Comparative chloroplast genomes of Paris Sect. Marmorata: insights into repeat regions and evolutionary implications
Species of Paris Sect. Marmorata are valuable medicinal plants to synthesize steroidal saponins with effective pharmacological therapy. However, the wild resources of the species are threatened by plundering exploitation before the molecular genetics studies uncover the genomes and evolutionary significance. Thus, the availability of complete chloroplast genome sequences of Sect. Marmorata is necessary and crucial to the understanding the plastome evolution of this section and facilitating future population genetics studies. Here, we determined chloroplast genomes of Sect. Marmorata, and conducted the whole chloroplast genome comparison.
This study presented detailed sequences and structural variations of chloroplast genomes of Sect. Marmorata. Over 40 large repeats and approximately 130 simple sequence repeats as well as a group of genomic hotspots were detected. Inverted repeat contraction of this section was inferred via comparing the chloroplast genomes with the one of P. verticillata. Additionally, almost all the plastid protein coding genes were found to prefer ending with A/U. Mutation bias and selection pressure predominately shaped the codon bias of most genes. And most of the genes underwent purifying selection, whereas photosynthetic genes experienced a relatively relaxed purifying selection.
Repeat sequences and hotspot regions can be scanned to detect the intraspecific and interspecific variability, and selected to infer the phylogenetic relationships of Sect. Marmorata and other species in subgenus Daiswa. Mutation and natural selection were the main forces to drive the codon bias pattern of most plastid protein coding genes. Therefore, this study enhances the understanding about evolution of Sect. Marmorata from the chloroplast genome, and provide genomic insights into genetic analyses of Sect. Marmorata.
KeywordsParis Sect. Marmorata Chloroplast genome Repeat sequence Codon usage Evolutionary rates
Coding DNA sequence
Conserved noncoding sequence
Dual Organellar GenoMe Annotator
Effective number of codons
GC content on the third synonymous codon position
Nonsynonymous substitutions per non-synonymous site
Synonymous substitutions per synonymous site
Large single copy
Polymorphic information content
Relative synonymous codon usage
Single nucleotide polymorphism
Small single copy
Simple sequence repeat
Tandem Repeats Finder
Herbal medicine is currently becoming increasingly popular to be used in complementary and alternative treatments all over the world. Moreover, herbal medicine is still a major source of healthcare, especially in developing countries, which have limited access to modern medical care . However, the wild resources of plant species are threatened by plundering exploitation with the population growth, particularly the increasing demand for herbal medicine with significant economic value.
The species of Paris are famous herbal essence for the elements like steroidal saponins with effective pharmacological therapy. Here, we selected the rare species of the genus Paris Section Marmorata H. Li to explore the chloroplast genome analyses. The Sect. Marmorata comprises two species of perennial medicinal herbs, i.e., P. marmorata Stearn and P. luquanensis H. Li. P. marmorata is mainly distributed in Southwest China (i.e., Yunnan, Sichuan, and Tibet), Nepal, and Bhutan, while P. luquanensis is mainly distributed throughout Yunnan (i.e., Luquan and Pingbian) and Sichuan (i.e., Huidong, Puge and Yuexi) . Their morphological characteristics are different from other Paris members, as these two species have variegated leaves, grow more slowly and thus are shorter than other Paris plants. As the other plants of Paris, Sect. Marmorata species have been used in oriental medicine for a long time. They contain Rhizoma Paridis saponins including diosgenyl and pennogenyl saponins as active ingredients, which are typically used in the treatment of tumors, hemostasis, and inflammation [3, 4, 5]. Thus, wild resources of P. marmorata and P. luquanensis are rapidly declining as consequence of their slow growth and low levels of artificial cultivation, but overexploitation for the economic value. What is more serious is that the wild plants are hard to find, but little is known about the sequence diversity and structure divergence of their chloroplast genomes.
Chloroplasts are essential plant organelles that originated from Cyanobacteria by endosymbiosis with the precursor of a nucleated ancestral cell more than 1.2 billion years ago . The circular, double stranded chloroplast genome encodes a set of proteins involved in photosynthesis and other biochemical pathways that are important for plant growth and development, even plant evolution, such as biosynthesis of starch, fatty acids, and pigments . Chloroplast genomes of plants are known to be predominantly uniparentally inherited and highly conserved in both gene order and gene contents . They typically have a quadripartite organization, consisting of two IR regions separated by two regions of unique DNA, LSC region and SSC region . Substitution rates of chloroplast genomes are much lower than those of nuclear DNA, which are even more substantially reduced in the IR regions . Low levels of recombination and primarily uniparental inheritance make chloroplast genomes a valuable source of genetic markers for phylogenetic analyses and useful tools for DNA barcoding [11, 12]. High proportion of SSRs has aroused considerable interest due to the ability to generate highly informative DNA markers . In the light of higher levels of allelic variation of SNPs, SSRs make their use as indicators for species identification, hybridization and introgression analyses [13, 14, 15], and they have been widely applied to investigating population differentiation and other plant science studies [16, 17, 18, 19]. Therefore, genome-wide comparative analysis of SSRs distribution in chloroplast genomes will lay the foundation for further monitoring gene flow, population differentiation and cytoplasmic diversity of Paris plants with intricate hybridization.
Previous study was focus on the phylogeny of Paris , but lacking of detailed information about genetic variation and molecular structural diversity in these species. The structural and nucleotide sequence variations among chloroplast genomes of Sect. Marmorata can be exposed by combining the chloroplast genomes of this section aligned to different reference chloroplast genomes (e.g., P. verticillata and P. polyphylla var. yunnanensis). Thus, chloroplast genomes of Sect. Marmorata were sequenced using the Illumina sequencing platform. The comparative analyses of chloroplast genomes will contribute to further investigating genetic diversity and evolution of this section to support conservation management strategies, and assist in the exploration and utilization of Paris Sect. Marmorata in herbal medicine.
Herein, the aims of this study enable us: (1) to examine sequence variations and screen for hotspot regions in Sect. Marmorata chloroplast genomes; (2) to characterize global structural patterns of chloroplast genomes of Sect. Marmorata; and (3) to explore codon usage patterns and substitution rates of protein coding genes from chloroplast genomes of Sect. Marmorata species.
Results and discussion
Chloroplast genome assembly, organization, and features
The chloroplast genomes of Sect. Marmorata encoded an identical set of 133 predicted functional genes, 113 of which were unique, and 20 were duplicated and located in the IR regions. The 113 unique genes were comprised of 30 tRNA genes, 4 rRNA genes, and 79 protein-coding genes, respectively (Additional file 1: Table S1). Fifteen distinctive genes, including aptF, ndhA, petB, rpl2, and trnA-UGC, contained the single introns; while the genes clpP, rps12, and ycf3 contained two introns (Additional file 1: Table S2). These introns share the same splicing mechanism as group II introns . However, different assembly strategies and different reference chloroplast genomes (i.e., P. verticillata versus P. polyphylla var. yunnanensis) led to some differences in gene contents. In the light of sequence similarity, the chloroplast genome of P. polyphylla var. yunnanensis was regarded as the most similar genome for Sect. Marmorata via BLAST in NCBI. In addition, Sect. Marmorata species and P. polyphylla var. yunnanensis are closely related, and they belong to the subgenus Daiswa. Notably, comparison of gene contents revealed that two genes were unique to chloroplast genomes sequenced in this study, and another four genes were unique to chloroplast genomes sequenced by Huang et al, respectively (Additional file 2: Figure S1).
A total of 59.41–60.03% of Sect. Marmorata chloroplast genomes were protein-coding regions. Overall, 1.83%, 5.74%, and 52.02–52.73% of the genome sequences encoded tRNAs, rRNAs, and proteins, respectively. The remaining sequences consisted of noncoding regions filled with introns, intergenic spacers, and a pseudogene. The ycf1-like (ycf1Ψ) gene in the IRb/SSC junction was found to be the only pseudogene, resulting from an incomplete duplication of the normal functional copy of ycf1 in the IRa/SSC junction. Similar to the chloroplast genomes of P. verticillata and P. polyphylla var. yunnanensis, the chloroplast genomes of Sect. Marmorata were AT-rich with an overall AT content of 62.6%. AT content of LSC (64.25–64.28%), SSC (68.70–70.10%), and IR (58.08–61.10%) regions varied slightly. AT content of genomic regions is probably associated with dynamics of repeat sequences and codon bias of chloroplast protein-coding genes [22, 23]. In general, the chloroplast genome features of Sect. Marmorata were similar in the terms of gene content, gene order, introns, intergenic spacers, and AT content.
Repeat sequence analyses
Repeat regions play an important role in recombination and genomic rearrangements [24, 25]. Eleven sets of repeats were identified in chloroplast genome sequences of P. marmorata and P. luquanensis using TRF with a 100% match criterion in repeat copies. With a > 90% match criterion, another 10 sets of repeats were identified, yielding 21 total sets detected in P. marmorata, with 12 in CDS regions, 8 in intergenic regions and 1 in a span spacer of a CDS region. Similarly, with a > 90% match criterion, another 8 sets of repeats, yielding 19 total sets were detected in P. luquanensis, with 9 in CDS regions, 9 in intergenic regions, and 1 in a span spacer of tRNA. The repeats were scattered around the LSC (6–7), SSC (6–8), and IRs (6–7) regions, and they mainly located in the intergenic regions and protein coding regions, including accD, rbcL, ycf1 and ycf2 (Additional file 1: Table S3).
Meanwhile, a total of 30 repeats were identified in chloroplast genome sequences of both P. marmorata and P. luquanensis, and the sizes of repeats ranged from 63 to 139 bp. Those repeats were scattered around SSC and IRs regions, and they mainly located in the intergenic regions, protein coding regions (ycf1 and ycf2) and ycf1Ψ (Additional file 1: Table S4). There were 14 and 21 repeats with 0 hamming distance in P. marmorata and P. luquanensis, respectively, that is, these were repeats with 100% identity. The output of REPuter was compared with the one of TRF, and the tandem repeats and dispersed repeats (i.e., forward and palindromic) were separately analyzed. The total numbers of those repeats were 51 and 49 for P. marmorata and P. luquanensis, respectively, in which their copy numbers ranged from 2 to 16. Among the coding regions, the richest in repeats was the ycf1 gene, which contained 31 and 30 repeats in P. marmorata and P. luquanensis, respectively. As reported in the other chloroplast genomes, ycf2 was also rich in repeats, carrying 4 to 5 repeats. These two protein-coding genes and divergent regions are demonstrated to be often associated with repeat events . The above-mentioned repeats can provide valuable information on developing markers for phylogenetic research and population studies.
Chloroplast genome comparison of sect. Marmorata
Codon usage pattern
Most protein-coding genes employed the standard initiator codon AUG; however, six unusual start codons were also identified, such as TTG (cemA) and GTG (rps19). Similar noncanonical start codons have been detected in other angiosperms and tree fern plants [41, 42, 43]. Furthermore, the codon usage patterns were determined for 71 distinct protein-coding genes in Sect. Marmorata chloroplast genomes. Codons of chloroplast genes of Sect. Marmorata with A/U at the third position nucleotide were used more frequently than those ending with G/C, according to RSCU values (with a threshold of RSCU > 1). As observed in chloroplast genomes of most land plants, codon usage patterns of this section are likely driven by the composition bias towards the high A/T content.
Evolutionary rates of sect. Marmorata chloroplast genes
Complete chloroplast genome sequences of Sect. Marmorata, i.e., P. marmorata and P. luquanensis were assembled, annotated and explored subsequent genome-wide comparative analyses. The chloroplast genomes exhibit a typical quadripartite structure of LSC and SSC regions separated by a pair of IRs, and they share similar features in the terms of gene organization and AT-rich content. Large repeats, polymorphic SSR loci, as well as genes and intergenic regions with high levels of variability were determined. Those repeat motifs and hotspot regions can be selected to study both intraspecific and interspecific variability, and they also can aid in inferring phylogenetic relationships of Sect. Marmorata and other species of subgenus Daiswa. Non-coding regions like some intergenic spacers exhibited significantly higher sequence divergence than most coding regions, and the divergent lengths of noncoding regions affected chloroplast genome size variation among species. Almost all the chloroplastic protein-coding genes bias ended with A/U. Mutations and selection forces, particularly natural selection, shaped the codon bias pattern of most genes. Most of these mentioned genes were predicted to have a signature of purifying selection, whereas photosynthetic genes experienced a relatively relaxed purifying selection, whose codon bias and evolutionary rate were also driven by other factors such as gene expression level and gene length. Additionally, chloroplast genomes sequenced in this study and those simultaneously sequenced did have differences in both gene content and structure, which brought different results of quadripartite boundaries, substitution rates, and selective pressure. Nevertheless, chloroplast genomes sequenced together with the ones sequenced simultaneously can provide insights into both the genetic relationships among Sect. Marmorata and the other species of Paris. Moreover, the results aid to expand the current understanding of the evolutionary history of Paris, particularly Sect. Marmorata.
Taxon sampling, DNA extraction and sequencing
P. marmorata and P. luquanensis analyzed in this study were cultivated and collected in green house (Kunming) of Xishuangbanna Tropical Botanical Garden. No specific permits are required for sampling. Total genomic DNA was extracted from 100 mg of fresh healthy leaves using a modified CTAB method [52, 53], and the quality of each sample was assessed by agarose gel electrophoresis. The whole chloroplast genomes were amplified using long-range PCR and nine universal primer pairs according to the procedure outlined by Yang et al . Then, six μg of purified PCR products was mixed and fragmented to construct short insert libraries (measuring 200–500 bp in length) according to the procedures outlined in the Illumina manual. The paired-end libraries were then sequenced using Illumina MiSeq 2000 platform (Illumina, San Diego, CA, USA) at Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences.
Assembly and annotation
Raw reads were filtered with the quality control program NGSQCToolkit v2.3.3 to obtain high quality Illumina data (the cut-off value for percentage of read length was 80, and the cut-off value for PHRED quality score was 30) and adaptor-free reads. Filtered reads were then assembled into contigs using SPAdes v3.6.1. Outputted contigs were aligned with the reference P. polyphylla var. yunnanensis chloroplast genome (Genbank accession No. KT805945) Contigs were then aligned with the reference genome to assemble each chloroplast genome sequence using Geneious v4.8.4. Assembled genome sequences were annotated using the online tool DOGMA and Geneious v4.8.4 [54, 55], and then annotated sequences were manually edited for start and stop codons. All tRNA genes were further confirmed by the online tRNAscan-SE search server . The annotated chloroplast genomes were deposited in GenBank with accession numbers: P. marmorata (MF495705) and P. luquanensis (MF417768). The annotated GenBank files of the two Paris chloroplast genomes were uploaded to obtain gene maps using the online tool GenomeVx .
Repeat sequence identification
Repeat elements in chloroplast genomes of P. marmorata and P. luquanensis were investigated using three different programs. The position and type of SSR were ascertained using the microsatellite identification tool MISA v1.0 , and each repeat sequence length was screened to be ≥10 bp. SSRs were identified with thresholds of 10, 5, 4, 3, 3, and 3 repeat units for mono-, di-, tri-, tetra-, penta-, and hexa-nucleotides, respectively. Diversity of chloroplast SSR markers were further estimated using the Shannon-Winener index and PIC. Tandem repeat sequences (> 10 bp in length) were identified with TRF v4.09 , with parameters of 2, 7, and 7 for matches, mismatches and indels, respectively. The minimum alignment score and maximum period size were set to 50 and 500, respectively. Meanwhile, the size and location of both forward and inverted/palindromic repeats were determined using REPuter v1.0 . The parameters were set with a minimal repeat size of 30 bp, hamming distance of 3 kb, and 90% sequence identity threshold. Gene and intergenic spacer regions harbored repeat sequence were extracted, on the basis of the loci of repeats. These regions were then applied to infer phylogenetic relationships with Neighbor-Joining algorithm in MEGA v6.06 and Maximum Likelihood algorithm of RAxML v7.2.6.
Comparison of chloroplast genome sequences
To investigate the sequence divergence among Sect. Marmorata chloroplast genomes, several released chloroplast genomes were retrieved from NCBI: P. marmorata (NC_033516, denoted by PMa), P. luquanensis (NC_033514, denoted by PLa), and P. verticillata (NC_024560, denoted by PV). Four chloroplast genome sequences of Sect. Marmorata were aligned using MAFFT v7.305b  and were manually adjusted using Se-Al v2.0 . A sliding window analysis was conducted to compare π among the chloroplast genomes of Sect. Marmorata, using DnaSP v5.0 . The window length was 600 bp with a 200 bp step size. To reveal both inter- and intra-specific variations, the full alignments of chloroplast genome sequences of Sect. Marmorata species and P. verticillata were visualized with Shuffle-LAGAN mode in mVISTA program .
Codon usage and substitution rate calculation
RSCU, GC3s, and ENc for 71 protein-coding genes were calculated using CodonW v1.4.4 . Then, the relationships between ENc and GC3s were analyzed. Ka, Ks and their ratios Ka/Ks were estimated with ParaAT v2.0 and KaKs_caculator v2.0 [66, 67]. These substitution analyses of 77 conserved protein-coding genes from chloroplast genomes of Sect. Marmorata species were implemented, using alignments with P. verticillata. Boxplots were constructed for each functional category/group, and plotted with SigmaPlot v13.0.
We are very grateful to Junbo Yang, Zhirong Zhang and Zhanshan He (Germplasm Bank of Wild Species in Kunming Institute of Botany) for their help with experiments and data analyses. We also thank the Public Technology Service Center at Xishuangbanna Tropical Botanical Garden for providing the computer resources.
This research was supported by National Natural Science Foundation of China (No. 31800273, 31471220, 91440113); Start-up Fund from Xishuangbanna Tropical Botanical Garden; ‘Top Talents Program in Science and Technology’ from Yunnan Province; the CAS “Light of West China” Program. The funders had no role in study design, data collection, analysis and interpretation, or preparation of the manuscript. Publication costs are funded by National Natural Science Foundation of China (No. 31800273), the CAS “Light of West China” Program.
Availability of data and materials
The chloroplast genomes generated during the current study were deposited in NCBI with accession number MF495705 (P. marmorata) and MF417768 (P. luquanensis).
About this supplement
This article has been published as part of BMC Genomics Volume 19 Supplement 10, 2018: Proceedings of the 29th International Conference on Genome Informatics (GIW 2018): genomics. The full contents of the supplement are available online at https://bmcgenomics.biomedcentral.com/articles/supplements/volume-19-supplement-10.
CNL and XYG designed the experiments. XZ, JL performed the experiments. XYG, XZ, DZ analyzed data. XYG, XZ, HHM wrote the paper. All authors reviewed and approved the manuscript.
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