Characterization of the complete chloroplast genome of Elaeagnus pungens (elaeagnaceae) and phylogeny within elaeagnaceae.

Elaeagnus pungens is an evergreen shrub with high medicinal values. In this study, the complete chloroplast (cp) genome of E. pungens was characterized. The chloroplast genome of E. pungens is 152,218 bp in length, consisting of two 25,876 bp inverted repeats, 18,231 bp small single copy region, and 82,235 bp large single copy region. The chloroplast genome contains 113 unique genes, including 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Phylogenomic analysis revealed that E. pungens and species from Elaeagnus formed a monophyletic clade sister to the clade consisting of species from Hippophae.

Belonging to the Elaeagnaceae family, Elaeagnus Linn. 1754 is a genus consisting of ∼60 species distributed in different continents worldwide (Heywood et al. 2007). Elaeagnus pungens Thunb. 1784, an evergreen shrub native to Japan and South China, is a member of the Elaeagnus genus. The leaves of E. pungens have been well-recognized as a traditional medicinal material. They are mainly used in prescriptions for the treatment of respiratory and digestive system diseases such as cough, tracheitis, hematemesis, dysentery, and enteritis. Previous studies showed that water extracts of E. pungens leaves were rich in flavonoids with antiasthmatic, antitussive, and expectorant effects (Ge et al. 2009). However, the current studies of E. pungens have been limited to its chemical component, pharmacological effect, and cultivation strategy. The genetic background of E. pungens has been largely ignored. In this study, we characterized the complete chloroplast genome of E. pungens to provide genomic and genetic resources for future molecular analysis.

Total genomic DNA was extracted from silica-dried leaves of one E. pungens plant individual cultivated in Hangzhou, Zhejiang Province, China (30.26 N, 120.12 E) using modified CTAB method (Doyle and Doyle, 1987). Collection of the plant material is in compliance with Plant Material Collection Guidelines of Zhejiang Shuren University. The study did not involve any endangered or protected species. The sample collection site is neither privately owned nor protected for which no specific permission was required. The voucher specimen (No. MQ20-20008) was deposited in the herbarium of Zhejiang Shuren University (Contact: Qing Ma, maqing90@live.cn). The chloroplast genome was first sequenced using the Illumina Hiseq Platform (Illumina, San Diego, CA) at BGI (Shenzhen, Guangdong, China). The obtained raw reads were preliminarily screened to remove adaptor sequences and low-quality sequences. Next, de novo assembly of filtered paired-end reads were conducted using the GetOrganelle software (Jin et al. 2020) and SPAdes 3.10.1 (Bankevich et al. 2012). Genome annotation was performed with Geneious R11 (https://www.geneious.com) using the chloroplast genome of Elaeagnus umbellata (GenBank accession number: LC522506) as the reference. The putative starts, stops, and boundaries between exons and introns were manually checked by comparison with homologues genes of published Elaeagnaceae species. The tRNA genes were verified using tRNAscan-SE v1.21 (Schattner et al. 2005). The complete chloroplast genome sequence of E. pungens after annotation was deposited to GeneBank under the accession No. MW145133.

The chloroplast genome size of E. pungens is 152,218 bp, which included two 25,876 bp inverted repeats (IRs), a 18,231 bp small single copy (SSC) region, and a 82,235 bp large single copy (LSC) region. The guanine and cytosine (GC) contents of the LSC, SSC, and IR regions were 34.9%, 30.6%, and 42.7%, respectively. The whole chloroplast genome contains a total of 113 unique genes, including 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Five protein-coding (ndhB, rps7, rpl2, rpl23, and ycf2), eight tRNA (trnA-UGC, trnH-GUG, trnI-CAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG, and trnV-GAC), and four rRNA genes (rrn4.5, rrn5, rrn16, and rrn23) are duplicated and located in the IR regions.

The evolutionary relationships of E. pungens and other 13 related species from Elaeagnaceae were analyzed using the whole chloroplast genome sequences. One species, Barbeya oleoides from Barbeyaceae (NC_040984) was selected as the outgroup according to the results of Choi et al. (2015). Sequence alignment was conducted using MAFFT version 7.0 (Katoh et al., 2017) and phylogenomic analysis was conducted using the maximum likelihood (ML) and Bayesian inference (BI) methods with the RAxML-HPC v8.1.11 and MrBayes v3.2.3 online tools implemented on the CIPRES Science Gateway (http://www.phylo.org/; Ronquist and Huelsenbeck 2003; Miller et al. 2010; Stamatakis, 2014). Both the ML and BI analyses were performed using GTR + I + G as the best-fit nucleotide substitution model. All the 13 species from Elaeagnaceae form one clade with full support on all the nodes. The nine Elaeagnus species formed a monophyletic clade sister to the clade consisting of four Hippophae species. Both the Elaeagnus and Hippophae clades were fully supported by ML and Bayesian analyses (Figure 1). The topology of phylogenetic tree obtained in this study is largely consistent with previous phylogenetic study of Elaeagnaceae based on ITS sequences of nuclear rDNA, which showed that Elaeagnus glabra was clustered with Elaeagnus macrophylla, while Elaeagnus multiflora was clustered with Elaeagnus umbellate (Son et al. 2014). In the future, more species and more samples from each species are necessary to fully resolve the phylogeny of Elaeagnaceae.

Figure 1.

Phylogenetic tree reconstruction of Elaeagnus and Hippophae in Elaeagnaceae using ML based on whole chloroplast genome sequences. The newly sequenced species in the study is indicated using bold letter and an asterisk. GenBank accession numbers of all the chloroplast genome used for phylogenomic analysis are indicated in the brackets. Numbers above the branches represent bootstrap values from maximum likelihood analyses (before slash) and posterior probabilities from Bayesian inference (after slash), respectively.