Integrated analysis of three newly sequenced fern chloroplast genomes: Genome structure and comparative analysis

BackgroundSome ferns have medicinal properties and are used in therapeutic interventions. However, the classification and phylogenetic relationships of ferns remain incompletely reported. Considering that chloroplast genomes provide ideal information for species identification and evolution, in this study, three unpublished and one published ferns were sequenced and compared with other ferns to obtain comprehensive information on their classification and evolution.Materials and MethodsThe complete chloroplast genomes of Dryopteris goeringiana (Kunze) Koidz, D. crassirhizoma Nakai, Athyrium brevifrons Nakai ex Kitagawa, and Polystichum tripteron (Kunze) Presl were sequenced using the Illumina HiSeq 4,000 platform. Simple sequence repeats (SSRs), nucleotide diversity analysis, and RNA editing were investigated in all four species. Genome comparison and inverted repeats (IR) boundary expansion and contraction analyses were also performed. The relationships among the ferns were studied by phylogenetic analysis based on the whole chloroplast genomes.ResultsThe whole chloroplast genomes ranged from 148,539 to 151,341 bp in size and exhibited typical quadripartite structures. Ten highly variable loci with parsimony informative (Pi) values of > 0.02 were identified. A total of 75–108 SSRs were identified, and only six SSRs were present in all four ferns. The SSRs contained a higher number of A + T than G + C bases. C‐to‐U conversion was the most common type of RNA editing event. Genome comparison analysis revealed that single‐copy regions were more highly conserved than IR regions. IR boundary expansion and contraction varied among the four ferns. Phylogenetic analysis showed that species in the same genus tended to cluster together with and had relatively close relationships.ConclusionThe results provide valuable information on fern chloroplast genomes that will be useful to identify and classify ferns, and study their phylogenetic relationships and evolution.     

The chloroplast genome of mulberry: complete nucleotide sequence, gene organization and comparative analysis

The complete nucleotide sequence of mulberry (Morus indica cv. K2) chloroplast genome (158,484 bp) has been determined using a combination of long PCR and shotgun-based approaches. This is the third angiosperm tree species whose plastome sequence has been completely deciphered. The circular double-stranded molecule comprises of two identical inverted repeats (25,678 bp each) separating a large and a small single-copy region of 87,386 bp and 19,742 bp, respectively. A total of 83 protein-coding genes including five genes duplicated in the inverted repeat regions, eight ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids, were assigned on the basis of homology to predicted genes from other chloroplast genomes. The mulberry plastome lacks the genes infA, sprA, and rpl21 and contains two pseudogenes ycf15 and ycf68. Comparative analysis, based on sequence similarity, both at the gene and genome level, indicates Morus to be closer to Cucumis and Lotus, phylogenetically. However, at genome level, inclusion of non-coding regions brings it closer to Eucalyptus, followed by Cucumis. This may reflect differential selection pressure operating on the genic and intergenic regions of the chloroplast genome.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Communicated by Y. Tsumura     

The development and evaluation of consensus chloroplast primer pairs that possess highly variable sequence regions in a diverse array of plant taxa

Although universal or consensus chloroplast primers are available, they are limited by their number and genomic distribution. Therefore, a set of consensus chloroplast primer pairs for simple sequence repeats (ccSSRs) analysis was constructed from tobacco (Nicotiana tabacum L.) chloroplast sequences. These were then tested for their general utility in the genetic analysis of a diverse array of plant taxa. In order to increase the number of ccSSRs beyond that previously reported, the target sequences for SSR motifs was set at A or T (n 7) mononucleotide repeats. Each SSR sequence motif, along with ±200-bp flanking sequences from the first of each mononucleotide base repeat, was screened for homologies with chloroplast DNA sequences of other plant species in GenBank databases using BLAST search procedures. Twenty three putative marker loci that possessed conserved flanking sequence spans were selected for consensus primer pair construction using commercially available computer algorithms. All primer pairs produced amplicons after PCR employing genomic DNA from members of the Cucurbitaceae (six species) and Solanaceae (four species). Sixteen, 22 and 19 of the initial 23 primer pairs were successively amplified by PCR using template DNA from species of the Apiaceae (two species), Brassicaceae (one species) and Fabaceae (two species), respectively. Twenty of 23 primer pairs were also functional in three monocot species of the Liliaceae [onion (Allium cepa L.) and garlic (Allium sativum L.)], and the Poaceae [oat (Avena sativa L.)]. Sequence analysis of selected ccSSR fragments suggests that ccSSR length and sequence variation could be useful as a tool for investigating the genetic relationships within a genus or closely related taxa (i.e., tribal level). In order to provide for a marker system having significant coverage of the cucumber chloroplast genome, ccSSR primers were strategically "recombined" and named recombined consensus chloroplast primers (RCCP) for PCR analysis. Successful amplification after extended-length PCR of 16 RCCP primer pairs from cucumber (Cucumis sativus L.) DNA suggested that the amplicons detected are representative of the cucumber chloroplast genome. These RCCP pairs, therefore, could be useful as an initial molecular tool for investigation of traits related to a chloroplast gene(s) in cucumber, and other closely related species.Communicated by C. Möllers     

Complete chloroplast genome sequence of Magnolia grandiflora and comparative analysis with related species

Magnolia grandiflora is an important medicinal,ornamental and horticultural plant species.The chloroplast(cp) genome of M.grandiflora was sequenced using a 454 sequencing platform and the genome structure was compared with other related species.The complete cp genome of M.grandiflora was 159623 bp in length and contained a pair of inverted repeats(IR) of 26563 bp separated by large and small single copy(LSC,SSC) regions of 87757 and 18740 bp,respectively.A total of 129 genes were successfully annotated,18 of which included introns.The identity,number and GC content of M.grandiflora cp genes were similar to those of other Magnoliaceae species genomes.Analysis revealed 218 simple sequence repeat(SSR) loci,most composed of A or T,contributing to a bias in base composition.The types and abundances of repeat units in Magnoliaceae species were relatively conserved and these loci will be useful for developing M.grandiflora cp genome vectors.In addition,results indicated that the cp genome size in Magnoliaceae species and the position of the IR border were closely related to the length of the ycf1 gene.Phylogenetic analyses based on 66 shared genes from 30 species using maximum parsimony(MP) and maximum likelihood(ML) methods provided strong support for the phylogenetic position of Magnolia.The availability of the complete cp genome sequence of M.grandiflora provides valuable information for breeding of desirable varieties,cp genetic engineering,developing useful molecular markers and phylogenetic analyses in Magnoliaceae.     

Sequencing and phylogenetic analysis of the chloroplast genome of Pseudosasa japonica f. Akebonosuji

Pseudosasa japonica f. Akebonosuji H. Okamura is a bamboo species with variable leaf colors, including albino, green, and green-white stripes. To determine whether variation in leaf color is due to mutations in the chloroplast genome, we sequenced the chloroplast genomes of green and albino leaves of P. japonica f. Akebonosuji. The results indicated that the chloroplast genome included 86 protein-coding genes, seven ribosomal RNA genes, and 31 tRNA genes. The similarity of chloroplast genomes for the two leaf types was 99.98%, with variation between genes encoding for trnfM and trnT. We observed that the relative expression patterns of trnfM and trnT were reversed in green and albino leaves. Whether the differential expression of trnfM and trnT is involved in leaf color variation among P. japonica f. Akebonosuji remains unclear.With many bamboo chloroplast genomes available, we aligned the chloroplast genomes of 28 bamboo species, including P. japonica f. Akebonosuji, to analyze polymorphisms. This comparison revealed that noncoding regions possessed more nucleotide polymorphisms than coding regions. Chloroplast genomes and the nuclear gene “granule-bound starch synthase I” (GBSSI) of 28 bamboo species were used to construct evolutionary trees. Both evolutionary trees indicated that P. japonica f. Akebonosuji was clustered into Subtrib. Arundinariinae.     

Complete chloroplast genomes of Liliaceae (s.l.) species: comparative genomic and phylogenetic analyses

We first report the complete chloroplast (cp) genome of Fritillaria taipaiensis and determine its characteristics, sequence divergence and phylogenetic relationships by comparing it with complete cp genomes of Liliaceae s.l. (including e.g. Nartheciaceae, Amaryllidaceae and Asparagaceae) species obtained from NCBI Genbank. We show that the ycf1, ycf15 and infA genes have become pseudogenes or are lost in some of the seventeen Liliaceae species, and that dispersed repeats are prevailing among the four types of repeats (dispersed, palindromic, complement and tandem repeats). The number of simple sequence repeats ranged from 53 to 84 in the seventeen species, with mononucleotide repeats being the most abundant, followed by dinucleotides. A total of nine genes with positive selection sites were identified (atpB, atpE, ndhF, ndhH, petB, rpl2, rpl20, rpl22 and ycf2). Furthermore, we examined 19 mutational hotspot regions, including three coding regions (rps16, infA and rpl22) and sixteen non-coding regions. A phylogenetic analysis of the complete cp genomes and protein-coding sequences showed that Fritillaria is most closely related to Lilium. Moreover, Asparagus and Polygonatum, Hosta and Yucca are closely related to the Liliaceae. These results will contribute to further study of evolutionary patterns and phylogenetic relationships in Liliaceae s.l.     

The complete chloroplast genome sequences of Solanum tuberosum and comparative analysis with Solanaceae species identified the presence of a 241-bp deletion in cultivated potato chloroplast DNA sequence

The complete nucleotide sequence of the chloroplast genome of potato Solanum tuberosum L. cv. Desiree was determined. The circular double-stranded DNA, which consists of 155,312 bp, contains a pair of inverted repeat regions (IRa, IRb) of 25,595 bp each. The inverted repeat regions are separated by small and large single copy regions of 18,373 and 85,749 bp, respectively. The genome contains 79 proteins, 30 tRNAs, 4 rRNAs, and unidentified genes. A comparison of chloroplast genomes of seven Solanaceae species revealed that the gene content and their relative positions of S. tuberosum are similar to the other six Solanaceae species. However, undefined open reading frames (ORFs) in LSC region were highly diverged in Solanaceae species except N. sylvestris. Detailed comparison was identified by numerous indels in the intergenic regions that were mostly located in the LSC region. Among them, a single large 241-bp deletion, was not associated with direct repeats and found in only S. tuberosum, clearly discriminates a cultivated potato from wild potato species Solanum bulbocastanum. The extent of sequence divergence may provide the basis for evaluating genetic diversity within the Solanaceae species, and will be useful to examine the evolutionary processes in potato landraces.     

Complete sequence and organization of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome

The nucleotide sequence of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome was completed (DQ119058). The circular double-stranded DNA, consisting of 155,527 bp, contained a pair of inverted repeat regions (IRa and IRb) of 25,187 bp each, which were separated by small and large single copy regions of 86,879 and 18,274 bp, respectively. The presence and relative positions of 113 genes (76 peptide-encoding genes, 30 tRNA genes, four rRNA genes, and three conserved open reading frames) were identified. The major portion (55.76%) of the C. sativus chloroplast genome consisted of gene-coding regions (49.13% protein coding and 6.63% RNA regions; 27.81% LSC, 9.46% SSC and 18.49% IR regions), while intergenic spacers (including 20 introns) made up 44.24%. The overall G-C content of C. sativus chloroplast genome was 36.95%. Sixteen genes contained one intron, while two genes had two introns. The expansion/contraction manner of IR at IRb/LSC and IR/SSC border in Cucumis was similar to that of Lotus and Arabidopsis, and the manner at IRa/LSC was similar to Lotus and Nicotiana. In total, 56 simple sequence repeats (more than 10 bases) were identified in the C. sativus chloroplast genome.     

Complete nucleotide sequence of the sugarcane (Saccharum officinarum) chloroplast genome: a comparative analysis of four monocot chloroplast genomes.

The complete nucleotide sequence of the chloroplast genome of sugarcane (Saccharum officinarum) has been determined. It is a circular double-stranded DNA molecule, 141,182 bp in size, and is composed of a large single copy of 83,048 bp, a small single copy of 12,544 bp, and a pair of inverted repeat regions of 22,795 bp each. A comparative analysis among monocots showed that the sugarcane chloroplast genome was very similar to maize but not to rice or wheat. Between sugarcane and maize at the rps16-trnQ (UUG) region, however, a length polymorphism was identified. With regard to insertions/deletions equal to or longer than 5 bp, a total of 53 insertion and 31 deletion events were identified in the sugarcane chloroplast genome. Of the 84 loci identified, a pair of direct repeat sequences was located side by side in a tandem fashion in 47 loci (56.0%). A recombination event during plant evolution is discussed at two sites between the sugarcane and tobacco chloroplast genomes.     

Complete chloroplast genome sequences from Korean ginseng (Panax schinseng Nees) and comparative analysis of sequence evolution among 17 vascular plants.

The nucleotide sequence of Korean ginseng (Panax schinseng Nees) chloroplast genome has been completed (AY582139). The circular double-stranded DNA, which consists of 156,318 bp, contains a pair of inverted repeat regions (IRa and IRb) with 26,071 bp each, which are separated by small and large single copy regions of 86,106 bp and 18,070 bp, respectively. The inverted repeat region is further extended into a large single copy region which includes the 5' parts of the rpsl9 gene. Four short inversions associated with short palindromic sequences that form stem-loop structures were also observed in the chloroplast genome of P. schinseng compared to that of Nicotiana tabacum. The genome content and the relative positions of 114 genes (75 peptide-encoding genes, 30 tRNA genes, 4 rRNA genes, and 5 conserved open reading frames [ycfs]), however, are identical with the chloroplast DNA of N. tabacum. Sixteen genes contain one intron while two genes have two introns. Of these introns, only one (trnL-UAA) belongs to the self-splicing group I; all remaining introns have the characteristics of six domains belonging to group II. Eighteen simple sequence repeats have been identified from the chloroplast genome of Korean ginseng. Several of these SSR loci show infra-specific variations. A detailed comparison of 17 known completed chloroplast genomes from the vascular plants allowed the identification of evolutionary modes of coding segments and intron sequences, as well as the evaluation of the phylogenetic utilities of chloroplast genes. Furthermore, through the detailed comparisons of several chloroplast genomes, evolutionary hotspots predominated by the inversion end points, indel mutation events, and high frequencies of base substitutions were identified. Large-sized indels were often associated with direct repeats at the end of the sequences facilitating intra-molecular recombination.     

Through a genome, darkly: comparative analysis of plant chromosomal DNA

Plant nuclear genomes encompass a wide range of variation in size and nucleotide composition with diverse arrangements of chromosomal segments, repetitive sequences and distribution of genes. Comparative genomic analysis may be undertaken at different levels of organisation, which are reflected in this review, together with a focus on the genetic and functional significance of the observed variation. Patterns of genome organisation have been revealed which reflect the different underlying mechanisms and constraints driving change. Thus comparative issues of genome size, nucleotide sequence composition and genome heterogeneity are provided as a background to understanding the different levels of segmental and repetitive sequence duplication and distribution of genes. The extent of synteny and collinearity revealed by recent genetic and sequence comparisons is discussed, together with a consideration of problems associated with such analyses. The possible origins and mechanisms of variation in genome size and organisation are covered, including the prevalence of duplication at different levels of organisation. The likely genetic, functional and adaptive consequences of replicated loci are discussed with evidence from comparative studies. The scope for comparative analysis of epigenetic plant genome variation is considered. Finally, opportunities for applying comparative genomics to isolating genes and understanding complex crop genomes are addressed.     

The complete chloroplast genome sequence of the medicinal plant Abrus pulchellus subsp. cantoniensis: genome structure,comparative and phylogenetic relationship analysis

Journal of Plant Research - Abrus pulchellus subsp. cantoniensis, an endemic medicinal plant in southern China, is clinically used to treat jaundice hepatitis, cholecystitis, stomachache and breast...     

Chloroplast genome characteristics and phylogenetic analysis of the medicinal plant Blumea balsamifera (L.) DC

Blumea balsamifera (L.) DC., a medicinal plant with high economic value in the Asteraceae family, is widely distributed in China and Southeast Asia. However, studies on the population structure or phylogenetic relationships with other related species are rare owing to the lack of genome information. In this study, through high-throughput sequencing, we found that the chloroplast genome of B. balsamifera was 151,170 bp in length, with a pair of inverted repeat regions (IRa and IRb) comprising 24,982 bp, a large single-copy (LSC) region comprising 82,740 bp, and a small single-copy (SSC) region comprising 18,466 bp. A total of 130 genes were identified in the chloroplast genome of B. balsamifera, including 85 protein-coding, 37 transfer RNA, and 8 ribosomal RNA genes; furthermore, sequence analysis identified 53 simple sequence repeats. Whole chloroplast genome comparison indicated that the inverted regions (IR) were more conserved than large single-copy and SSC regions. Phylogenetic analysis showed that B. balsamifera is closely related to Pluchea indica. Conclusively, the chloroplast genome of B. balsamifera was helpful for species identification and analysis of the genetic diversity and evolution in the genus Blumea and family Asteraceae.     

Genetic diversity and population structure of the endangered medicinal plant Phellodendron amurense in China revealed by SSR markers

Phellodendron amurense is an endangered tree with important medicinal and economic value in China. In this study, eight nuclear SSR primer pairs were employed to assess the genetic diversity and structure of 22 natural populations, including 516 individuals. A total of 66 alleles were detected with an average of 8.3 alleles per locus ranging from 3 to 17. The expected heterozygosity (He) of each SSR locus varied from 0.347 to 0.877 (average 0.627). Analysis of molecular variance (AMOVA) revealed that the main variation component existed within populations (95.11%) rather than among populations (4.89%). The Wilcoxon's sign-rank tests did not show any recent bottleneck effect in any population. A Mantel test displayed a significant correlation between the geographic distances and genetic distances for all populations (r = 0.566, P = 0.0001), indicating conformity to the isolation by distance model. Bayesian clustering and UPGMA supported grouping the populations into two groups. The present genetic structure of P. amurense may be explained by geographical isolation. The lack of genetic structure and genetic diversity decreased with increasing latitude within the Northeast China group may be due to postglacial northward expansion from a single refugium. Proper conservation measures are proposed for this species.     

The chloroplast genome of Phalaenopsis aphrodite (Orchidaceae): comparative analysis of evolutionary rate with that of grasses and its phylogenetic implications

Whether the Amborella/Amborella-Nymphaeales or the grass lineage diverged first within the angiosperms has recently been debated. Central to this issue has been focused on the artifacts that might result from sampling only grasses within the monocots. We therefore sequenced the entire chloroplast genome (cpDNA) of Phalaenopsis aphrodite, Taiwan moth orchid. The cpDNA is a circular molecule of 148,964 bp with a comparatively short single-copy region (11,543 bp) due to the unusual loss and truncation/scattered deletion of certain ndh subunits. An open reading frame, orf91, located in the complementary strand of the rrn23 was reported for the first time. A comparison of nucleotide substitutions between P. aphrodite and the grasses indicates that only the plastid expression genes have a strong positive correlation between nonsynonymous (Ka) and synonymous (Ks) substitutions per site, providing evidence for a generation time effect, mainly across these genes. Among the intron-containing protein-coding genes of the sampled monocots, the Ks of the genes are significantly correlated to transitional substitutions of their introns. We compiled a concatenated 61 protein-coding gene alignment for the available 20 cpDNAs of vascular plants and analyzed the data set using Bayesian inference, maximum parsimony, and neighbor-joining (NJ) methods. The analyses yielded robust support for the Amborella/Amborella-Nymphaeales-basal hypothesis and for the orchid and grasses together being a monophyletic group nested within the remaining angiosperms. However, the NJ analysis using Ka, the first two codon positions, or amino acid sequences, respectively, supports the monocots-basal hypothesis. We demonstrated that these conflicting angiosperm phylogenies are most probably linked to the transitional sites at all codon positions, especially at the third one where the strong base-composition bias and saturation effect take place.     

Complete chloroplast genome sequences of Schisandra chinensis:genome structure,comparative analysis,and phylogenetic relationship of basal angiosperms

正Dear Editor,Schisandra chinensis(Turcz.)Baill.belongs to family Schisandraceae.Its fruit called"Wu Wei Zi"in Chinese is a well-known medicinal material,which is used to treat chronic cough and dyspnea,nocturnal emission,enuresis,etc.(National Pharmacopoeia Committee,2015).Except for S.chinensis,many species of Schisandraceae,such as S.sphenanthera,S.lancifolia and S.rubriflora,are used as the original plants of folk medicines.Although these species have similar traditional effects,modern studies     

Complete sequence of the chloroplast genome from pear (Pyrus pyrifolia): genome structure and comparative analysis

The chloroplast genome of Pyrus was found to be 159,922?bp in length which included a pair of inverted repeats (IRs) of 26,392?bp, separated by a small single-copy region of 19,237?bp and a large single-copy region (LSC) of 87,901?bp. A total of 130 predicted genes (113 unique genes and 17 genes, which were duplicated in the IR) including 79 protein-coding genes, four ribosomal RNA genes and 30 tRNA genes were identified based on similarity to homologs from the chloroplast genome of Nicotiana tabacum. Genome organization was very similar to the inferred ancestral angiosperm chloroplast genome. Comparisons between Pyrus, Malus, and Prunus in Rosaceae revealed 220 indels (??10?bp). Excluding ycf1 and ycf2, which contained deletions in the coding region, all of these were detected in the spacer or intron regions. Three insertions and 13 deletions were detected in Pyrus compared to the same loci in Malus and Prunus. After comparing 89 noncoding chloroplast DNA regions in Pyrus and Malus, highly variable regions such as ndhC-trnV and trnR-atpA were identified. In Pyrus and Malus, the IR/LSC borders were 62?bp shorter than those of Prunus. In addition, there were length mutations at the IRa/LSC junction and in trnH. A total of 67 simple sequence repeats (more than 10 repeated motifs) were identified in the Pyrus chloroplast genome. The indels and simple sequence repeats will be useful evolutionary tools at both intra- and interspecific levels. Phylogenetic analysis demonstrated a close relationship between Pyrus and Prunus in the Rosaceae.     

Complete chloroplast genome sequences of Dipterygium glaucum and Cleome chrysantha and other Cleomaceae Species,comparative analysis and phylogenetic relationships

This current study presents, for the first time, the complete chloroplast genome of two Cleomaceae species: Dipterygium glaucum and Cleome chrysantha in order to evaluate the evolutionary relationship. The cp genome is 158,576 bp in length with 35.74% GC content in D. glaucum and 158,111 bp with 35.96% GC in C. chrysantha. Inverted repeats IR 26,209 bp, 26,251 bp each, LSC of 87,738 bp, 87,184 bp and SSC of 18,420 bp, 18,425 bp respectively. There are 136 genes in the genome, which includes 80 protein coding genes, 31 tRNA genes and four rRNA genes were observed in both chloroplast genomes. 117 genes are unique while the remaining 19 genes are duplicated in IR regions. The analysis of repeats shows that the cp genome includes all types of repeats with more frequent occurrences of palindromic; Also, this analysis indicates that the total number of simple sequence repeats (SSR) were 323 in D. glaucum, and 313 in C. chrysantha, of which the majority of the SSRs in these plastid genomes were mononucleotide repeats A/T which are located in the intergenic spacer. Moreover, the comparative analysis of the four cp sequences revealed four hotspot genes (atpF, rpoC2, rps19, and ycf1), these variable regions could be used as molecular makers for the species authentication as well as resources for inferring phylogenetic relationships of the species. All the relationships in the phylogenetic tree are with high support, this indicate that the complete chloroplast genome is a useful data for inferring phylogenetic relationship within the Cleomaceae and other families. The simple sequence repeats identified will be useful for identification, genetic diversity, and other evolutionary studies of the species. This study reported the first cp genome of the genus Dipterygium and Cleome. The finding of this study will be beneficial for biological disciplines such as evolutionary and genetic diversity studies of the species within the core Cleomaceae.