaluation of Chinese pepper genetic resources are important for their effective utilization and preservation.

Plant chloroplasts are the principal sites of photosynthesis and carbon fixation [3]. The genetic information in the chloroplast genomes contains valuable information for molecular evolution, phylogeny, and species identification [4–6]. Chloroplast genomes in angiosperms typically contain a quadripartite circular structure with one large copy (LSC), one small copy (SSC), and two copies of inverted repeats (IR) [7]. In addition, chloroplast genomes can also be mapped rapidly, efficiently, and affordably using next-generation sequencing techniques nowadays.

There are a number of species of Zanthoxylum in the Rutaceae family, a group of subshrub or arboreal plants found across Asia, Africa, Oceania, and North America, including approximately 250 species [8]. Among them, 45 species and 13 varieties are found in China, which is considered one of the major diversity centers for Zanthoxylum [9]. Among these, five wild Chinese pepper resources are the most common in China, including Zanthoxylum bungeanum Maxim., Z. armatum DC., Z. nitidum (Roxb.) DC. Z. ailanthoides Sied. et. Zucc. and Z. piasezkii Maxim. The fruits of Zanthoxylum have been used as spices throughout eastern Asia for centuries. In traditional Chinese medicine, the roots, stems and leaves of many Zanthoxylum species are used to treat toothache, stomachache, neuralgia, rheumatic arthralgia, sore throat and snakebites [10]. In particular, Z. nitidum has a variety of physiological activities that make it more useful, and it even has anticancer and anti-inflammatory properties [11].

Currently, the majority of Zanthoxylum species research is focused on its chemical components and pharmacological properties [10], and phylogenetic analysis using chloroplast markers including trnL-trnF and matK [12–14], but chloroplast whole-genome comparative and phylogenetic analyses of Zanthoxylum species, however, are less common. Therefore, the complete chloroplast genomes of the five widespread Zanthoxylum species were obtained by sequencing, assembly and comparison and analysis of these data with those from fifteen previously published Zanthoxylum species. The genomes of the different species differ clearly at the molecular level and can be used to identify and divide them. Identifying repeated sequences and observing the overall structure of chloroplast genomes may help develop SSR markers that can be used to analyze the genetic diversity of Zanthoxylum species. In addition, by analyzing the entire chloroplast genome, we can better resolve deep-level relationships within the plant community, which is of great significance for species identification. Our findings will supply rich fundamental data on chloroplast genetic diversity and evolutionary relationships and contribute to Zanthoxylum species genetic resources for further research in this genus.

Plant material

Fresh leaves of Z. piasezkii, Z. bungeanum and Z. armatum were collected from Sichuan Academy of Botanical Engineering, Neijiang City, Sichuan Province, China, and leaves from Z. ailanthoides and Z. nitidum were obtained from South China Botanical Garden in Guangzhou, China. The fresh leaves were collected and dried with silica gel desiccants. Zexiong Chen (Chongqing University of Arts and Sciences, Chongqing, China; Email: [email protected]) and Xia Gong (Sichuan Academy of Botanical Engineering, Neijiang, Sichuan Province, China; Email: [email protected]) undertook the formal identification of the plant material used in our study. Voucher specimens of the five Zanthoxylum species were preserved in the herbarium of Chongqing University of Arts and Sciences, Chongqing, China. Voucher numbers of Z. piasezkii, Z. bungeanum, Z. armatum, Z. nitidum, and Z. ailanthoides are 18LCS1, 18LHJ, 19L-ZY, 19L-LMZ23, and 19LCY1, respectively. All samples are collected with the permission of the relevant institutions.

DNA Extraction and Sequencing

Using an improved CTAB protocol [15], 100 mg leaf tissue was used to isolate the genome. After DNA isolation and quality testing, DNA fragments were used to build 350 bp short-insert libraries, which were then sequenced on the BGISEQ-500 using PE 150 bp (BGI, Shenzhen, China) following the manufacturer’s protocol.

Chloroplast Genomes Assembly and Annotation

High-quality clean paired-end reads were mapped to the reference chloroplast of Rutaceae obtained from GenBank using Bowtie 2 v.2.3.4.3 [16] with default parameters. NOVOPlasty v.2.6.2 [17] used three coding gene sequences with the greatest coverage for de novo assembly of the chloroplast genome. Genes annotation and analysis were performed using the GeSeq [18] and CPGAVAS2[19] software. The tRNA genes were detected using tRNAscan-SE v2.0 [20], and then manually adjusted and confirmed using Geneious v9.1.8 [21]. The circular genome map was drawn by Organellar GenomeDRAW tool (OG-DRAW) v.1.3.1 [22] for further comparison of the gene order and content with default settings. A similar method was applied to the annotation of the other genomes obtained from GenBank for comparative analysis. Complete chloroplast genome sequences of five newly assembled and annotated Zanthoxylum species have been submitted to GenBank with the accession numbers MW206785, MW206786, MW602887, MW602879, and MW478808.

Chloroplast Genomes Comparison and Sequence Variation Analysis

Codonw software [23] was used to calculate the Relative Synonymous Codon Usage (RSCU) for each codon in each genome. These plastome junction regions were compared using the IRscope + online program [24] to determine the position of their junctions between IR, SSC, and LSC. Shuffle-LAGAN mode [25] in mVISTA 2.0 [26] was used to visualize divergent regions with the Z. ailanthoides (MW478808) genome as a reference. The nucleotide sequence polymorphism was determined using DnaSP v6.12.03 [27] with a 15 bp step size and a 200 bp window length. The program REPuter [28] with adjusted parameters was used to identify long repeat sequences (forward, palindrome, complement and reverse repeats) in the five plastomes. The minimum repeat size was determined to be 25 bp, and the two repeat copies had greater than 90% similarity. SSRs with minimal repeat numbers of 8, 5, 3, 3, 3, and 3 were detected for mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide repeats, respectively, and were used in MISA v1.01 [29].

Phylogenetic Analysis

The whole chloroplast genome sequences of fifteen Zanthoxylum species were downloaded from the NCBI database( and combined with the five newly assembled Zanthoxylum chloroplast genomes were used to analyze the phylogenetic relationships; Phellodendron chinense and Tetradium ruticarpum were set as the outgroups. The accession information for samples used in the study are provided in the supplementary files (Table S1).

Alignment was performed using MAFFT v7.0 with default parameters [30], and then manual adjustment of multiple sequence alignment was performed with BioEdit software [31]. PhyML v2.4.4 [32] and MrBayes v3.2.6 [33] were used to construct the phylogenetic relationships. Nucleotide substitution models were determined using jModelTest v2.1.1 [34]. ML analyses were conducted using a GTR + I + G model with 1000 replicates.

The Chloroplast Genome Structures of Zanthoxylum Species

Five Zanthoxylum species were sequenced, revealing a typcial quadripatite structure, with a range in length from 157,231 (Z. ailanthoides) to 158,728 bp (Z. piasezkii) (Fig. 1, Table 1). There are two inverted repeat (IR) regions in the plastid genome, IRa and IRb, each separated by a large single-copy sequence (LSC, 84, 368–86, 122 bp) and a small single-copy sequence (SSC, 17, 603–18, 293 bp). There was a slight variation in the GC content of plastomes among the five species, ranging from 38.4–38.5% (Table 1). Most of Zanthoxylum species possess 132 genes encoded by their plastomes, which include 87 protein-coding, 37 transfer RNA (tRNA) and eight ribosomal RNA (rRNA) genes (Fig. 1a, Table 1, Table 2), while there were 133 genes (88 protein-coding genes) in Z. piasezkii (Fig. 1b, Table 1). The main difference was that there were two rpl22 genes in Z. piasezkii, which were located on the IR side of the junction between IR and LSC. The length of rpl22 in Z. piasezkii was relatively short, only 168 bp, while that in other four species were relatively long, ranging from 255 to 450 bp, which is only located across the LSC/IRb junction.

Among these genes, nine protein-coding genes (rps19, rps7, rpl2, rpl23, rpl22, ycf2, ycf15, ndhB, and ycf1), seven tRNA genes (trnR-ACG, trnN-GUU, trnA-UGC, trnV-GAC, trnL-GAU, trnL-CAA, and trnL-CAU) and four rRNA genes (rrn5, rrn16, rrn4.5, and rrn23) were duplicated in the IR regions. Additionally, 19 genes, including 13 protein-coding genes and six tRNA genes, had two exons, while four protein-coding genes (pafI, clpP1, and two rps12) had three exons (Table S2 ).

Table 1

New sequencing and annotation of the complete chloroplast genomes of *Zanthoxylum* species.
Species	Accession Number	Plastome (bp)	LSC (bp)	SSC (bp)	IRs (bp)	Total genes	Protein-coding genes	tRNA genes	rRNA genes	Overall GC content (%)
Z. bungeanum	MW206786	158,401	85, 898	17, 611	27, 466	132	87	37	8	38.5
Z. piasezkii	MW206785	158,728	85, 918	17, 612	27, 599	133	88	37	8	38.4
Z. armatum	MW602887	158,558	85, 759	17, 603	27, 598	132	87	37	8	38.5
Z. nitidum	MW602879	157,304	84, 368	17, 634	27, 651	132	87	37	8	38.5
Z. ailanthoides	MW478808	157,231	86, 122	18, 293	26, 408	132	87	37	8	38.4

Table 2

Annotated gene classification of the chloroplast genome of *Zanthoxylum* species
Category	Genes Group	Genes Name	Number
Self-replication	tRNA genes	trnH-GUG、trnK-UUU、trnQ-UUG、trnS-GCU、trnG-UCC、trnR-UCU、trnC-GCA、trnD-GUC、trnY-GUA、trnE-UUC、trnT-GGU、trnS-UGA、trnG-GCC、trnfM-CAU、trnS-GGA、trnT-UGU、trnL-UAA、trnF-GAA、trnV-UAC、trnM-CAU、trnW-CCA、trnP-UGG、trnI-GAU(2)、trnL-CAA(2)、trnV-GAC(2)、trnI-GAU(2)、trnA-UGC(2)、trnR-ACGc、trnN-GUU(2)、trnL-UAG	37
	rRNA genes	rrn5(2)、rrn4.5(2)、rrn16(2)、rrn23(2)	8
	DNA-dependent RNA polymerase	rpoC1、rpoC2、rpoA、rpoB*	4
	Ribosomal small subunit	rps16、rps2、rps14、rps4、rps18、rps12(2)、rps11、rps8、rps3、rps19(2)、rps15、rps7(2)	15
	Ribosomal large subunit	rpl33、rpl20、rpl36、rpl14、rpl16、rpl22(2)、rpl2(2)、rpl23(2)、rpl32、*	11
photosynthesis	Photosystem I	psaA、psaB、psaC、psaI、pafI、pafII、psaJ	7
	Photosystem II	psbA、psbB、psbC、psbD、psbK、psbI、psbM、psbZ、psbJ、psbL、psbF、psbE、psbT、psbN、psbH	15
	Cytochrome b/f complex	petN、petA、petL、petG、petB、petD	6
	ATP synthase	atpE、atpB、atpA、atpF、atpH、atpI*	6
	Protease	clpP**	1
	Large subunit of rubisco	rbcL	1
	NADH dehydrogenase	ndhJ、ndhK、ndhC、ndhB(2)、ndhF、ndhD、ndhE、ndhG、ndhI、ndhA、ndhH	12
Others	Maturase	matK	1
	Envelope membrane protein	cemA	1
	Subunit of acetyl-CoA carboxylase	accD	1
	Cytochrome c synthesis	ccsA	1
Function unknown	Open reading frames	ycf1(2)、ycf2(2)、ycf15(2)	6

Note: *Gene contains one intron; **Gene contains two introns; (2) indicates with two copies of the gene.

All of the protein-coding genes were composed of 25,825–26,512 codons in the chloroplast genomes of the five species of Zanthoxylum (Fig. 2, Table S3). Among these codons, leucine, arginine and serine represent the most abundant amino acids, whereas methionine (1.10–1.15%) has the lowest abundance. Based on the relative synonymous codon usage (RSCU) statistical analysis all amino acids have more than one synonymous codon, except for methionine (AUG) and tryptophan (UGG) (RSCU = 1). Moreover, half of the codons had RSCU > 1, and most of those (29/31, 93.5%) ended with A or U. The rest of the codons had RSCU < 1, and most of those (28/31, 90.3%) ended with G or C (Table S3).

Comparative Genomic Divergence and Hotspots Regions

The sequence divergence of the cpDNA of five species (Z. nitidum, Z. bungeanum, Z. armatum, Z. ailanthoides and Z. piasezkii) was drawn by mVISTA, with Z. armatum (MW602887) as a reference (Fig. 3). Z. ailanthoides’ entire chloroplast sequence revealed sequence variation different from the four other species. The comparison among the five cpDNAs showed that the intensity of variation in the IR region was low, and the most variation was observed in the LSC regions and in the combined sites of the IR and LSC regions. Furthermore, the sequence divergence among the five entire cp genomes was evaluated for the nucleotide diversity (Pi) value (Fig. 4). In terms of rps19-psbA, rps16-psbK, psbL-atpA, rpoB-petN, rsbZ-rps14, an ndhF displayed the highest Pi values, whereas rsbZ-rps14 displayed the most variability. Using these divergent sites, DNA markers could be developed for identifying species and reconstructing phylogenetic trees in the genus Zanthoxylum.

IR Contraction and Expansion

The IR regions of the five chloroplast genomes ranged in size from 26,408 bp (Z. ailanthoides) to 27,651 bp (Z. nitidum) (Table 1, Fig. 1). We compared the IR borders in five widespread Zanthoxylum species, and the results showed that the IR junction regions showed slight changes (Fig. 5). In contrast, the rps3 and rps19 genes were fully located in the LSC and IR regions for five Zanthoxylum species, respectively. It is worth noting that the rpl22 gene was fully situated in the LSC/IRb border for Z. ailanthoides, Z. nitidum, Z. bungeanum, and Z. armatum, while the rpl22 gene of Z. piasezkii was truncated in the IRa and IRb regions (Fig. 5). The ycf1 was fully located in the SSC/IR borders with the same length 5487 bp expanded into the IRa region, while ycf1 gene was situated in IRb/SSC boundary have equal size 205 bp expanded into the SSC region for Z. nitidum, Z. bungeanum, Z. armatum, and Z. piasezkii, only have 4 bp expanded into the SSC region for Z. ailanthoides. The photosynthetic ndhF gene was fully situated in the SSC region in Z. ailanthoides, whereas Z. nitidum, Z. piasezkii, Z. bungeanum, and Z. armatum had an identical distance (23 bp long) from the SSC to the IRb regions, indicating a closer genetic relationship between them. The rpl2 gene were all in the IRa region for the five species. The gaps in the trnH sequences in the LSC region were 5bp, 36bp, 211bp, 52bp and 53 bp away from the IRa/LSC border in the five Zanthoxylum chloroplast genomes.

Analyses of Repeat Structure and Simple Sequence Repeats

For the repeat structure analysis in the five chloroplast genomes (Fig. 6 and Table S4), palindromic repeats were not found in all five species. Most repeats were between 20 and 39 bp in length, while the longest forward repeats were 73 bp in length and found in the LSC region in the chloroplast genomes of the five Zanthoxylum species. The total number of SSRs was also identified in the chloroplast genomes of the five species (Fig. 7 and Table S5). Only four types of SSRs (mononucleotide to tetranucleotide repeat motifs) were identified in the five species. Most of these SSRs had mononucleotide repeats, with A/T repeats being the most common, with 97.1% and 96.7% found in Z. nitidum and Z. armatum, respectively, while Z. nitidum, Z. pinasezkii and Z. bungeanum are all characterized by AT/TA repeats in their dinucleotide repeat motifs (all 100%). The five species of Zanthoxylum all comprised AAAT/ATTT and ACAT/ATGT repeats.

Phylogenetic Relationship Analyses

The maximum likelihood (ML) and Bayesian inference (BI) methods were used to construct a phylogenetic tree using 20 Zanthoxylum plastid genome sequences (160,430 bp). Phellodendron chinense and Tetradium ruticarpum were used as outgroups, which resulted in similar topology with only slight differences in the support values in some branches (Fig. 8). All Zanthoxylum species formed a high-resolution clade. Z. paniculatum and Z. madagascariense were in the top position and gathered together to form a single branch and then sisters to six subgenus Fagara species of Zanthoxylum. Z. tragodes is the sister species of seven subgenus Zanthoxylum species. However, it is worth noting that Z. nitidum and Z. nitidum var. tomentosum belongs to subgenus Fagara based on the record in the Flora of China [8], but rather than being grouped together subgenus Fagara branch, they were sister to subgenus Zanthoxylum species.

The Features of Zanthoxylum Species Chloroplast Genomes

This study examined the chloroplast genomes of five Zanthoxylum species. It was found that all five species had a typical structure and order of characteristics, which were consistent with the characteristics of angiosperms [35]. There was less divergence among the coding regions than among the noncoding regions, and the inverted repeat regions (IRs) were more conserved than the single-copy (SC) regions. Similar results have also been found in the chloroplast genomes of other genera, including Saposhnikovia [36], Gynostemma [37], Fritillaria [38], Rehmannia [4], and Aconitum [39]. Zanthoxylum chloroplast genomes exhibited a lower GC content than AT content, a phenomenon seen in other angiosperm chloroplast genomes [40–42]. In addition, the results showed that the GC content was highest in IR regions, possibly due to the large quantities of rRNA present there.
Despite their uniparental inheritance and high level of conservation, SSRs (simple sequence repeats) are widely used to study plant populations, identify species, and infer their evolutionary history [43, 44]. Li et al. [14] developed SSRs derived from the chloroplast genome (cpSSRs) to analyze the genetic diversity among Zanthoxylum species. In our study, 246–268 SSRs were identified in the five plastid genomes (Fig. 7 and Table S5). The number of poly(C)/(G) SSRs in the Zanthoxylum cp genome is much lower than that of poly (A)/(T) (Table S5), which is consistent with previous findings (Xue et al., 2019; Zhao et al., 2020). In addition, Zanthoxylum's phylogenetic relationships were revealed using effective markers designed based on hotspot regions within the five chloroplast genomes. Overall, Zanthoxylum species will benefit from these newly identified cpSSR markers, contributing to understanding their diversity, genetic structure, and differentiation in the future.

IR Expansion and Contraction

Border contractions and expansions within the IR region, a dominant area for changes in the length of the chloroplast genomes and the shifts in the IR region borders, are common evolutionary events [45–47], are prevalent in many species, and play essential roles in plant evolution [48, 49]. Many reports have indicated that IR regions are the most conserved regions of the chloroplast genome in most plant species [50], but some studies have also reported that many chloroplast genome sequences in most plant species are rearranged, including the inversions and reinversions in the LSC and SSC regions [51–53]. In our study, the length of the chloroplast genomes of the five Zanthoxylum species did not change significantly by approximately 158 kb. Although the IR junction regions of the five Zanthoxylum species were not significantly different, our results showed that except for the photosynthetic ndhF genes of subgenus Zanthoxylum, which exhibited an identical (23 bp) distance from the SSC to the IRb regions for Z. bungeanum, Z. armatum, and Z. piasezkii (Fig. 5), the ndhF genes of Z. nitidum in subgenus Fagara also has this characteristic. The trnH sequence gaps in LSC have significantly different distances away from the IRa/LSC border in the five Zanthoxylum chloroplast genomes. Hence, the psbA-trnH intergenic spacer region was selected as a candidate DNA barcode sequence to distinguish similar species [54] and even at the family level [55, 56]. Therefore, the psbA-trnH sequence region to develop DNA barcoding for Z. nitidum, Z. bungeanum, Z. armatum, and Z. piasezkii.

It is noteworthy that a stop codon appeared in the coding region of the rpl22 gene in Z. piasezkii, resulting in truncation of the rpl22 gene in the IRb and IRa regions in our study (Fig. 5). To verify whether truncation of the rpl22 gene sequence was actually present, we matched the sequencing reads to the assembled chloroplasts and found that this locus was a homozygous DNA locus (about 1000x coverage depth) by read alignment (Fig. 9a), indicating that there is no full-length rpl22 gene in the chloroplast genome of Z. piasezkii. In addition, we intersected 20 bp sequences upstream and downstream of this locus and compared them in the Genebank database to further determine whether truncation of the rpl22 gene is common in the chloroplast genome of plant species. We found that the truncated rpl22 gene with the same stop codon (TGA) existed in chloroplasts of two Citrus species, Citrus × polytrifolia Govaerts (GenBank no. NC_045403) and Citrus trifoliata L. (GenBank no. MN102360) (Fig. 9b). Mutations in this locus leading to the emergence of a truncated rpl22 gene may be universal, at least within Rutaceae (Zanthoxylum and Citrus). In fact, we compared with rpl22 gene in other chloroplasts of Zanthoxylum species, the rpl22 gene in Z. bungeanum (MW206786) chloroplast showed 156 bp deletion (with the same start and stop codon), which resulted in the truncated rpl22 gene. In order to further analyze the authenticity and heterozygosity of deletion, the reads and homologous sequences alignment were also carried out, and it was also found the deletion under the around 800×coverage depth. The homologous sequence alignment showed that deletion occurred in both Z. bungeanum and Z. piasezkii species. So the long sequence insertion/deletion mutation of rpl22 gene is a common phenomenon in Zanthoxylum. As a result of this intriguing finding, we may obtain comprehensive understanding the structure of plastomes within Zanthoxylum and will gain valuable insights into its evolution history, classification, and phylogeny.

Phylogeny and Species Identification of Zanthoxylum

Chloroplast genome sequencing, especially the application of next-generation sequencing, has become simpler and easier with its continuous development [57]. Currently, researchers have widely used the entire chloroplast genome sequence to conduct evolution, classification, and phylogenetic studies of angiosperms [58–60]. Our study included 20 Zanthoxylum species with robust phylogenetic relationships based on ML and BI. The results showed that almost all relationships inferred from chloroplast genome data were generally highly supported based on the two methods (Fig. 8). Zanthoxylum species were all clustered together to form a single clade with high resolution, which was almost completely consistent with the classification record in the Flora of China [8]. However, it is noteworthy that Z. nitidum, Z. nitidum var. tomentosum in subgenus Fagara and Z. tragodes clustered together to form a single clade and sister to other subgenus Zanthoxylum species, supported Z. nitidum belonging to subgenus Zanthoxylum instead of subgenus Fagara, which showed some differences from the traditional classification system. In addition, Z. paniculatum and Z. madagascariense were in the top position gathered together to form a single branch and then were sisters to other Zanthoxylum species. The results proposed that the possibility of the classification of the genus Zanthoxylum should be further updated. Overall, our research provides robust phylogenetic relationships of Zanthoxylum based on the entire chloroplast genome, which might provide insights into the genetic diversity, molecular breeding, and plastome evolution of the genus Zanthoxylum.

In this study, we analyzed five species that are widely distributed and cultivated in China. The results showed that the genome length and gene content of the genus Zanthoxylum were comparatively conserved, while the IR-SC boundary regions were variable between the chloroplast genomes of the five Zanthoxylum species. The whole rpl22 gene in the Z. piasezkii species, which was different from other Zanthoxylum species and could be used as a marker for accurate identify Z. piasezkii. We identified SSR sites and seven variable regions that may be used to develop tools to study Zanthoxylum species in the future. A phylogenetic tree was constructed with the whole chloroplast genomes to better understand the genetic relationships of the Zanthoxylum species, and the results support that Z. nitidum belongs to subgenus Zanthoxylum instead of subgenus Fagara, which seems to suggest that the earlier intrageneric classifications need to be further refined. Briefly, our research provides extensive resources for studying the chloroplast genome of prickly ash and provides valuable reference information for species identification and selection of parents with closer genetic relationships in Zanthoxylum.

SSR: Simple sequence repeats; RSCU: Relative synonymous codon usage; BI: Bayesian Inference; ML: Maximum likelihood; Pi: Nucleotide diversity; tRNA: transfer RNA; rRNA: ribosomal RNA; CP: Chloroplast; LSC: Large single copy region; SSC Small single copy region; IR: Inverted repeat region; SC: Single copy; CTAB: Hexadecyltrimethy ammonium bromide.

Acknowledgments

The authors special thanks to the staff of Kunming Botanical Garden (Yunnan, China), Shenzhen Xianhu Botanical Garden (Guangdong, China), and South China Botanical Garden (Guangdong, China) for collecting plant material used for experiments. The authors would like to thank Yinming Wu and his team (Sichuan Academy of Botanical Engineering) for help with fieldwork.

Authors’ Contributions

Conceptualization: XL and ZXC. Resource: XL, XXZ and CS. Software: HL, XL and QQH. Formal analysis: XL. Data curation: MZL. Writing original draft preparation: XL. Visualization: HML. Supervision: ZXC. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (31901324), Youth Projects of Chongqing Municipal Education Commission (KJQN202201337; KJQN202101320; KJQN202201332).

Availability of data and materials

The data presented in this study are openly available in NCBI database (https://www.ncbi.nlm.nih.gov/). The GeneBank numbers of five newly sequenced samples (Z. ailanthoides, MW478808; Z. armatum, MW602887; Z. bungeanum, MW206786; Z. piasezkii, MW206785; Z. nitidum, MW602879)

Ethics approval and consent to participate

No specific permits were required, materials collection and molecular experiments were carried on following current Chinese regulations.

Consent for publication

Not applicable.

Competing interests

The authors declare that there have no competing interests.

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No competing interests reported.

Comparative Chloroplast Genomes and Phylogenetic Analyses of Zanthoxylum L. Provide Insights into Species Delimitation and Phylogenetic Relationships

Status:

Version 1

Abstract

Figures

Background

Materials And Methods

Plant material

DNA Extraction and Sequencing

Chloroplast Genomes Assembly and Annotation

Chloroplast Genomes Comparison and Sequence Variation Analysis

Phylogenetic Analysis

Results

The Chloroplast Genome Structures of Zanthoxylum Species

Comparative Genomic Divergence and Hotspots Regions

IR Contraction and Expansion

Analyses of Repeat Structure and Simple Sequence Repeats

Phylogenetic Relationship Analyses

Discussion

The Features of Zanthoxylum Species Chloroplast Genomes

IR Expansion and Contraction

Phylogeny and Species Identification of Zanthoxylum

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1