Next Article in Journal
Biodiversity and Litter Breakdown in a Karstic Freshwater System (Doberdò Lake, Northeast Italy) in Relation to Water Level Fluctuations and Environmental Features
Previous Article in Journal
Not All That Glitters Is Gold: Barcoding Effort Reveals Taxonomic Incongruences in Iconic Ross Sea Sea Stars
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Diversity
Volume 14
Issue 6
10.3390/d14060458
diversity-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Comparative Analysis and Phylogenetic Study of the Chloroplast Genome Sequences of Two Korean Endemic Primula Varieties

by
Sang-Chul Kim
,
Young-Ho Ha
,
Dong-Kap Kim
,
Dong Chan Son
,
Hyuk-Jin Kim
* and
Kyung Choi
Division of Forest Biodiversity, Korea National Arboretum, 509 Gwangneungsumogwon-ro, Soheul-eup, Pocheon-si 11186, Korea
*
Author to whom correspondence should be addressed.
Diversity 2022, 14(6), 458; https://doi.org/10.3390/d14060458
Submission received: 13 April 2022 / Revised: 3 June 2022 / Accepted: 6 June 2022 / Published: 7 June 2022
Browse Figures
Versions Notes

Abstract

:
Primula comprises many species of horticultural value. In Korea, six species grow in the wild. Yamazaki classified the variety Primula modesta var. fauriei into P. modesta var. hannasanensis and P. modesta var. koreana based on the differences in leaf morphology. We compared the chloroplast genome sequences of the two Korean endemic Primula varieties and found that both had the typical quadripartite structure of angiosperms. The chloroplast genome size of P. modesta var. hannasanensis is 154,772 bp, including an 85,238 bp large single-copy region and a 17,790 bp small single-copy region, whereas that of P. modesta var. koreana is 154,667 bp, including an 85,152 bp large single-copy region and a 17,771 bp small single-copy region. The inverted repeat region is 25,872 bp in both varieties. We predicted 129 genes—84 protein-coding genes, 8 rRNAs, and 37 tRNAs. We identified 536 single-nucleotide polymorphisms and 501 indels between the varieties. Phylogenetic analysis revealed that the two varieties formed a sister group in the clade P. knuthiana–P. stenocalyx. This study will contribute to phylogenetic, taxonomic, and evolutionary studies of the genus Primula; it will also contribute to the analysis of the genetic diversity of the two varieties, and to the development of identification markers.

1. Introduction

The genus Primula L. comprises approximately 430 species worldwide. It is widespread in the northern hemisphere in the highlands of Asia, North America, Europe, and the Eastern Shino Himalayas [1]. Many Primula species are popular as garden species owing to their appealing flowers and long flowering duration. Molecular phylogenetic studies of Primula have been carried out primarily with nuclear and plastid genes and fragments in China, which has the largest distribution area of the species. This has considerably improved the comprehension of the phylogenetic history of Primula. However, the exact phylogenetic history remains uncertain given the occurrence of frequent hybridization events and interspecies morphological variations caused by translocation [2,3,4].
In Korea, there are six Primula species [5]. Among them, Yamazaki divided Primula modesta var. fauriei (Franch.) Takeda into P. modesta var. koreana, which grows wild in the mainland, and P. modesta var. hannasanensis, which grows wild in Jeju Island, based on variations in the morphology of the leaf base [6,7,8,9]. However, comparative genetic information analysis of the two varieties has not yet been conducted.
Chloroplasts (CPs) play important roles in photosynthesis. Plastid genomes are generally 115–165 kb long. They have a quadripartite molecular structure containing two inverted repeat (IR) regions (20–28 kb long) linking the large single-copy (LSC; 80–90 kb long) and small single-copy regions (SSC; 16–27 kb long; [10,11]). In most terrestrial plant lineages, the CP genomes are similar in gene order, gene content, structure, and intron content. Owing to their relatively high substitution rates, CP genomes are an important source of genetic markers for the phylogenetic identification of species and population genetics [12].
In this study, we analyzed the CP genomes of two Korean endemic Primula variants, P. modesta var. koreana and P. modesta var. hannasanensis, which have not yet been reported. We compared the two variants to examine single nucleotide polymorphisms (SNPs), indels, and simple sequence repeat (SSR) polymorphisms to identify markers useful for DNA barcodes and phylogenetic analysis. Our findings may not only contribute to further studies on the evolutionary history, lineage, and taxonomy of the genus Primula, but also aid in helping to better understand its CP genome.

2. Materials and Methods

2.1. Material Collection, DNA Extraction, and Next-Generation Genome Sequencing

Samples of the two varieties (P. modesta var. koreana and P. modesta var. hannasanensis) were collected from Sinbul Mountain (Ulsan-si, Gyeongsangnam-do, Korea; N: 35°31′26.7″ E: 129°03′01.5″) and Halla Mountain (Jeju, Korea; N: 33°22′48.9″ E: 126°34′52.049″), respectively. The total genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA) following the manufacturer’s instructions. DNA quality was evaluated using a NanoDrop 2000 microspectrophotometer (Thermo Fisher Inc., Waltham, MA, USA), and the quantity was confirmed using 1% agarose gel electrophoresis. Voucher specimens of the two Primula accessions have been deposited at the Herbarium of the Korea National Arboretum (P. modesta var. koreana, ESK20-040; P. modesta var. hannasanensis, ESK20-138). Next-generation paired-end sequencing was performed using the Illumina MiSeq platform (TruSeq DNA PCR-Free) according to the manufacturer’s protocol (Macrogen Inc., Seoul, Korea).

2.2. CP Genome Assembly and Annotation

The CP DNA sequence data were filtered from the whole genome data using GetOrganelle [13]. The two CP genomes were assembled using Geneious Prime (Biomatters, Auckland, New Zealand) [14] and annotated using GeSeq [15]. Unannotated portions, such as exons and introns, were manually edited. Transfer (t)RNA sequences were confirmed using tRNAscan-SE v1.21 [16]. Genome maps were drawn using OrganellarGenomeDRAW (OGDRAW; [17]).

2.3. Genome Comparison

The complete CP genomes of the two Primula varieties were aligned using MAFFT [18] and compared using m-VISTA (http://genome.lbl.gov/vista/index.shtml, accessed on 1 August 2021) in the shuffle-LAGAN mode [19]. Genome junctions were visualized and compared using IRscope [20].

2.4. Divergent Hotspot Identification

CP genome polymorphisms were analyzed using DNA Sequence Polymorphism (DnaSP) v6 [21] to determine the nucleotide diversity (Pi) values and confirm highly variable sites. The sequences were aligned using MAFFT in Geneious Prime [14,18]. The variations (SNPs, insertions, and deletions (indels)) between the varieties were analyzed using Geneious Prime based on a minimum variant frequency criterium of 0.25. The compartments were separated into coding sequences (CDSs), tRNA, ribosomal (r)RNA, and intergenic spacers (IGSs).

2.5. Relative Synonymous Codon Usage Analysis

Relative synonymous codon usage (RSCU) was computed from the CDSs of the two Primula CP genomes. The DAMBE program was employed for the RSCU and codon frequency analyses [22].

2.6. SSR and Long Repeat Sequence Analysis

SSRs within the two Primula CP genomes were analyzed using the MISA Perl script (MIcroSAtellite; [23]) based on the following minimum repeats criteria: mononucleotide repeats, 10; dinucleotide repeats, 5; trinucleotide repeats, 4; and tetra-, penta-, and hexa-nucleotide repeats, 3. REPuter was used to locate four repeat types (forward, reverse, complementary, and palindromic) based on the following criteria: a minimum repeat size of 30 bp and sequence identity of 90% [24].

2.7. Phylogenetic Analysis

The complete CP genome sequences of 76 Primulaceae and 9 other Ericales (4 Sapotaceae, 4 Pentaphylacaceae, and 1 Polemoniaceae) species were obtained from the NCBI database and used for a maximum likelihood (ML) phylogenetic analysis (Table S1). In total, 77 CDSs from 85 species were aligned using MAFFT in PhyloSuite [25]. ModelFinder within the PhyloSuite program was used to determine the optimal alternative model [26]. The ML analysis was performed using IQ-Tree software (http://iqtree.cibiv.univie.ac.at/, accessed on 4 August 2021).

3. Results

3.1. Common Features of the CP Genomes

The CP genome lengths of the two variants were in similar ranges: P. modesta var. koreana, 154,667 bp (LSC; 85,152 bp, SSC; 85,152 bp, and IRs; 25,872 bp); P. modesta var. hannasanensis, 154,772 bp (LSC; 85,238 bp, SSC; 17,790 bp, and IRs; 25,872 bp). Moreover, they had typical quadripartite structures (Figure 1 and Table 1). The CP of P. modesta var. hannasanensis is the largest among the Primula CP genomes reported to date. The two CP genomes had identical gene contents (129 genes—84 protein-coding, 8 rRNA, and 37 tRNA genes; Table 2). Seventeen genes located in the IR regions included 6 protein-coding genes (rpl2, rpl23, rps7, rps12, ndhB, and ycf2) and 4 rRNA genes (rrn4.5, rrn5, rrn16, and rrn23). Eight tRNA genes (trnA-UGC, trnG-UCC, trnK-UUU, trnI-GAU, trnI-CAU, trnL-UAA, trnN-GUU, trnR-ACG, and trnV-UAC) and 10 protein-coding genes (atpF, ndhA, ndhB, rpl2, rpl16, rps12, rps16, rpoC1, petB, and petD) contained one intron, and two protein-coding genes (clpP1 and pafI) contained two introns (Table 2). rps12 was confirmed to be a trans-spliced gene consisting of three exons: exon 1, found in the LSC region, and exons 2 and 3, located in the IR regions.

3.2. Comparison of the CP Genomes of the Two Primula Varieties

We compared the gene sequence and CP genome content between the varieties via m-VISTA, and found that the CP genomes were nearly identical with coding and IR regions more conserved than non-coding, LSC, and SSC regions (Figure 2). The boundary structures of the two genomes were compared with those of four other Primula CP genomes located in the same group in the ML tree (see Section 3.6) The overall identity of the CP genomes was confirmed at all junctions, including JLB (LSC/IRb), JSB (IRb/SSC), JSA (SSC/IRa), and JLA (IRa/LSC). However, trnH-GUG was located in the LSC region 3 bp away from the JLA junction in four Primula species, and 11 and 1 bp away in P. pulchella Franch. and P. knuthiana Pax, respectively. A 978 bp region of ycf1 was located in IRa and a 7 bp region of ndhF was integrated into IRb. At the JLB junction, a 41 bp region of rps19 was present in the IR region of all five species, except that of P. knuthiana (100 bp in the IR) (Figure 3).

3.3. Divergent Hotspots in the Primula CP genomes

Overall, 536 SNPs were found in the CP genomes of the two Primula varieties. In total, 205 (38.2%) SNPs were located in the CDS regions, and 331 (61.8%) were located in the IGS regions and introns. In addition, 501 indels were identified. The level of sequence divergence was determined by calculating the Pi values for the CP genomes of the two varieties (Figure 4 and Table S2). The Pi values for SNPs in the CDSs ranged from 0.00045 (psaB) to 0.01465 (rps15), with an average of 0.0023. The Pi values for SNPs in the IGSs ranged from 0.00146 (ndhB intron) to 0.03318 (psbA~trnK-UUU), with an average of 0.005. SNPs were identified in the trnY-GUA, 16S rRNA, and 23S rRNA genes. CDSs with a Pi value higher than 0.008 were identified as cemA, rpl33, rps11, and rps15. The regions with the highest Pi values in the IGSs were identified as the psbA~trnK-UUU, psbZ~trnG-GCC, ndhD~psaC, and rps15~ycf1 regions (average Pi value: 0.0259).

3.4. Relative Synonymous Codon Usage Analysis

A total of 78 CDSs from the CP genomes of the two Primula varieties were used to estimate the frequency of relative synonymous codon usage (except for the stop codons UAA, UAG, and UGA). In total, 22,696 codons were detected in P. modesta var. koreana and 22,691 in P. modesta var. hannasanensis. The two varieties showed similar results. Leucine was the most abundant amino acid (10.65%), whereas cysteine was the least abundant (1.06%, P. modesta var. koreana; 1.07%, P. modesta var. hannasanensis). The most used codon was AUU (998), encoding isoleucine, and the least used codon was UGC (57), which encoded cysteine in both varieties. The RSCU frequency analyses of the two CP genomes revealed a bias in codon usage; 29 amino acids had RSCU > 1. Two amino acids, methionine (AUG) and tryptophan (UGG), did not display codon usage bias (RSCU = 1.00). In both varieties, the highest RSCU value was recorded for GCU (1.879, P. modesta var. koreana; 1.872, P. modesta var. hannasanensis), which encodes alanine, and the lowest for UAC (0.359, P. modesta var. koreana; 0.355, P. modesta var. hannasanensis), which encodes tyrosine (Figure 5).

3.5. SSR and Long Repeat Analyses

In total, 58 SSRs were identified in P. modesta var. hannasanensis and 53 in P. modesta var. koreana. Both varieties had a high number of mononucleotide repeats. There were 4 (P. modesta var. koreana) and 6 (P. modesta var. hannasanensis) dinucleotide repeats and 2 identical tetranucleotide and hexanucleotide repeats; a single pentanucleotide repeat was identified in P. modesta var. koreana. Most SSRs consisted of the A/T motif rather than the G/C motif (Table 3 and Table S3).
The long repeat analysis identified more forward and palindromic repeats than reverse and complementary repeats in the two Primula varieties. A total of 30 long repeats were identified in P. modesta var. koreana and 32 in P. modesta var. hannasanensis. Only one reverse repeat was found in P. modesta var. hannasanensis. The length of most repeats ranged from 30 to 39 bp, whereas the largest repeat was 50 bp long (P. modesta var. koreana). The locations and numbers of iterations of long repeats are listed in Table 4 and Table S4.

3.6. Phylogenetic Analysis

The phylogenetic analysis was performed using the ML method and 77 genes from 85 Ericales CP genomes (Figure 6 and Table S1). The best-fit model according to ModelFinder was GTR + F + R3. The resulting phylogeny showed that the monophyly of the Primulaceae clade was highly bootstrap supported (BS = 100). Within the family, Maesa montana A. DC. branched first to form the basal group (BS = 100). Lysimachia L., Aegiceras Gaertn., Myrsine L., Embelia Burm. f., Elingamita G.T.S. Baylis, Parathesis (A. DC.) Hook. f., Tapeinosperma Hook. f., and Ardisia Sw. showed close relationships and formed a monophyletic group (BS = 100). The genus Androsace formed a sister group with the Primula and Bryocarpum Hook clades. f. and Thomson (BS = 100). The genus Primula was divided into two clades (BS = 100). The clade containing P. modesta var. koreana and P. modesta var. hannasanensis was further divided into two clades (BS = 100). In this clade, P. veris branched first, followed by P. denticulata subsp. sinodenticulata and P. pulchella. Two groups were then formed: one comprising P. modesta var. koreana and P. modesta var. hannasanensis and the other comprising P. stenocalyx and P. knuthiana (BS = 100).

4. Discussion

Most Primula species are distributed in highlands. Some species are threatened by anthropogenic disturbances, such as collection for horticultural purposes, and the wild population is rapidly declining [3,27]. Through genome analyses, it is possible to provide basic information to evaluate the genetic diversity of species and develop identification markers. Although Yamazaki distinguished two varieties endemic to Korea (P. modesta var. koreana and P. modesta var. hannasanensis), genetic analyses of these varieties have not yet progressed. Therefore, we compared the CP genomes of the two varieties using next-generation sequencing. Herein, we reported the CP genome structures of the two Korean Primula varieties and presented the results of a comparative genome study.
The CP genomes of the two Primula varieties were well conserved and had equal gene numbers, gene orders, and typical quadripartite molecular structures. Our findings are consistent with those reported for the CP genomes of other Primula species [28,29,30,31,32,33,34,35,36,37,38,39,40,41,42]. The genome size of P. modesta var. koreana and P. modesta var. hannasanesis is 154,667 and 154,772 bp, respectively (Figure 1). It was found that infA, which encodes translation initiation factor 1 and aids in the assembly of the translation initiation complex, was not present in these varieties, but it has been identified in other Primula CP genomes [43]. We found that the varieties were located in the same clade (Figure 6). In recent studies, ycf3 and ycf4—encoding the photosystem assembly factors that act in the photosystem I complex—were renamed pafI and pafII, respectively [44], and photosystem biogenesis factor 1 (psbN) was renamed pbf1 [45]. Here, we used the new names. An analysis using MAFFT revealed a 99.3% sequence identity between the varieties. In addition, 536 SNPs and 501 indels were identified. Our results revealed a significant number of structural variations (SNPs and indels) in the cp genome. These results were less than the structural variations in Commiphora gileadensis [46]. It may be the result of subspecies taxa.
SSRs have codominant and highly polymorphic features, and they are used as markers for phylogenetic and population genetics studies [47,48,49]. The SSRs discovered in this study had a high A/T content; accordingly, most of the confirmed mononucleotide repeats were composed of A/T (P. modesta var. koreana: 97.7% and P. modesta var. hannasanensis: 100%). The CDS region with the highest number of SSRs was found in ycf1, and this is consistent with the findings of a previous study [43]. The SSRs identified here may be useful as molecular markers for studies on Primula species. In total, 62 long repeat sequences were discovered in both varieties and were identical to those of Primula species of the same order. Most repeats were in the 30–50 bp range, which was slightly smaller than previously reported ranges for Primula species [43]. This may be additional evidence that Primula species have not undergone rearrangement events [50]. Information on structural variation and SSR obtained in this study can be used to select effective molecular markers for the identification of inter- and intra-specific polymorphisms.
The CP genomes have been widely used in species verifications and phylogenetic studies on terrestrial plants [51,52]. In the present study, we performed an ML analysis to construct a phylogenetic tree. Primulaceae formed a monophyletic group that was divided into three subfamilies. Maesoideae branched first within the family, and Myrsinoideae and Primuloideae formed a monophyletic group. Primula formed a strong monophyletic group and was closely related to Bryocarpum in Primulaceae. The genus Primula was divided into two clades. The first clade comprised the subgenus Auganthus, except for species included in Ranunculoides (P. cicutariifolia, P. merrilliana, P. jiugongshanensis, P. hubeiensis, and P. ranunculoides) [1,53]. The second clade comprised the subgenus Aleuritia, Primula, and Ranunculoides of the subgenus Auganthus and was further divided into two clades. The varieties in this study, P. modesta var. koreana and P. modesta var. hannasanensis, formed a sister group with the clades P. knuthiana and P. stenocalyx. The phylogenetic tree was similar to those in previous molecular studies, but it did not follow traditional subgenus taxonomy [1,54]. Owing to insufficient information on taxa belonging to Theophrastoideae within Primulaceae, a more reliable relationship within the family could not be obtained. However, the genetic differences between the varieties were clearly identified and support the results of previous studies [6,8]. The identification of species-level differences is essential for the ongoing conservation of vulnerable members of the genus Primula.

5. Conclusions

Herein, we reported the complete CP genomic sequences of P. modesta var. koreana and P. modesta var. hannasanensis for the first time. The comprehensive genetic information presented herein can form a basis for the identification of Primula species and the analysis of genetic differences at the individual level. Phylogenetically, Primula was found to be related to Bryocarpum belonging to Primulaceae. We also obtained important genetic information on SNPs, SSRs, long repeats, divergent hotspot regions, and phylogeny. The results of our CP genome analysis provide a valuable resource to facilitate phylogenetic, taxonomic, and evolutionary studies of this genus. Our findings will further contribute to the analysis of the genetic diversity of both species and development of identification markers.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/d14060458/s1: Table S1: NCBI data used in the ML Analysis; Table S2: Single-nucleotide polymorphisms (SNPs) identified in the chloroplast genome of the two Primula varieties; Table S3: SSR information of the two Primula varieties; Table S4: Information of long repeats of the two Primula varieties.

Author Contributions

Validation, H.-J.K., D.C.S. and Y.-H.H.; formal analysis, S.-C.K.; investigation, D.-K.K. and Y.-H.H.; writing—original draft preparation, S.-C.K.; writing—review and editing, H.-J.K., D.C.S. and S.-C.K.; visualization, H.-J.K.; supervision, K.C.; funding acquisition, H.-J.K. and K.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Scientific Research Grants (KNA1-1-13, 14–1) from the Korea National Arboretum.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The genome sequence data that support the findings of this study are openly available in the GenBank of NCBI at https://www.ncbi.nlm.nih.gov (Registered Date on 13 August 2021) under accession nos. MZ779112 (P. modesta var. hannasanensis) and MZ779113 (P. modesta var. koreana).

Acknowledgments

We thank Sa-Bum Jang, Hee Young Gil, and Eun-Ho Lee for sampling and laboratory assistance throughout the study.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript or in the decision to publish the results.

References

  1. Xu, W.; Xia, B.; Li, X. The complete chloroplast genome sequences of five pinnate-leaved Primula species and phylogenetic analyses. Sci. Rep. 2020, 10, 20782. [Google Scholar] [CrossRef] [PubMed]
  2. Yan, H.F.; He, C.H.; Peng, C.I.; Hu, C.M.; Hao, G. Circumscription of Primula subgenus Auganthus (Primulaceae) based on chloroplast DNA sequences. J. Syst. Evol. 2010, 48, 123–132. [Google Scholar] [CrossRef]
  3. Mast, A.R.; Kelso, S.; Richards, A.J.; Lang, D.J.; Feller, D.M.; Conti, E. Phylogenetic relationships in Primula L. and related genera (Primulaceae) based on noncoding chloroplast DNA. Int. J. Plant Sci. 2001, 162, 1381–1400. [Google Scholar] [CrossRef] [Green Version]
  4. Singh, S.; Ali, S.; Singh, M. Biological screening of plants extract showing hypoglycaemic and wound healing properties: Capparis zeylanica and Primula denticulata. Am. J. Phytomed. Clin. Ther. 2014, 12, 1338–1345. [Google Scholar]
  5. Korea National Arboretum. Checklist of Vascular Plants in Korea (Native Plants); Korea National Arboretum: Pocheon, Korea, 2020. [Google Scholar]
  6. Chung, J.M.; Son, S.W.; Kim, S.Y.; Park, G.W.; Kim, S.S. Genetic diversity and geographic differentiation in the endangered Primula farinosa subsp. modesta, a subalpine endemic to Korea. Korean J. Plant Taxon. 2013, 43, 236–243. [Google Scholar] [CrossRef]
  7. Korea National Arboretum. Rare Plants Data Book in Korea; Geobook: Seoul, Korea, 2008; p. 148. (In Korean) [Google Scholar]
  8. Chung, G.Y.; Chang, K.S.; Chung, J.M.; Choi, H.J.; Paik, W.K.; Hyun, J.O. A checklist of endemic plants on the Korean Peninsula. Korean J. Plant Taxon. 2017, 47, 264–288. [Google Scholar] [CrossRef] [Green Version]
  9. Yamazaki, T. Intraspecific taxa in Primula farinosa L. subsp. modesta (Bisset and Moore) Pax. J. Jpn. Bot. 2003, 78, 295–299. (In Japanese) [Google Scholar]
  10. Jansen, R.K.; Raubeson, L.A.; Boore, J.L.; de Pamphilis, C.W.; Chumley, T.W.; Haberle, R.C.; Wyman, S.K.; Alverson, A.J.; Peery, R.; Herman, S.J.; et al. Methods for obtaining and analyzing whole chloroplast genome sequences. Methods Enzymol. 2005, 395, 348–384. [Google Scholar]
  11. Daniell, H.; Lin, C.S.; Yu, M.; Chang, W.J. Chloroplast genomes: Diversity, evolution, and applications in genetic engineering. Genome Biol. 2016, 17, 134. [Google Scholar] [CrossRef] [Green Version]
  12. Drouin, G.; Daoud, H.; Xia, J. Relative rates of synonymous substitutions in the mitochondrial, chloroplast and nuclear genomes of seed plants. Mol. Phylogenet. Evol. 2008, 49, 827–831. [Google Scholar] [CrossRef]
  13. Jin, J.J.; Yu, W.B.; Yang, J.B.; Song, Y.; Yi, T.S.; Li, D.Z. GetOrganelle: A simple and fast pipeline for de novo assembly of a complete circular chloroplast genome using genome skimming data. BioRxiv 2018, 4, 256479. [Google Scholar]
  14. Kearse, M.; Moir, R.; Wilson, A.; Stones-Havas, S.; Cheung, M.; Sturrock, S.; Buxton, S.; Cooper, A.; Markowitz, S.; Duran, C.; et al. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 2012, 28, 1647–1649. [Google Scholar] [CrossRef] [PubMed]
  15. Tillich, M.; Lehwark, P.; Pellizzer, T.; Ulbricht-Jones, E.S.; Fischer, A.; Bock, R.; Greiner, S. GeSeq—Versatile and accurate annotation of organelle genomes. Nucleic Acids Res. 2017, 45, W6–W11. [Google Scholar] [CrossRef] [PubMed]
  16. Lowe, T.M.; Chan, P.P. TRNAscan-SE On-Line: Integrating search and context for analysis of transfer RNA genes. Nucleic Acids Res. 2016, 44, W54–W57. [Google Scholar] [CrossRef] [PubMed]
  17. Lohse, M.; Drechsel, O.; Bock, R. OrganellarGenomeDRAW (OGDRAW): A tool for the easy generation of high-quality custom graphical maps of plastid and mitochondrial genomes. Curr. Genet. 2007, 52, 267–274. [Google Scholar] [CrossRef]
  18. Katoh, K.; Kuma, K.I.; Toh, H.; Miyata, T. MAFFT Version 5: Improvement in accuracy of multiple sequence alignment. Nucleic Acids Res. 2005, 33, 511–518. [Google Scholar] [CrossRef]
  19. Mayor, C.; Brudno, M.; Schwartz, J.R.; Poliakov, A.; Rubin, E.M.; Frazer, K.A.; Pachter, L.S.; Dubchak, I. VISTA: Visualizing global DNA sequence alignments of arbitrary length. Bioinformatics 2000, 16, 1046–1047. [Google Scholar] [CrossRef] [Green Version]
  20. Amiryousefi, A.; Hyvönen, J.; Poczai, P. IRscope: An online program to visualize the junction sites of chloroplast genomes. Bioinformatics 2018, 34, 3030–3031. [Google Scholar] [CrossRef]
  21. Librado, P.; Rozas, J. DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics 2009, 25, 1451–1452. [Google Scholar] [CrossRef] [Green Version]
  22. Xia, X.; Xie, Z. DAMBE: Software package for data analysis in molecular biology and evolution. J. Hered. 2001, 92, 371–373. [Google Scholar] [CrossRef] [Green Version]
  23. Thiel, T.; Michalek, W.; Varshney, R.K.; Graner, A. Exploiting EST databases for the development and characterization of gene-derived SSR-Markers in barley (Hordeum vulgare L.). Theor. Appl. Genet. 2003, 106, 411–422. [Google Scholar] [CrossRef] [PubMed]
  24. Kurtz, S.; Choudhuri, J.V.; Ohlebusch, E.; Schleiermacher, C.; Stoye, J.; Giegerich, R. REPuter: The manifold applications of repeat analysis on a genomic scale. Nucleic Acids Res. 2001, 29, 4633–4642. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Zhang, D.; Gao, F.; Jakovlić, I.; Zou, H.; Zhang, J.; Li, W.X.; Wang, G.T. PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Mol. Ecol. Resour. 2020, 20, 348–355. [Google Scholar] [CrossRef] [PubMed]
  26. Kalyaanamoorthy, S.; Minh, B.Q.; Wong, T.K.; Von Haeseler, A.; Jermiin, L.S. ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat. Methods 2017, 14, 587–589. [Google Scholar] [CrossRef] [Green Version]
  27. Richards, J. Primula; Timber: Portland, OR, USA, 1993. [Google Scholar]
  28. Zhou, T.; Zhao, J.; Chen, C.; Meng, X.; Zhao, G. Characterization of the complete chloroplast genome sequence of Primula veris (Ericales: Primulaceae). Conserv. Genet. Resour. 2016, 8, 455–458. [Google Scholar] [CrossRef]
  29. Sun, H.Y.; Zhong, L.; Gan, Q.L.; Zhang, T.; Wu, Z.K. The complete chloroplast genome of an endangered endemic herb species in China, Primula filchnerae (Primulaceae). Mitochondrial DNA B Resour. 2019, 4, 2746–2747. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  30. Zhang, C.Y.; Liu, T.J.; Yan, H.F.; Ge, X.J.; Hao, G. The complete chloroplast genome of a rare candelabra primrose Primula stenodonta (Primulaceae). Conserv. Genet. Resour. 2017, 9, 123–125. [Google Scholar] [CrossRef]
  31. Zhang, L.; Chen, X.; Huang, Y.; Wu, Z. The complete chloroplast genome of Primula helodoxa, a species endemic to China. Mitochondrial DNA B Resour. 2020, 5, 194–195. [Google Scholar] [CrossRef] [Green Version]
  32. Chen, X.; Zhang, L.; Li, W.; Huang, Y.; Wu, Z. The complete chloroplast genome of Primula bulleyana, a popular ornamental species. Mitochondrial DNA B Resour. 2019, 4, 3673–3674. [Google Scholar] [CrossRef] [Green Version]
  33. Zhang, C.; Yuan, X.; Yang, T.; Yan, H.; Liu, T. The complete chloroplast genome of Primula obconica (Primulaceae). Mitochondrial DNA B Resour. 2019, 4, 2189–2190. [Google Scholar] [CrossRef] [Green Version]
  34. Sun, H.Y.; Zhong, L.; Guo, Y.J.; Zhou, W.; Wu, Z.K. The complete chloroplast genome of a distylous-homostylous species, Primula homogama (Primulaceae). Mitochondrial DNA B Resour. 2021, 6, 393–394. [Google Scholar] [CrossRef] [PubMed]
  35. Wu, Z.; Chen, X.; Zhang, L.; Huang, Y. The complete chloroplast genome of Primula beesiana, an ornamental alpine plant from SW China. Mitochondrial DNA B Resour. 2020, 5, 182–183. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Liu, T.J.; Zhang, C.Y.; Yan, H.F.; Zhang, L.; Ge, X.J.; Hao, G. Complete plastid genome sequence of Primula sinensis (Primulaceae): Structure comparison, sequence variation and evidence for accD transfer to nucleus. PeerJ 2016, 4, e2101. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Chen, S.; Yan, X.; Hao, G.; Xu, Y. The complete chloroplast genome of Primula tsiangii WW Smith (Primulaceae): A karst endemic primrose in Southwest China. Mitochondrial DNA B Resour. 2019, 4, 2627–2628. [Google Scholar] [CrossRef] [Green Version]
  38. Lu, Y.; Liu, P.L.; Sun, Y.F.; Li, S.F. The complete chloroplast genome sequence of Primula filchnerae Knuth (Primulaceae), an endangered species in China. Mitochondrial DNA B Resour. 2020, 5, 2047–2048. [Google Scholar] [CrossRef]
  39. Zhang, C.Y.; Liu, T.J.; Yan, H.F.; Xu, Y. The complete chloroplast genome of Primula persimilis (Primulaceae). Conserv. Genet. Resour. 2017, 9, 189–191. [Google Scholar] [CrossRef]
  40. Zhang, C.Y.; Liu, T.J.; Xu, Y.; Yan, H.F.; Hao, G.; Ge, X.J. Characterization of the whole chloroplast genome of an endangered species Primula kwangtungensis (Primulaceae). Conserv. Genet. Resour. 2017, 9, 87–89. [Google Scholar] [CrossRef]
  41. Wang, J.; Zhang, R.; Ren, T.; Han, K.; Zeng, S.; Biffin, E.; Liu, Z.L. Characterization of the complete chloroplast genome of the Cortusa matthioli subsp. pekinensis (Primulaceae). Conserv. Genet. Resour. 2017, 9, 603–605. [Google Scholar] [CrossRef]
  42. Zhang, C.Y.; Liu, T.J.; Xu, Y.; Yan, H.F. Characterization of the whole chloroplast genome of a rare candelabra primrose Primula chrysochlora (Primulaceae). Conserv. Genet. Resour. 2017, 9, 361–363. [Google Scholar] [CrossRef]
  43. Ren, T.; Yang, Y.; Zhou, T.; Liu, Z.L. Comparative plastid genomes of Primula species: Sequence divergence and phylogenetic relationships. Int. J. Mol. Sci. 2018, 19, 1050. [Google Scholar] [CrossRef] [Green Version]
  44. Wicke, S.; Schneeweiss, G.M.; Depamphilis, C.W.; Müller, K.F.; Quandt, D. The evolution of the plastid chromosome in land plants: Gene content, gene order, gene function. Plant Mol. Biol. 2011, 76, 273–297. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Krech, K.; Fu, H.Y.; Thiele, W.; Ruf, S.; Schöttler, M.A.; Bock, R. Reverse genetics in complex multigene operons by co-transformation of the plastid genome and its application to the open reading frame previously designated psbN. Plant J. 2013, 75, 1062–1074. [Google Scholar] [CrossRef] [PubMed]
  46. Khan, A.; Asaf, S.; Khan, A.L.; Al-Harrasi, A.; Al-Sudairy, O.; AbdulKareem, N.M.; Khan, A.; Shehzad, T.; Alsaady, N.; Al-Lawati, A.; et al. First complete chloroplast genomics and comparative phylogenetic analysis of Commiphora gileadensis and C. foliacea: Myrrh producing trees. PLoS ONE 2019, 14, e0208511. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Grassi, F.; Labra, M.; Scienza, A.; Imazio, S. Chloroplast SSR markers to assess DNA diversity in wild and cultivated grapevines. Vitis 2002, 41, 157–158. [Google Scholar]
  48. He, S.L.; Wang, Y.S.; Volis, S.; Li, D.Z.; Yi, T.S. Genetic diversity and population structure: Implications for conservation of wild soybean (Glycine soja Sieb. et Zucc) based on nuclear and chloroplast microsatellite variation. Int. J. Mol. Sci. 2012, 13, 12608–12628. [Google Scholar] [CrossRef] [Green Version]
  49. Xue, J.; Wang, S.; Zhou, S.L. Polymorphic chloroplast microsatellite loci in Nelumbo (Nelumbonaceae). Am. J. Bot. 2012, 99, e240–e244. [Google Scholar] [CrossRef]
  50. Kim, S.C.; Lee, J.W.; Choi, B.K. Seven complete chloroplast genomes from Symplocos: Genome organization and comparative analysis. Forests 2021, 12, 608. [Google Scholar] [CrossRef]
  51. Särkinen, T.; George, M. Predicting plastid marker variation: Can complete plastid genomes from closely related species help? PLoS ONE 2013, 8, e82266. [Google Scholar] [CrossRef] [Green Version]
  52. Ma, P.F.; Zhang, Y.X.; Zeng, C.X.; Guo, Z.H.; Li, D.Z. Chloroplast phylogenomic analyses resolve deep-level relationships of an intractable bamboo tribe Arundinarieae (Poaceae). Syst. Biol. 2014, 63, 933–950. [Google Scholar] [CrossRef] [Green Version]
  53. Shao, J.W.; Wu, Y.F.; Kan, X.Z.; Liang, T.J.; Zhang, X.P. Reappraisal of Primula ranunculoides (Primulaceae), an endangered species endemic to China, based on morphological, molecular genetic and reproductive characters. Bot. J. Linn. Soc. 2012, 169, 338–349. [Google Scholar] [CrossRef] [Green Version]
  54. Gang, H.A.O.; Chi-Ming, H.U.; Nam-Sook, L.E.E. Circumscriptions and phylogenetic relationships of Primula Sects. Auganthus and Ranunculoides: Evidence from nrDNA ITS sequences. J. Integr. Plant Biol. 2002, 44, 72. [Google Scholar]
Diversity 14 00458 g001 550
Figure 1. Complete chloroplast (CP) genome map of Primula modesta var. koreana and P. modesta var. hannasanensis. Genes drawn outside and inside the circle are transcribed counterclockwise and clockwise, respectively. Functional categories of various genes are marked in color. Light gray corresponds to AT content and dark gray in the inner circle corresponds to GC content.
Figure 1. Complete chloroplast (CP) genome map of Primula modesta var. koreana and P. modesta var. hannasanensis. Genes drawn outside and inside the circle are transcribed counterclockwise and clockwise, respectively. Functional categories of various genes are marked in color. Light gray corresponds to AT content and dark gray in the inner circle corresponds to GC content.
Diversity 14 00458 g001
Diversity 14 00458 g002 550
Figure 2. Comparison of the CP genomes of the two Korean endemic Primula varieties using m-VISTA. Gray arrows above the alignments indicate gene orientation, purple bars represent coding sequences (CDSs) and exons, and blue bars represent RNAs. The y-axis shows the identity from 50% to 100%.
Figure 2. Comparison of the CP genomes of the two Korean endemic Primula varieties using m-VISTA. Gray arrows above the alignments indicate gene orientation, purple bars represent coding sequences (CDSs) and exons, and blue bars represent RNAs. The y-axis shows the identity from 50% to 100%.
Diversity 14 00458 g002
Diversity 14 00458 g003 550
Figure 3. Comparison of the boundary distances between the adjacent genes and junctions of large single-copy (LSC), small single-copy (SSC), and two inverted repeat (IR) regions among the CP genomes of six Primula species. Genes are displayed as colored boxes. The figure is not representative of sequence length but shows the relative differences in the IR/SC borders.
Figure 3. Comparison of the boundary distances between the adjacent genes and junctions of large single-copy (LSC), small single-copy (SSC), and two inverted repeat (IR) regions among the CP genomes of six Primula species. Genes are displayed as colored boxes. The figure is not representative of sequence length but shows the relative differences in the IR/SC borders.
Diversity 14 00458 g003
Diversity 14 00458 g004 550
Figure 4. Sliding window analysis of the complete CP genome nucleotide diversity (Pi) between the Korean Primula varieties.
Figure 4. Sliding window analysis of the complete CP genome nucleotide diversity (Pi) between the Korean Primula varieties.
Diversity 14 00458 g004
Diversity 14 00458 g005 550
Figure 5. Relative synonymous codon usage (RSCU) analysis of 20 amino acids in all CDSs of the complete CP genomes of the two Korean Primula varieties.
Figure 5. Relative synonymous codon usage (RSCU) analysis of 20 amino acids in all CDSs of the complete CP genomes of the two Korean Primula varieties.
Diversity 14 00458 g005
Diversity 14 00458 g006 550
Figure 6. Maximum likelihood (ML) phylogenetic tree based on 77 protein-coding genes from 85 Ericales species. Bootstrap support values are shown at the nodes.
Figure 6. Maximum likelihood (ML) phylogenetic tree based on 77 protein-coding genes from 85 Ericales species. Bootstrap support values are shown at the nodes.
Diversity 14 00458 g006
Table
Table 1. Information of the two Korean Primula CP genomes.
Table 1. Information of the two Korean Primula CP genomes.
FeatureP. modesta var. koreanaP. modesta var. hannasanensis
Accession numberMZ779113MZ779112
Genome size [GC(%)]154,667 [37.0]154,772 [36.9]
LSC [GC(%)]85,152 [34.8]85,238 [34.8]
SSC [GC(%)]17,771 [30.3]17,790 [30.3]
IR [GC(%)]25,872 [42.7]25,872 [42.7]
Table
Table 2. CP genome gene content and functional classification in Primula modesta var. koreana and P. modesta var. hannasanensis.
Table 2. CP genome gene content and functional classification in Primula modesta var. koreana and P. modesta var. hannasanensis.
Gene CategoryGene GroupGene Names
Self-replicationLarge subunit ribosomal proteinsrpl2(×2) *, rpl14, rpl16 *, rpl20, rpl22, rpl23(×2), rpl32, rpl33, rpl36
DNA dependent RNA polymeraserpoA, rpoB, rpoC1 *, rpoC2
Small subunit ribosomal proteinsrps2, rps3, rps4, rps7(×2), rps8, rps11, rps12(×2) *, rps14, rps15, rps16 *, rps18, rps19
rRNAsrrn4.5S(×2), rrn5S(×2), rrn16S(×2), rrn23S(×2)
tRNAstrnA-UGC(×2) *, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-GCC, trnG-UCC *, trnH-GUG, trnI-GAU(×2) *, trnI-CAU(×2), trnK-UUU *, trnL-CAA(×2), trnL-UAA *, trnL-UAG, trnM-CAU, trnN-GUU(×2), trnP-UGG, trnQ-UUG, trnR-ACG(×2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC(×2), trnV-UAC *, trnW-CCA, trnY-GUA
PhotosynthesisSubunits of ATP synthaseatpA, atpB, atpE, atpF *, atpH, atpI
Subunits of NADH-dehydrogenasendhA *, ndhB(×2) *, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Subunits of cytochrome b/f complexpetA, petB *, petD, petG, petL, petN
Subunits of photosystem IpsaA, psaB, psaC, psaI, psaJ
Subunits of photosystem IIpsbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbT, psbZ
Subunit of rubiscorbcL
Photosystem assembly factorspafI **, pafII
Photosystem biogenesis factorpbf1
Other genesSubunit of acetyl-CoA-carboxylaseaccD
C-type cytochrome synthesis geneccsA
Envelop membrane proteincemA
ATP-dependent protease subunit PclpP1 **
MaturasematK
Unknown functionConserved open reading framesycf1, ycf2(×2)
Note: (×2), two gene copies in IRs; *, gene containing a single intron; **, gene containing two introns.
Table
Table 3. Information on the types and numbers of SSRs in the CP genomes of the two Korean Primula varieties.
Table 3. Information on the types and numbers of SSRs in the CP genomes of the two Korean Primula varieties.
SSR TypeRepeat UnitPrimula modesta var. koreanaPrimula modesta var. hannasanensisTotal
MononucleotideA/T434892
C/G10
DinucleotideAT/AT4610
TetranucleotideAGAT/ATCT224
PentanucleotideAAAGT/ACTTT101
HexanucleotideAAATAG/ATTTCT114
AAGATG/ATCTTC11
Total 5358111
Table
Table 4. Types and numbers of repeats in the CP genomes of the two Primula varieties, as identified using REPuter.
Table 4. Types and numbers of repeats in the CP genomes of the two Primula varieties, as identified using REPuter.
Type of RepeatPrimula modesta var. koreanaPrimula modesta var. hannasanensis
Forward1213
Reverse01
Palindromic1818
Total3032
Length of repeat (bp)
30–392224
40–4978
50–591
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Kim, S.-C.; Ha, Y.-H.; Kim, D.-K.; Son, D.C.; Kim, H.-J.; Choi, K. Comparative Analysis and Phylogenetic Study of the Chloroplast Genome Sequences of Two Korean Endemic Primula Varieties. Diversity 2022, 14, 458. https://doi.org/10.3390/d14060458

AMA Style

Kim S-C, Ha Y-H, Kim D-K, Son DC, Kim H-J, Choi K. Comparative Analysis and Phylogenetic Study of the Chloroplast Genome Sequences of Two Korean Endemic Primula Varieties. Diversity. 2022; 14(6):458. https://doi.org/10.3390/d14060458

Chicago/Turabian Style

Kim, Sang-Chul, Young-Ho Ha, Dong-Kap Kim, Dong Chan Son, Hyuk-Jin Kim, and Kyung Choi. 2022. "Comparative Analysis and Phylogenetic Study of the Chloroplast Genome Sequences of Two Korean Endemic Primula Varieties" Diversity 14, no. 6: 458. https://doi.org/10.3390/d14060458

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop