Characterization of the complete chloroplast genome of Gypsophila huashanensis Y. W. Tsui & D. Q. Lu, an endemic herb species in China

Abstract

Gypsophila huashanensis Y. W. Tsui & D. Q. Lu (Caryophyllaceae) is an endemic herb species to the Qinling Mountains in China. In this study, we characterized its whole plastid genome using the Illumina sequencing platform. The complete plastid genome of G. huashanensis is 152,457 bp in length, including a large single-copy DNA region of 83,476 bp, a small single-copy DNA region of 17,345 bp, and a pair of inverted repeat DNA sequences of 25,818 bp. The genome contains 130 genes comprising 85 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Evolutionary analysis showed that the non-coding regions of Caryophyllaceae exhibit a higher level of divergence than the exon regions. Gene site selection analysis suggested that 11 coding protein genes (accD, atpF, ndhA, ndhB, petB, petD, rpoCl, rpoC2, rps16, ycfl, and ycf2) have some sites under protein sequence evolution. Phylogenetic analysis showed that G. huashanensis is most closely related to the congeneric species G. oldhamiana. These results are very useful for studying phylogenetic evolution and species divergence in the family Caryophyllaceae.

1. Introduction

Gypsophila huashanensis Y. W. Tsui & D. Q. Lu (Caryophyllaceae) is an endemic herb species in central China (Figure 1) (Lu 1994), and it is currently only distributed in the Huashan and Qinling Mountains in Shaanxi, China. G. huashanensis grows on mountain slopes, valleys, roadside grasslands, and rock crevices at 600–2600 m above sea level. Previous studies of species in the genus Gypsophila mainly focused on their chemical constituents and pharmacological effects (Xie et al. 2015; Zhu et al. 2016), whereas few have investigated genomic evolution in this genus. Acquiring chloroplast genome data is conducive to identifying further species and phylogenetic studies (Mehmood et al. 2020a, 2020b, 2020c).

Figure 1.

Plant characteristics image of Gypsophila huashanensis. The flower characteristics of G. huashanensis is the corymbose cymes terminal or borne in distal leaf axils, in subcapitate clusters; petals pinkish white, oblong-oblanceolate, ca. 5 mm, apex retuse; filaments exserted, linear, flat, unequal, shorter than to longer than petals, base broad. The photograph was taken by the authors in the Qinling Mountains (108°55′23.115756″N, 34°14′58.102116″E, altitude 394.7 m).

2. Materials

The fresh G. huashanensis leaf tissues used in this study were sampled from the Qinling Mountains in China (108°55′23.115756″N, 34°14′58.102116″E, altitude 394.7 m). A plant voucher specimen (GHLZH2020113523) was deposited in the Laboratory of Plant Evolution and Ecology, Northwestern University (Xi’an, China) (Contact: Zhonghu Li, lizhonghu@nwu.edu.cn).

3. Methods

Total genomic DNA was isolated from G. huashanensis using a modified version of the hexadecyltrimethylammonium bromide method (Doyle and Doyle 1990). After DNA quality and quantity testing, a paired-end library with an insert size of 350 bp was constructed and sequenced using the Illumina NovaSeq 6000 platform. The NGSQC Toolkit_v.2.3.3 was used to filter the raw sequencing reads (Patel and Jain 2012). De novo assembly was performed with SPAdes software (Bankevich et al. 2012). The assembly accuracy and efficiency were further improved using the GetOrganelle program (Jin et al. 2020). The circular plastome was obtained by using Bandage (Wick et al. 2015) and Geneious v.9.0.2 (https://www.geneious.com/) with G. oldhamiana (NC_058757) as the reference. The complete chloroplast genome of G. huashanensis was automatically annotated by PGA (Qu et al. 2019), and adjusted and confirmed in Geneious. Finally, a chloroplast genome map was drawn for G. huashanensis using CPGView (Figure 2) (Liu et al. 2023).

Figure 2.

Circular map of the complete chloroplast genome of Gypsophila huashanensis. The center of the figure provides the specific information (genome length, GC content, and number of genes) of the G. huashanensis complete chloroplast genome sequence. From the center to the outside, the first track uses different colors to show the large single-copy (LSC) region (deep blue), small single-copy (SSC) region (light blue), and two inverted repeat (IRa and IRb) regions (gray). The GC content throughout the genome is plotted in the second track. Genes are indicated in the outermost track and color coded according to their functional classifications. The directions of transcription for the inner and outer genes are clockwise and anticlockwise, respectively. Different colors represent different gene types, the detailed gene types are listed in the captions.

To infer the phylogenetic position of G. huashanensis in the family Caryophyllaceae, the complete chloroplast genome sequences of 21 plant species (including eight Colobanthus species, five Pseudostellaria species, three Silene species, and two outgroups belonging to Phytolaccaceae) were used to reconstruct their evolutionary relationships. First, the data matrices were aligned using the MAFFT v7 program (Katoh and Standley 2013). Second, maximum-likelihood (ML) and maximum parsimony (MP) phylogenetic trees were generated based on a concatenated data matrix of 21 complete chloroplast sequences. The ML tree was generated with the RAxML v8 program (Stamatakis 2014) under the GTR + G evolutionary model with 1000 bootstrap replicates. The MP tree was produced using the PAUP v.4 program (Swofford 2004) with 1000 bootstrap replicates.

In order to detect evolutionary variation in the chloroplast genomes in Caryophyllaceae, sequence alignment was conducted for the complete chloroplast genomes in Caryophyllaceae by using the mVISTA program (Frazer et al. 2004). We also performed gene site selection analysis for the protein-coding genes in Caryophyllaceae plastid genomes using the PAML 4.7 program (Yang et al. 2005). In order to choose the most reliable model, we have conducted likelihood ratio tests for evolutionary selection analysis.

4. Results and discussion

The complete chloroplast genome sequence of G. huashanensis (GenBank accession: OP094658) is 152,457 bp in length, with a GC content of 36.5% and it has four regions comprising two inverted repeat regions (IRs, 25,818 bp) separated by a large single-copy region (LSC, 83,476 bp) region and a small single-copy region (SSC, 17,345 bp). The read coverage depth map is shown in Supplementary Figure S1. The chloroplast genome contains 130 genes comprising 85 protein-coding genes, 37 tRNA genes, and eight rRNA genes. The GC contents of the chloroplast genome, LSC region, SSC region, and IR region are 34.1, 30.0, and 42.5%, respectively. Fourteen genes contain one intron (rps16, atpF, rpoCl, petB, petD, rpl16, ndhB, ndhA, trnK-UUU, trnG-UCC, trnL-UAA, trnl-GAU, trnA-UGC, and trnI-GAU), and three genes contain two introns (rps12, pafI, and clpPl). In addition, we mapped the structures of genes that are difficult to annotate in the chloroplast genome of G. huashanensis (Supplementary Figure S2).

Figure 3 shows that the ML and MP phylogenetic trees have similar topological structures. Phylogenetic analysis indicated that all of the Caryophyllaceae species considered in the present study clustered into a monophyletic evolutionary clade with high bootstrap support. Gypsophila oldhamiana was recovered as a sister taxon of G. huashanensis. The results obtained in this study extend our understanding of chloroplast genome evolution in the genus Gypsophila.

Figure 3.

Phylogenetic relationships among Gypsophila huashanensis inferred from (a) maximum-likelihood (ML) method, and (b) maximum parsimony method based on concatenated complete chloroplast genome sequence of 21 species with two outgroups (Phytolaccaceae). *Newly sequenced plastid genome of Gypsophila huashanensis. The number on the branch represents bootstrap support. GenBank accession numbers of the following sequences were used: G. oldhamiana NC058757 (Jeong et al. 2021^a), A. githago NC023357 (Sloan et al. 2014), S. chalcedonica NC023359 (Sloan et al. 2014), S. paradoxa NC023360 (Sloan et al. 2014), S. conoidea NC023358 (Sloan et al. 2014), P. setulosa NC041462 (Kim and Park 2019^a), P. heterantha NC058231 (Kim et al. 2021^a), P. palibiniana MK120981 (Kim et al. 2021^a), P. longipedicellata MH373593 (Kim et al. 2021^a), P. okamotoi NC039974 (Kim et al. 2019^a), C. lycopodioides NC053721 (Androsiuk et al. 2020), C. acicularis NC053724 (Androsiuk et al. 2020), C. nivicola NC053720 (Androsiuk et al. 2020), C. pulvinatus NC053719 (Androsiuk et al. 2020), C. apetalus NC036424 (Androsiuk et al. 2017^a), C. affinis NC053722 (Androsiuk et al. 2020), C. subulatus NC053723 (Androsiuk et al. 2020), C. quitensis NC028080 (Lee et al. 2015^a), Monococcus echinophorus MH286317 (Yao et al. 2019), Phytolacca insularis NC041113 (Yang et al. 2019). ^aDirect submission to NCBI, unpublished.

Sequence evolution analysis showed that the non-coding regions of the chloroplast genomes of Caryophyllaceae species exhibit higher levels of genetic divergence than the exon regions (Figure 4). This result is consistent with the evolutionary characteristics of most angiosperm chloroplast genomes (Khakhlova and Bock 2006). We also detected 11 coding genes with some sites under positive selection (p < 0.001, Supplementary Table S1) comprising accD, atpF, ndhA, ndhB, petB, petD, rpoC1, rpoC2, rps16, ycf1, and ycf2. In particular, the accD, ndhA, petD, rpoC2, rps16, ycf1, and ycf2 genes were found to harbor multiple sites under evolutionary selection. The accD gene encoding acetyl-CoA carboxylase subunit is necessary for plant leaf development and it has important impacts on the leaf life and seed yield (Madoka et al. 2002; Kode et al. 2005). In addition, the ndhA gene encodes the NADH dehydrogenase subunit, which is involved in the electron transport chain and plant chlororespiration. The petD gene encodes cytochrome b6/f subunit IV, which plays important roles in linear and cyclic electron transport functions (Xiao et al. 2012). Moreover, the ycf1 and ycf2 genes are the largest genes in plastid genomes, and they encode part of the chloroplast inner envelope membrane protein translocon (Kikuchi et al. 2013). These genes might have played important roles in environmental adaptation by G. huashanensis.

Figure 4.

Sequence alignment of chloroplast genomes from 19 Caryophyllaceae species. Chloroplast genome sequences were aligned and compared with mVISTA software. The X-axis and Y-axis indicate the coordinates within the chloroplast genome and percentage identity (ranging from 50 to 100%), respectively. The grey arrows indicate the gene directions in the chloroplast genomes. Purple and pink bars represent exons and conserved non-coding sequences in chloroplast genomes, respectively.

5. Conclusions

The complete chloroplast genome sequence of G. huashanensis was assembled and annotated in the present study. G. oldhamiana was found to be most closely related to G. huashanensis. Some genes under positive selection were identified in the chloroplast genome, and they might have played key roles in environmental adaptation by G. huashanensis. These results provide the basis for further studies of molecular evolution in Caryophyllaceae plants.

Ethical approval

Gypsophila huashanensis was not listed as a protected herb plant in China nor a threatened plant species on the IUCN Red List. Therefore, no specific permissions were needed for the sampling collections of G. huashanensis for scientific research purpose according to the regulations of the People’s Republic of China on the protection and management of wild plants. During the field collecting process, we followed the local collecting guideline to ensure no substantial harm to the collecting wild plant individual.

Funding Statement

This work was financially supported by the Key Program of Research and Development of Shaanxi Province [2022ZDLSF06-02].

Author contributions

Conception and design: Li ZH and Fang MF; software, analysis and interpretation of the data: Guan TX, Lu ZP, Liu ML, and Xun LL; the drafting of the paper, revising it critically for intellectual content: Guan TX, Lu ZP, and Liu ML; the final approval of the version to be published: Guan TX, Fang MF, and Li ZH. All authors agree 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/nuccore/OP094658/ under the accession no. OP094658. The associated BioProject, SRA, and Bio-Sample numbers are: PRJNA895000, SRR22100483, and SAMN31487758, respectively.