Long-reads reveal that the chloroplast genome exists in two distinct versions in most plants

AbstractThe chloroplast genome usually has a quadripartite structure consisting of a large single copy region and a small single copy region separated by two long inverted repeats. It has been known for some time that a single cell may contain at least two structural haplotypes of this structure, which differ in the relative orientation of the single copy regions. However, the methods required to detect and measure the abundance of the structural haplotypes are labour-intensive, and this phenomenon remains understudied. Here we develop a new method, Cp-hap, to detect all possible structural haplotypes of chloroplast genomes of quadripartite structure using long-read sequencing data. We use this method to conduct a systematic analysis and quantification of chloroplast structural haplotypes in 61 land plant species across 19 orders of Angiosperms, Gymnosperms and Pteridophytes. Our results show that there are two chloroplast structural haplotypes which occur with equal frequency in most land plant individuals. Nevertheless, species whose chloroplast genomes lack inverted repeats or have short inverted repeats have just a single structural haplotype. We also show that the relative abundance of the two structural haplotypes remains constant across multiple samples from a single individual plant, suggesting that the process which maintains equal frequency of the two haplotypes operates rapidly, consistent with the hypothesis that flip-flop recombination mediates chloroplast structural heteroplasmy. Our results suggest that previous claims of differences in chloroplast genome structure between species may need to be revisited.Significance StatementChloroplast genome consists of a large single copy region, a small single copy region, and two inverted repeats. Some decades ago, a discovery showed that there are two types chloroplast genome in some plants, which differ the way that the four regions are put together. However, this phenomenon has been shown in just a small number of species, and many open questions remain. Here, we develop a fast method to measure the chloroplast genome structures, based on long-reads. We show that almost all plants we analysed contain two possible genome structures, while a few plants contain only one structure. Our findings hint at the causes of the phenomenon, and provide a convenient new method with which to make rapid progress.

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Complete Chloroplast Genome Sequence of Malus hupehensis: Genome Structure, Comparative Analysis, and Phylogenetic Relationships

Molecules ◽

10.3390/molecules23112917 ◽

2018 ◽

Vol 23 (11) ◽

pp. 2917 ◽

Cited By ~ 4

Author(s):

Xin Zhang ◽

Chunxiao Rong ◽

Ling Qin ◽

Chuanyuan Mo ◽

Lu Fan ◽

...

Keyword(s):

Chloroplast Genome ◽

Genome Structure ◽

Single Copy ◽

Nucleotide Composition ◽

Complete Chloroplast Genome ◽

Sequencing Platform ◽

Malus Hupehensis ◽

Chloroplast Genomes ◽

Cp Genome ◽

Rna Genes

Malus hupehensis belongs to the Malus genus (Rosaceae) and is an indigenous wild crabapple of China. This species has received more and more attention, due to its important medicinal, and excellent ornamental and economical, values. In this study, the whole chloroplast (cp) genome of Malus hupehensis, using a Hiseq X Ten sequencing platform, is reported. The M. hupehensis cp genome is 160,065 bp in size, containing a large single copy region (LSC) of 88,166 bp and a small single copy region (SSC) of 19,193 bp, separated by a pair of inverted repeats (IRs) of 26,353 bp. It contains 112 genes, including 78 protein-coding genes (PCGs), 30 transfer RNA genes (tRNAs), and four ribosomal RNA genes (rRNAs). The overall nucleotide composition is 36.6% CG. A total of 96 simple sequence repeats (SSRs) were identified, most of them were found to be mononucleotide repeats composed of A/T. In addition, a total of 49 long repeats were identified, including 24 forward repeats, 21 palindromic repeats, and four reverse repeats. Comparisons of the IR boundaries of nine Malus complete chloroplast genomes presented slight variations at IR/SC boundaries regions. A phylogenetic analysis, based on 26 chloroplast genomes using the maximum likelihood (ML) method, indicates that M. hupehensis clustered closer ties with M. baccata, M. micromalus, and M. prunifolia than with M. tschonoskii. The availability of the complete chloroplast genome using genomics methods is reported here and provides reliable genetic information for future exploration on the taxonomy and phylogenetic evolution of the Malus and related species.

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Complete Chloroplast Genome Sequence and Phylogenetic Inference of the Canary Islands Dragon Tree (Dracaena draco L.)

Forests ◽

10.3390/f11030309 ◽

2020 ◽

Vol 11 (3) ◽

pp. 309

Author(s):

Konrad Celiński ◽

Hanna Kijak ◽

Justyna Wiland-Szymańska

Keyword(s):

Chloroplast Genome ◽

Canary Islands ◽

Genome Structure ◽

Gc Content ◽

Phylogenetic Inference ◽

Single Copy ◽

Genetic Identification ◽

Chloroplast Genomes ◽

Mutational Hotspots ◽

Dracaena Draco

Dracaena draco, which belongs to the genus Dracaena, is an endemic succulent of the Canary Islands. Although it is one of the most popular and widely grown ornamental plants in the world, little is known about its genomic variability. Next generation sequencing, especially in combination with advanced bioinformatics analysis, is a new standard in taxonomic and phylogenetic research. Therefore, in this study, the complete D. draco chloroplast genome (cp) was sequenced and analyzed in order to provide new genomic information and to elucidate phylogenetic relationships, particularly within the genus Dracaena. The D. draco chloroplast genome is 155,422 bp, total guanine-cytosine (GC) content is 37.6%, and it has a typical quadripartite plastid genome structure with four separate regions, including one large single copy region of 83,942 bp length and one small single copy region of 18,472 bp length, separated by two inverted repeat regions, each 26,504 bp in length. One hundred and thirty-two genes were identified, 86 of which are protein-coding genes, 38 are transfer RNAs, and eight are ribosomal RNAs. Seventy-seven simple sequence repeats were also detected. Comparative analysis of the sequence data of various members of Asparagales revealed mutational hotspots potentially useful for their genetic identification. Phylogenetic inference based on 16 complete chloroplast genomes of Asparagales strongly suggested that Dracaena species form one monophyletic group, and that close relationships exist between D. draco, D. cochinchinensis and D. cambodiana. This study provides new and valuable data for further taxonomic, evolutionary and phylogenetic studies within the Dracaena genus.

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Comparative analysis of chloroplast genome structure and molecular dating in Myrtales

BMC Plant Biology ◽

10.1186/s12870-021-02985-9 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Xiao-Feng Zhang ◽

Jacob B. Landis ◽

Hong-Xin Wang ◽

Zhi-Xin Zhu ◽

Hua-Feng Wang

Keyword(s):

Chloroplast Genome ◽

Genome Structure ◽

Divergence Time ◽

Gc Content ◽

Single Copy ◽

Molecular Dating ◽

Rrna Genes ◽

Chloroplast Structure ◽

Chloroplast Genomes ◽

Part Structure

Abstract Background Myrtales is a species rich branch of Rosidae, with many species having important economic, medicinal, and ornamental value. At present, although there are reports on the chloroplast structure of Myrtales, a comprehensive analysis of the chloroplast structure of Myrtales is lacking. Phylogenetic and divergence time estimates of Myrtales are mostly constructed by using chloroplast gene fragments, and the support for relationships is low. A more reliable method to reconstruct the species divergence time and phylogenetic relationships is by using whole chloroplast genomes. In this study, we comprehensively analyzed the structural characteristics of Myrtales chloroplasts, compared variation hotspots, and reconstructed the species differentiation time of Myrtales with four fossils and one secondary calibration point. Results A total of 92 chloroplast sequences of Myrtales, representing six families, 16 subfamilies and 78 genera, were obtained including nine newly sequenced chloroplasts by whole genome sequencing. Structural analyses showed that the chloroplasts range in size between 152,214–171,315 bp and exhibit a typical four part structure. The IR region is between 23,901–36,747 bp, with the large single copy region spanning 83,691–91,249 bp and the small single copy region spanning 11,150–19,703 bp. In total, 123–133 genes are present in the chloroplasts including 77–81 protein coding genes, four rRNA genes and 30–31 tRNA genes. The GC content was 36.9–38.9%, with the average GC content being 37%. The GC content in the LSC, SSC and IR regions was 34.7–37.3%, 30.6–36.8% and 39.7–43.5%, respectively. By analyzing nucleotide polymorphism of the chloroplast, we propose 21 hypervariable regions as potential DNA barcode regions for Myrtales. Phylogenetic analyses showed that Myrtales and its corresponding families are monophyletic, with Combretaceae and the clade of Onagraceae + Lythraceae (BS = 100%, PP = 1) being sister groups. The results of molecular dating showed that the crown of Myrtales was most likely to be 104.90 Ma (95% HPD = 87.88–114.18 Ma), and differentiated from the Geraniales around 111.59 Ma (95% HPD = 95.50–118.62 Ma). Conclusions The chloroplast genome structure of Myrtales is similar to other angiosperms and has a typical four part structure. Due to the expansion and contraction of the IR region, the chloroplast genome sizes in this group are slightly different. The variation of noncoding regions of the chloroplast genome is larger than those of coding regions. Phylogenetic analysis showed that Combretaceae and Onagraceae + Lythraceae were well supported as sister groups. Molecular dating indicates that the Myrtales crown most likely originated during the Albian age of the Lower Cretaceous. These chloroplast genomes contribute to the study of genetic diversity and species evolution of Myrtales, while providing useful information for taxonomic and phylogenetic studies of Myrtales.

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Complete Chloroplast Genomes of Acanthochlamys bracteata (China) and Xerophyta (Africa) (Velloziaceae): Comparative Genomics and Phylogenomic Placement

Frontiers in Plant Science ◽

10.3389/fpls.2021.691833 ◽

2021 ◽

Vol 12 ◽

Author(s):

Vincent Okelo Wanga ◽

Xiang Dong ◽

Millicent Akinyi Oulo ◽

Elijah Mbandi Mkala ◽

Jia-Xin Yang ◽

...

Keyword(s):

Chloroplast Genome ◽

Genome Structure ◽

Single Copy ◽

Average Score ◽

African Species ◽

Complete Chloroplast Genome ◽

Coding Regions ◽

Chloroplast Genomes ◽

Genomic Characterization ◽

Cp Genome

Acanthochlamys P.C. Kao is a Chinese endemic monotypic genus, whereas XerophytaJuss. is a genus endemic to Africa mainland, Arabian Peninsula and Madagascar with ca.70 species. In this recent study, the complete chloroplast genome of Acanthochlamys bracteata was sequenced and its genome structure compared with two African Xerophyta species (Xerophyta spekei and Xerophyta viscosa) present in the NCBI database. The genomes showed a quadripartite structure with their sizes ranging from 153,843 bp to 155,498 bp, having large single-copy (LSC) and small single-copy (SSC) regions divided by a pair of inverted repeats (IR regions). The total number of genes found in A. bracteata, X. spekei and X. viscosa cp genomes are 129, 130, and 132, respectively. About 50, 29, 28 palindromic, forward and reverse repeats and 90, 59, 53 simple sequence repeats (SSRs) were found in the A. bracteata, X. spekei, and X. viscosa cp genome, respectively. Nucleotide diversity analysis in all species was 0.03501, Ka/Ks ratio average score was calculated to be 0.26, and intergeneric K2P value within the Order Pandanales was averaged to be 0.0831. Genomic characterization was undertaken by comparing the genomes of the three species of Velloziaceae and it revealed that the coding regions were more conserved than the non-coding regions. However, key variations were noted mostly at the junctions of IRs/SSC regions. Phylogenetic analysis suggests that A. bracteata species has a closer genetic relationship to the genus Xerophyta. The present study reveals the complete chloroplast genome of A. bracteata and gives a genomic comparative analysis with the African species of Xerophyta. Thus, can be useful in developing DNA markers for use in the study of genetic variabilities and evolutionary studies in Velloziaceae.

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A Circular Chloroplast Genome of Fagus sylvatica Reveals High Conservation between Two Individuals from Germany and One Individual from Poland and an Alternate Direction of the Small Single-Copy Region

Forests ◽

10.3390/f12020180 ◽

2021 ◽

Vol 12 (2) ◽

pp. 180

Author(s):

Bagdevi Mishra ◽

Bartosz Ulaszewski ◽

Sebastian Ploch ◽

Jaroslaw Burczyk ◽

Marco Thines

Keyword(s):

Chloroplast Genome ◽

Fagus Sylvatica ◽

Pollen Dispersal ◽

European Beech ◽

Single Copy ◽

Migration History ◽

Chloroplast Genomes ◽

Long Read ◽

The Individual ◽

Small Single Copy

Chloroplasts are difficult to assemble because of the presence of large inverted repeats. At the same time, correct assemblies are important, as chloroplast loci are frequently used for biogeography and population genetics studies. In an attempt to elucidate the orientation of the single-copy regions and to find suitable loci for chloroplast single nucleotide polymorphism (SNP)-based studies, circular chloroplast sequences for the ultra-centenary reference individual of European Beech (Fagus sylvatica), Bhaga, and an additional Polish individual (named Jamy) was obtained based on hybrid assemblies. The chloroplast genome of Bhaga was 158,458 bp, and that of Jamy was 158,462 bp long. Using long-read mapping on the configuration inferred in this study and the one suggested in a previous study, we found an inverted orientation of the small single-copy region. The chloroplast genome of Bhaga and of the individual from Poland both have only two mismatches as well as three and two indels as compared to the previously published genome, respectively. The low divergence suggests low seed dispersal but high pollen dispersal. However, once chloroplast genomes become available from Pleistocene refugia, where a high degree of variation has been reported, they might prove useful for tracing the migration history of Fagus sylvatica in the Holocene.

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A systematic comparison of eight new plastome sequences from Ipomoea L

PeerJ ◽

10.7717/peerj.6563 ◽

2019 ◽

Vol 7 ◽

pp. e6563

Author(s):

Jianying Sun ◽

Xiaofeng Dong ◽

Qinghe Cao ◽

Tao Xu ◽

Mingku Zhu ◽

...

Keyword(s):

Comparative Analysis ◽

Next Generation Sequencing ◽

Chloroplast Genome ◽

Dna Sequences ◽

Repetitive Sequences ◽

Single Copy ◽

Next Generation ◽

Variable Regions ◽

Chloroplast Genomes ◽

Generation Sequencing

Background Ipomoea is the largest genus in the family Convolvulaceae. The species in this genus have been widely used in many fields, such as agriculture, nutrition, and medicine. With the development of next-generation sequencing, more than 50 chloroplast genomes of Ipomoea species have been sequenced. However, the repeats and divergence regions in Ipomoea have not been well investigated. In the present study, we sequenced and assembled eight chloroplast genomes from sweet potato’s close wild relatives. By combining these with 32 published chloroplast genomes, we conducted a detailed comparative analysis of a broad range of Ipomoea species. Methods Eight chloroplast genomes were assembled using short DNA sequences generated by next-generation sequencing technology. By combining these chloroplast genomes with 32 other published Ipomoea chloroplast genomes downloaded from GenBank and the Oxford Research Archive, we conducted a comparative analysis of the repeat sequences and divergence regions across the Ipomoea genus. In addition, separate analyses of the Batatas group and Quamoclit group were also performed. Results The eight newly sequenced chloroplast genomes ranged from 161,225 to 161,721 bp in length and displayed the typical circular quadripartite structure, consisting of a pair of inverted repeat (IR) regions (30,798–30,910 bp each) separated by a large single copy (LSC) region (87,575–88,004 bp) and a small single copy (SSC) region (12,018–12,051 bp). The average guanine-cytosine (GC) content was approximately 40.5% in the IR region, 36.1% in the LSC region, 32.2% in the SSC regions, and 37.5% in complete sequence for all the generated plastomes. The eight chloroplast genome sequences from this study included 80 protein-coding genes, four rRNAs (rrn23, rrn16, rrn5, and rrn4.5), and 37 tRNAs. The boundaries of single copy regions and IR regions were highly conserved in the eight chloroplast genomes. In Ipomoea, 57–89 pairs of repetitive sequences and 39–64 simple sequence repeats were found. By conducting a sliding window analysis, we found six relatively high variable regions (ndhA intron, ndhH-ndhF, ndhF-rpl32, rpl32-trnL, rps16-trnQ, and ndhF) in the Ipomoea genus, eight (trnG, rpl32-trnL, ndhA intron, ndhF-rpl32, ndhH-ndhF, ccsA-ndhD, trnG-trnR, and pasA-ycf3) in the Batatas group, and eight (ndhA intron, petN-psbM, rpl32-trnL, trnG-trnR, trnK-rps16, ndhC-trnV, rps16-trnQ, and trnG) in the Quamoclit group. Our maximum-likelihood tree based on whole chloroplast genomes confirmed the phylogenetic topology reported in previous studies. Conclusions The chloroplast genome sequence and structure were highly conserved in the eight newly-sequenced Ipomoea species. Our comparative analysis included a broad range of Ipomoea chloroplast genomes, providing valuable information for Ipomoea species identification and enhancing the understanding of Ipomoea genetic resources.

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The complete chloroplast genome of Saxifraga sinomontana (Saxifragaceae) and comparative analysis with other Saxifragaceae species

Revista Brasileira de Botânica ◽

10.1007/s40415-019-00561-y ◽

2019 ◽

Vol 42 (4) ◽

pp. 601-611 ◽

Cited By ~ 1

Author(s):

Yan Li ◽

Liukun Jia ◽

Zhihua Wang ◽

Rui Xing ◽

Xiaofeng Chi ◽

...

Keyword(s):

Comparative Analysis ◽

Chloroplast Genome ◽

Phylogenetic Relationships ◽

De Novo ◽

Single Copy ◽

Bootstrap Support ◽

Protein Coding ◽

Complete Chloroplast Genome ◽

Protein Coding Genes ◽

Chloroplast Genomes

Abstract Saxifraga sinomontana J.-T. Pan & Gornall belongs to Saxifraga sect. Ciliatae subsect. Hirculoideae, a lineage containing ca. 110 species whose phylogenetic relationships are largely unresolved due to recent rapid radiations. Analyses of complete chloroplast genomes have the potential to significantly improve the resolution of phylogenetic relationships in this young plant lineage. The complete chloroplast genome of S. sinomontana was de novo sequenced, assembled and then compared with that of other six Saxifragaceae species. The S. sinomontana chloroplast genome is 147,240 bp in length with a typical quadripartite structure, including a large single-copy region of 79,310 bp and a small single-copy region of 16,874 bp separated by a pair of inverted repeats (IRs) of 25,528 bp each. The chloroplast genome contains 113 unique genes, including 79 protein-coding genes, four rRNAs and 30 tRNAs, with 18 duplicates in the IRs. The gene content and organization are similar to other Saxifragaceae chloroplast genomes. Sixty-one simple sequence repeats were identified in the S. sinomontana chloroplast genome, mostly represented by mononucleotide repeats of polyadenine or polythymine. Comparative analysis revealed 12 highly divergent regions in the intergenic spacers, as well as coding genes of matK, ndhK, accD, cemA, rpoA, rps19, ndhF, ccsA, ndhD and ycf1. Phylogenetic reconstruction of seven Saxifragaceae species based on 66 protein-coding genes received high bootstrap support values for nearly all identified nodes, suggesting a promising opportunity to resolve infrasectional relationships of the most species-rich section Ciliatae of Saxifraga.

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Complete Chloroplast Genome of Paphiopedilum delenatii and Phylogenetic Relationships among Orchidaceae

Plants ◽

10.3390/plants9010061 ◽

2020 ◽

Vol 9 (1) ◽

pp. 61 ◽

Cited By ~ 5

Author(s):

Huyen-Trang Vu ◽

Ngan Tran ◽

Thanh-Diem Nguyen ◽

Quoc-Luan Vu ◽

My-Huyen Bui ◽

...

Keyword(s):

Chloroplast Genome ◽

Inverted Repeat ◽

Gc Content ◽

Single Copy ◽

Rrna Genes ◽

Trna Genes ◽

Complete Chloroplast Genome ◽

Critically Endangered Species ◽

Plastid Genomes ◽

Chloroplast Genomes

Paphiopedilum delenatii is a native orchid of Vietnam with highly attractive floral traits. Unfortunately, it is now listed as a critically endangered species with a few hundred individuals remaining in nature. In this study, we performed next-generation sequencing of P. delenatii and assembled its complete chloroplast genome. The whole chloroplast genome of P. delenatii was 160,955 bp in size, 35.6% of which was GC content, and exhibited typical quadripartite structure of plastid genomes with four distinct regions, including the large and small single-copy regions and a pair of inverted repeat regions. There were, in total, 130 genes annotated in the genome: 77 coding genes, 39 tRNA genes, 8 rRNA genes, and 6 pseudogenes. The loss of ndh genes and variation in inverted repeat (IR) boundaries as well as data of simple sequence repeats (SSRs) and divergent hotspots provided useful information for identification applications and phylogenetic studies of Paphiopedilum species. Whole chloroplast genomes could be used as an effective super barcode for species identification or for developing other identification markers, which subsequently serves the conservation of Paphiopedilum species.

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The complete structure of the cucumber (Cucumis sativus L.) chloroplast genome: Its composition and comparative analysis

Cellular & Molecular Biology Letters ◽

10.2478/s11658-007-0029-7 ◽

2007 ◽

Vol 12 (4) ◽

Cited By ~ 24

Author(s):

Wojciech Pląder ◽

Yasushi Yukawa ◽

Masahiro Sugiura ◽

Stefan Malepszy

Keyword(s):

Chloroplast Genome ◽

Single Copy ◽

Trna Genes ◽

Inverted Repeat Region ◽

Cucumis Sativus L ◽

Stem Loop ◽

Protein Coding ◽

Chloroplast Genomes ◽

Trna Species ◽

Rrna Species

AbstractThe complete nucleotide sequence of the cucumber (C. sativus L. var. Borszczagowski) chloroplast genome has been determined. The genome is composed of 155,293 bp containing a pair of inverted repeats of 25,191 bp, which are separated by two single-copy regions, a small 18,222-bp one and a large 86,688-bp one. The chloroplast genome of cucumber contains 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes (4 rRNA species), and 37 tRNA genes (30 tRNA species), with 18 of them located in the inverted repeat region. Of these genes, 16 contain one intron, and two genes and one ycf contain 2 introns. Twenty-one small inversions that form stem-loop structures, ranging from 18 to 49 bp, have been identified. Eight of them show similarity to those of other species, while eight seem to be cucumber specific. Detailed comparisons of ycf2 and ycf15, and the overall structure to other chloroplast genomes were performed.

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