The complete mitochondrial genome of Camellia nitidissima (Theaceae)

Abstract The mitochondrial genome of Camellia nitidissima was sequenced by Illumina and Pacbio sequencing. The results of sequences showed that a total length was 949,915 bp, and the GC content was 45.7% in assembled mitochondrial genome of C. nitidissima. 71 unigenes had been found, including 36 coding proteins and 35 non-coding proteins. Subsequently, the phylogenetic tree was built on 24 plants with the maximum-likelihood method, which had high bootstrap value and fited to the angiosperm phylogeny group classification (APG IV). The study’s findings unravel the taxonomic status of C. nitidissima and benefit the evolution study.


Introduction
Camellia nitidissima C.W. Chi (1948), with golden flowers, belongs to Camellia genus of the family Theaceae, and it is mainly distributed in Guangxi, China. Camellia nitidissima is a kind of Chinese herbal medicine, whose flowers and leaves are particularly rich in saponins, flavonoids and polysaccharides (Hou et al. 2018), and C. nitidissima can be used in treatment of dysentery, hypertension, pharyngitis and hematochezia He et al. 2018). Recently, the studies of C. nitidissima mainly focus on the formation of flower color (Zhou et al. 2017;Li et al. 2019), the types of secondary compounds (Jiang et al. 2020), and the extraction of chemical substances (Lin et al. 2013), but the mitochondrial genome of C. nitidissima has not been reported. However, the mitochondrial genome of C. nitidissima benefits to clarify its evolutionary position, so we propose to sequence and assemble the complete mitochondrial genome of C. nitidissima, and provide valuable genomic information for phylogeny.

Materials
Camellia nitidissima is a shade-tolerant species and prefers warm, humid climate and acid soil with good drainage, and it is mainly propagated by seeds and cuttings. Camellia nitidissima were cultivated in the nursery of Yulin Normal University (N 22 40 0 58 00 , E 110 11 0 27 00 ), Guangxi, China ( Figure  1). The purple leaves of 7-year-old C. nitidissima plants were collected, cleaned, immediately frozen in liquid nitrogen, and stored at À80 C. The studied specimen and genomic DNA of C. nitidissima were stored in the Herbarium of Yulin Normal University (https://syy.ylu.cn/index.html, Yulin Zhu, gxzyl@163. com) under the voucher number YLU20210011.

Methods
Mitochondria of C. nitidissima was isolated from purple and young leaves with the density gradient centrifugation, and the contamination of genomic DNA was eliminated by DNase I (Promega, Madison, USA). DNA quality of mitochondria was checked by Qubit fluorometer (Thermo, Massachusetts, USA) and agarose gel electrophoresis. The sequencing library for Illumina was constructed using the NEBNext V R Ultra TM DNA Library Prep Kit (New England Biolabs, Suffolk, England) and sequenced by Illumina NovaSeq 6000 (Illumina, San Diego, USA). The SMRTbell libraries for Pacbio was constructed using the Express Template Prep Kit 2.0 (Pacific Biosciences, California, USA) according to the manufacturer's protocol and sequenced by Pacbio Sequel II (Pacific Biosciences, California, USA). All of the above were entrusted to Biozeron company (Biozeron, Shanghai, China).
We used two strategies to assemble the mitogenome of C. nitidissima. In the first strategy, the short clean reads were de novo assembled with GetOrganelle v1.6.4 (parameters: -k 21, 65, 105) (Jin et al. 2020). Then, in order to extract the potential mitochondrial contigs, the assembled mitochondrial protein-coding genes were alignment with the plant mitogenome database by BLAST. Subsequently, the Pacbio long mitochondrial reads, which were used as bait, were mapped to the potential mitochondrial contigs by BLASR v5.1 with default parametersand assembled by Canu v2.1.1 (parame- (Koren et al. 2017). In the second strategy, firstly, all Pacbio long reads were de novo assembled to get the draft contigs with Canu v2.1.1. Secondly, the short clean reads were mapped to the draft contigs by using BWA (parameters: bwa mem -t 4), and the draft contigs were improved by Pilon v1.22 (parameters: -fix all). Then, MUMmer 3.23 was used to check whether these contigs were circular. And finally, two corrected contigs, which were obtained from two assembly strategies, were align to each other by using MUMmer (parameters: nucmer -prefix). If these two contigs were identical, a master circle of the C. nitidissima mitogenome was correctly assembled. Mitochondrial genes of C. nitidissima were annotated by the GeSeq tool (Tillich et al. 2017), with the default parameters, to predict coding proteins, tRNA, and rRNA. And then those genes of functional annotations were performed to blast against (evalue < 1e-10) the non-redundant protein database (Nr), Swiss-Prot, Clusters of Orthologous Groups (COGs), and Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology terms (GO). Subsequently the position of each coding gene was determined using BLAST searches against ref mitochondrion genes in NCBI. The mitochondrial genome map was drawn by the OGDRAW tool. Based on 23 conserved protein-coding genes in the mitochondrial genomes, phylogenetic tree of C. nitidissima and 23 plants was constructed with maximum-likelihood estimation by PhyloSuite software with the best-fit model (GTR þ FþR2) and 1000 bootstrap replicates (Zhang et al. 2020).
(unpublished), Vitis vinifera NC_012119 (Goremykin et al. 2009), Ginkgo biloba NC_027976 (Guo et al. 2016). The Ginkgo biloba (NC_027976) used as outgroup was divided into one branch; the grape was divided into one branch alone; C. nitidissima and six plants were divided into one branch; two plants of Malvaceae and four plants of Cruciferous were divided into another branch and the remaining nine plants were divided into final branch (Figure 3). In the phylogenetic tree, C. nitidissima was clustered into a branch with the plants of Theaceae, Apocynaceae, Solanaceae and Compositae, which had high bootstrap value and fited to the angiosperm phylogeny group classification (APG IV). Moreover, C. nitidissima was closely related to Camellia sinensis, which all belonged to Camellia genus of the family Theaceae, with 100 bootstrap values. Therefore, it is thought that the phylogenetic analysis in this study can distinguish species of plant by using sequences of mitochondrial gene.

Discussion and conclusion
The event of horizontal gene transfer can be identified by comparing mitochondrial genome of C. nitidissima with the chloroplast genome in future studies, which will provide a basis for the functional study of horizontal gene transfer sequences. This complete mitochondrial genome of C. nitidissima will benefit the evolution study, germplasm identification and development of molecular markers.

Ethical approval
The data collection of plants was carried out with the permission of Yulin Normal University and complied with local (Yulin, Guangxin, China) legislation. The research involved Camellia nitidissima, which was an endangered species, so in this study we complied with the policies of the International Union for Conservation of Nature (IUCN), the Convention on Biological Diversity and the Convention on the Trade in Endangered Species of Wild Fauna and Flora, and tried ours best to protect the resources of C. nitidissima.