The complete chloroplast genome of Ziziphus mairei Dode 1908 (Rhamnaceae), an endangered perennial plant in Yunnan, China

Abstract Ziziphus mairei Dode 1908 (Rhamnaceae) is a rare and endangered perennial plant in Yunnan, China. In this study, we sequenced, assembled, and annotated the complete chloroplast genome of Z. mairei. The complete chloroplast genome was a closed circular molecule of 161,546 bp with a typical tetrad structure, containing a large single-copy (LSC) region of 89,252 bp, a small single-copy (SSC) region of 19,364 bp, and a pair of inverted repeat (IR) regions of 26,465 bp. A total of 128 genes have been annotated, including 83 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The GC content is 36.7%. Phylogenetic analysis revealed that Z. mairei is closely related to Z. hajarensis, Z. jujuba, and Z. jujuba var. spinosa. Our results provide useful genetic resources for further studies on the conservation and evolution of Z. mairei.


Introduction
Ziziphus mairei Dode 1908, a 15 m tall spinose tree (Figure 1), belongs to the genus Ziziphus (Rhamnaceae).It is mainly distributed in the thickets and forest margins along riverbanks at an altitude of 1900-2000 m in the central to northwestern Yunnan Province of China (Du and Fang 1989).Z. mairei is a good material for making furniture and charcoal because of its hard texture and high calorific value (Yue and Feng 1991).However, due to the narrow distribution and limited number of surviving individuals, Z. mairei is been listed as an endangered species on the IUCN Red List (Qin et al. 2017).The chloroplast genome is a powerful genetic tool for conservation research on endangered species (Song et al. 2022).Here, we report the first complete chloroplast genome of Z. mairei, which will provide potential genetic resources for further conservation and evolutionary studies of this species.

Materials and methods
The fresh leaves of Z. mairei were collected from Kunming Botanic Garden, Yunnan Province of China (25 � 14'56"N, 102 � 74'07"E).The voucher specimen was deposited in the herbarium of the School of Life Sciences, Yunnan Normal University (Kunming, China; Jianlin Hang, hjlyuun@163.com)under the accession number HY-23.Total genomic DNA was extracted from Z. mairei leaves using a modified CTAB method (Porebski et al. 1997).The genomic DNA was fragmented to construct a 300 bp short-insert library, and then paired-end sequenced on the Illumina Hiseq X Ten sequencing platform.The raw data (4.71G) was filtered using fastp v.0.23.2 software (https://github.com/OpenGene/fastp) to remove low-quality sequences, resulting in 4.68 G clean data, with Q20 of 92.98% and Q30 of 80.88%.We obtained 31,430,978 filtered reads (SRR21639213) and assembled the chloroplast genome of Z. mairei using NOVOPlasty v4.3.1 software (Dierckxsens et al. 2017).Annotation of the chloroplast genome was performed using Geneious v2020.1.1 software (Kearse et al. 2012), with Ziziphus jujuba (Genbank accession No. MW381776) as the reference genome.The annotated chloroplast genome of Z. mairei was submitted to Genebank with the accession number OP4880228.1, and we drew the circular map of the chloroplast genome using CPGView (http://www.1kmpg.cn//cpgview).The chloroplast genome of Z. mairei and 24 related chloroplast genomes downloaded from GenBank were aligned for phylogenetic analysis using the software MAFFT v7.47 (Katoh and Standley 2013).The 24 species included 22 species from 7 genera of Rhamnaceae and 2 species of Vitaceae as an outgroup, and then a maximum likelihood tree was constructed using the IQ-TREE v1.6.10 software (Nguyen et al. 2015) based on the substitution TVM þ FþR2 best-fit model according to the Bayesian information criterion (Kalyaanamoorthy et al. 2017).The branch supports were tested with 10,000 replicates using ultrafast bootstrap (UFBoot) (Hoang et al. 2018) and SH-like approximate likelihood ratio test (SH-aLRT) (Guindon et al. 2010) with a scale bar of 0.02.

Results
The complete chloroplast genome of Z. mairei is a closed circular molecule of 161,546 bp in length (Figure 2) with an average coverage of approximately 4809.0 (Supplementary Figure S1) and 36.7%G-C content (GenBank accession No. OP480228.1).The assembled chloroplast genome is a typical tetrad structure, containing a large single-copy (LSC) region of 89,252 bp (34.5% GC content), a small singlecopy (SSC) region of 19,364 bp (30.9% GC content), and a pair of inverted repeat (IR) regions of 26,465 bp (42.7% GC content).In total, the complete chloroplast genome consisted of 83 protein-coding genes, 37 tRNA genes, and 8 rRNA genes, including 76 unique protein-coding genes, 30 unique tRNA genes, and 4 unique rRNA genes.Furthermore, the 110 unique genes can be divided into four types according to their functions: photosynthesis, self-replication, other genes, and genes of unknown functions.In total, one trans-splicing gene and 11 cis-splicing genes (rpl2, ndhB, ndhA, rpl16, petD, petB, clpP, ycf3, rpoC1, atpF, rps16) were identified (Supplementary Figure S2).The phylogenetic tree divided the analyzed plants into 2 clades, all Rhamnaceae as 1 clade and the outgroup Vitaceae as 1 clade, with a total of 25 plants forming 23 nodes, with each node having a bootstrap value of 100.Meanwhile, phylogenetic analysis revealed that Z. mairei was closely related to Z. hajarensis, Z. jujuba, and Z. jujuba var.spinosa.8 species of Ziziphus formed a stable monophyletic group (Figure 3).

Discussion and conclusion
The structural features and genetic composition of the chloroplast genome of Z. mairei are consistent with previous chloroplast genome characteristics of flowering plants (Wang et al. 2012).The previous studies of Ziziphus showed that eight species of Ziziphus formed a stable monophylete, our results of phylogenetic analysis are consistent with this (Asaf et al. 2022).
In summary, this study was the chloroplast genome sequence of Z. mairei assembled and annotated for the first time, and it also clarifies the phylogenetic position of Z. mairei in the Ziziphus.Our results provide useful genetic resources for further studies about the conservation and evolution of Z. mairei.

Figure 1 .
Figure 1.Species image of Z. mairei.(A) Morphology of the whole plant.Z. mairei is a spinose tree of 15 m in height.(B) Morphological characteristics of leaves of Z. mairei.Leaves papery, ovate-lanceolate, and apically long acuminate.The photograph was taken by the corresponding author Yuan Huang at Kunming Botanic Garden, Yunnan Province of China (25 � 14'56"N, 102 � 74'07"E), on March 2023.