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

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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Stable and widespread structural heteroplasmy in chloroplast genomes revealed by a new long-read quantification method

10.1101/692798 ◽

2019 ◽

Author(s):

Weiwen Wang ◽

Robert Lanfear

Keyword(s):

Chloroplast Genome ◽

Single Copy ◽

Land Plant ◽

Inverted Repeats ◽

Equal Frequency ◽

Large Single Copy ◽

Chloroplast Genomes ◽

Long Read ◽

Small Single Copy ◽

Large Single Copy Region

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 Genomes of Fagus sylvatica L. Reveal Sequence Conservation in the Inverted Repeat and the Presence of Allelic Variation in NUPTs

Genes ◽

10.3390/genes12091357 ◽

2021 ◽

Vol 12 (9) ◽

pp. 1357

Author(s):

Bartosz Ulaszewski ◽

Joanna Meger ◽

Bagdevi Mishra ◽

Marco Thines ◽

Jarosław Burczyk

Keyword(s):

Fagus Sylvatica ◽

Inverted Repeat ◽

Allelic Variation ◽

Phylogenetic Analyses ◽

Geographic Distance ◽

European Beech ◽

Single Copy ◽

Chloroplast Genomes ◽

Efficient Suppression ◽

Broadleaved Tree Species

Growing amounts of genomic data and more efficient assembly tools advance organelle genomics at an unprecedented scale. Genomic resources are increasingly used for phylogenetic analyses of many plant species, but are less frequently used to investigate within-species variability and phylogeography. In this study, we investigated genetic diversity of Fagus sylvatica, an important broadleaved tree species of European forests, based on complete chloroplast genomes of 18 individuals sampled widely across the species distribution. Our results confirm the hypothesis of a low cpDNA diversity in European beech. The chloroplast genome size was remarkably stable (158,428 ± 37 bp). The polymorphic markers, 12 microsatellites (SSR), four SNPs and one indel, were found only in the single copy regions, while inverted repeat regions were monomorphic both in terms of length and sequence, suggesting highly efficient suppression of mutation. The within-individual analysis of polymorphisms showed >9k of markers which were proportionally present in gene and non-gene areas. However, an investigation of the frequency of alternate alleles revealed that the source of this diversity originated likely from nuclear-encoded plastome remnants (NUPTs). Phylogeographic and Mantel correlation analysis based on the complete chloroplast genomes exhibited clustering of individuals according to geographic distance in the first distance class, suggesting that the novel markers and in particular the cpSSRs could provide a more detailed picture of beech population structure in Central Europe.

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Complete chloroplast genome sequences of Dioscorea: Characterization, genomic resources, and phylogenetic analyses

PeerJ ◽

10.7717/peerj.6032 ◽

2018 ◽

Vol 6 ◽

pp. e6032 ◽

Cited By ~ 12

Author(s):

Zhenyu Zhao ◽

Xin Wang ◽

Yi Yu ◽

Subo Yuan ◽

Dan Jiang ◽

...

Keyword(s):

Chloroplast Genome ◽

Phylogenetic Analyses ◽

Single Copy ◽

Genome Sequences ◽

Complete Chloroplast Genome ◽

Genomic Resources ◽

Chloroplast Genomes ◽

The Family ◽

Small Single Copy

Dioscorea L., the largest genus of the family Dioscoreaceae with over 600 species, is not only an important food but also a medicinal plant. The identification and classification of Dioscorea L. is a rather difficult task. In this study, we sequenced five Dioscorea chloroplast genomes, and analyzed with four other chloroplast genomes of Dioscorea species from GenBank. The Dioscorea chloroplast genomes displayed the typical quadripartite structure of angiosperms, which consisted of a pair of inverted repeats separated by a large single-copy region, and a small single-copy region. The location and distribution of repeat sequences and microsatellites were determined, and the rapidly evolving chloroplast genome regions (trnK-trnQ, trnS-trnG, trnC-petN, trnE-trnT, petG-trnW-trnP, ndhF, trnL-rpl32, and ycf1) were detected. Phylogenetic relationships of Dioscorea inferred from chloroplast genomes obtained high support even in shortest internodes. Thus, chloroplast genome sequences provide potential molecular markers and genomic resources for phylogeny and species identification.

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Insights into chloroplast genome variation across Opuntioideae (Cactaceae)

10.1101/2020.03.06.981183 ◽

2020 ◽

Cited By ~ 1

Author(s):

Matias Köhler ◽

Marcelo Reginato ◽

Tatiana T. Souza-Chies ◽

Lucas C. Majure

Keyword(s):

Chloroplast Genome ◽

De Novo ◽

Single Copy ◽

Physiological Constraints ◽

Chloroplast Genomes ◽

The Family ◽

The Common ◽

And Inversion ◽

Small Single Copy ◽

High Support

AbstractChloroplast genomes (plastomes) are frequently treated as highly conserved among land plants. However, many lineages of vascular plants have experienced extensive structural rearrangements, including inversions and modifications to the size and content of genes. Cacti are one of these lineages, containing the smallest plastome known for an obligately photosynthetic angiosperm, including the loss of one copy of the inverted repeat (∼25 kb) and the ndh genes suite, but only a few cacti from the subfamily Cactoideae have been sufficiently characterized. Here, we investigated the variation of plastome sequences across the second-major lineage of the Cactaceae, the subfamily Opuntioideae, to address 1) how variable is the content and arrangement of chloroplast genome sequences across the subfamily, and 2) how phylogenetically informative are the plastome sequences for resolving major relationships among the clades of Opuntioideae. Our de novo assembly of the Opuntia quimilo plastome recovered an organelle of 150,347 bp in length with both copies of the inverted repeats and the presence of all the ndh genes suite. An expansion of the large single copy unit and a reduction of the small single copy was observed, including translocations and inversion of genes as well as the putative pseudogenization of numerous loci. Comparative analyses among all clades within Opuntioideae suggested that plastome structure and content vary across taxa of this subfamily, with putative independent losses of the ndh gene suite and pseudogenization of genes across disparate lineages, further demonstrating the dynamic nature of plastomes in Cactaceae. Our plastome dataset was robust in determining relationships among major clades and subclades within Opuntioideae, resolving three tribes with high support: Cylindropuntieae, Tephrocacteae and Opuntieae. A plastome-wide survey for highly informative phylogenetic markers revealed previously unused regions for future use in Sanger-based studies, presenting a valuable dataset with primers designed for continued evolutionary studies across Cactaceae. These results bring new insights into the evolution of plastomes in cacti, suggesting that further analyses should be carried out to address how ecological drivers, physiological constraints and morphological traits of cacti may be related with the common rearrangements in plastomes that have been reported across the family.

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Comparative Chloroplast Genomics in Phyllanthaceae Species

Diversity ◽

10.3390/d13090403 ◽

2021 ◽

Vol 13 (9) ◽

pp. 403

Author(s):

Umar Rehman ◽

Nighat Sultana ◽

Abdullah ◽

Abbas Jamal ◽

Maryam Muzaffar ◽

...

Keyword(s):

Chloroplast Genome ◽

De Novo ◽

Single Copy ◽

Protein Coding ◽

Coding Sequences ◽

Tropical Regions ◽

Chloroplast Genomes ◽

The Family ◽

Insight Into ◽

Small Single Copy

Family Phyllanthaceae belongs to the eudicot order Malpighiales, and its species are herbs, shrubs, and trees that are mostly distributed in tropical regions. Here, we elucidate the molecular evolution of the chloroplast genome in Phyllanthaceae and identify the polymorphic loci for phylogenetic inference. We de novo assembled the chloroplast genomes of three Phyllanthaceae species, i.e., Phyllanthus emblica, Flueggea virosa, and Leptopus cordifolius, and compared them with six other previously reported genomes. All species comprised two inverted repeat regions (size range 23,921–27,128 bp) that separated large single-copy (83,627–89,932 bp) and small single-copy (17,424–19,441 bp) regions. Chloroplast genomes contained 111–112 unique genes, including 77–78 protein-coding, 30 tRNAs, and 4 rRNAs. The deletion/pseudogenization of rps16 genes was found in only two species. High variability was seen in the number of oligonucleotide repeats, while guanine-cytosine contents, codon usage, amino acid frequency, simple sequence repeats, synonymous and non-synonymous substitutions, and transition and transversion substitutions were similar. The transition substitutions were higher in coding sequences than in non-coding sequences. Phylogenetic analysis revealed the polyphyletic nature of the genus Phyllanthus. The polymorphic protein-coding genes, including rpl22, ycf1, matK, ndhF, and rps15, were also determined, which may be helpful for reconstructing the high-resolution phylogenetic tree of the family Phyllanthaceae. Overall, the study provides insight into the chloroplast genome evolution in Phyllanthaceae.

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Assembly of chloroplast genomes with long- and short-read data: a comparison of approaches using Eucalyptus pauciflora as a test case

10.1101/320085 ◽

2018 ◽

Author(s):

weiwen wang ◽

Miriam Schalamun ◽

Alejandro Morales Suarez ◽

David Kainer ◽

Benjamin Schwessinger ◽

...

Keyword(s):

Chloroplast Genome ◽

Inverted Repeat ◽

Single Copy ◽

Test Case ◽

Short Read ◽

Short Reads ◽

Chloroplast Genomes ◽

Long Reads ◽

Long Read ◽

Hybrid Assemblies

AbstractBackgroundChloroplasts are organelles that conduct photosynthesis in plant and algal cells. Chloroplast genomes code for around 130 genes, and the information they contain is widely used in agriculture and studies of evolution and ecology. Correctly assembling complete chloroplast genomes can be challenging because the chloroplast genome contains a pair of long inverted repeats (10–30 kb). The advent of long-read sequencing technologies should alleviate this problem by providing sufficient information to completely span the inverted repeat regions. Yet, long-reads tend to have higher error rates than short-reads, and relatively little is known about the best way to combine long- and short-reads to obtain the most accurate chloroplast genome assemblies. Using Eucalyptus pauciflora, the snow gum, as a test case, we evaluated the effect of multiple parameters, such as different coverage of long (Oxford nanopore) and short (Illumina) reads, different long-read lengths, different assembly pipelines, and different genome polishing steps, with a view to determining the most accurate and efficient approach to chloroplast genome assembly.ResultsHybrid assemblies combining at least 20x coverage of both long-reads and short-reads generated a single contig spanning the entire chloroplast genome with few or no detectable errors. Short-read-only assemblies generated three contigs representing the long single copy, short single copy and inverted repeat regions of the chloroplast genome. These contigs contained few single-base errors but tended to exclude several bases at the beginning or end of each contig. Long-read-only assemblies tended to create multiple contigs with a much higher single-base error rate, even after polishing. The chloroplast genome of Eucalyptus pauciflora is 159,942 bp, contains 131 genes of known function, and confirms the phylogenetic position of Eucalyptus pauciflora as a close relative of Eucalyptus regnans.ConclusionsOur results suggest that very accurate assemblies of chloroplast genomes can be achieved using a combination of at least 20x coverage of long- and short-reads respectively, provided that the long-reads contain at least ~5x coverage of reads longer than the inverted repeat region. We show that further increases in coverage give little or no improvement in accuracy, and that hybrid assemblies are more accurate than long-read-only or short-read-only assemblies.

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Chloroplast Genome Evolution in Four Montane Zingiberaceae Taxa in China

Frontiers in Plant Science ◽

10.3389/fpls.2021.774482 ◽

2022 ◽

Vol 12 ◽

Author(s):

Qian Yang ◽

Gao-Fei Fu ◽

Zhi-Qiang Wu ◽

Li Li ◽

Jian-Li Zhao ◽

...

Keyword(s):

Chloroplast Genome ◽

Adaptive Evolution ◽

Related Species ◽

Single Copy ◽

Large Single Copy ◽

Closely Related Species ◽

Chloroplast Genomes ◽

Insight Into ◽

Small Single Copy ◽

Selection Of

Chloroplasts are critical to plant survival and adaptive evolution. The comparison of chloroplast genomes could provide insight into the adaptive evolution of closely related species. To identify potential adaptive evolution in the chloroplast genomes of four montane Zingiberaceae taxa (Cautleya, Roscoea, Rhynchanthus, and Pommereschea) that inhabit distinct habitats in the mountains of Yunnan, China, the nucleotide sequences of 13 complete chloroplast genomes, including five newly sequenced species, were characterized and compared. The five newly sequenced chloroplast genomes (162,878–163,831 bp) possessed typical quadripartite structures, which included a large single copy (LSC) region, a small single copy (SSC) region, and a pair of inverted repeat regions (IRa and IRb), and even though the structure was highly conserved among the 13 taxa, one of the rps19 genes was absent in Cautleya, possibly due to expansion of the LSC region. Positive selection of rpoA and ycf2 suggests that these montane species have experienced adaptive evolution to habitats with different sunlight intensities and that adaptation related to the chloroplast genome has played an important role in the evolution of Zingiberaceae taxa.

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The chloroplast genome evolution of Venus slipper (Paphiopedilum): IR expansion, SSC contraction, and highly rearranged SSC regions

BMC Plant Biology ◽

10.1186/s12870-021-03053-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yan-Yan Guo ◽

Jia-Xing Yang ◽

Ming-Zhu Bai ◽

Guo-Qiang Zhang ◽

Zhong-Jian Liu

Keyword(s):

Gene Order ◽

Inverted Repeat ◽

Single Copy ◽

Gene Content ◽

Comparative Genomic ◽

Substitution Rates ◽

Plastome Evolution ◽

Chloroplast Genomes ◽

Ideal System ◽

Small Single Copy

Abstract Background Paphiopedilum is the largest genus of slipper orchids. Previous studies showed that the phylogenetic relationships of this genus are not well resolved, and sparse taxon sampling documented inverted repeat (IR) expansion and small single copy (SSC) contraction of the chloroplast genomes of Paphiopedilum. Results Here, we sequenced, assembled, and annotated 77 plastomes of Paphiopedilum species (size range of 152,130 – 164,092 bp). The phylogeny based on the plastome resolved the relationships of the genus except for the phylogenetic position of two unstable species. We used phylogenetic and comparative genomic approaches to elucidate the plastome evolution of Paphiopedilum. The plastomes of Paphiopedilum have a conserved genome structure and gene content except in the SSC region. The large single copy/inverted repeat (LSC/IR) boundaries are relatively stable, while the boundaries of the inverted repeat and small single copy region (IR/SSC) varied among species. Corresponding to the IR/SSC boundary shifts, the chloroplast genomes of the genus experienced IR expansion and SSC contraction. The IR region incorporated one to six genes of the SSC region. Unexpectedly, great variation in the size, gene order, and gene content of the SSC regions was found, especially in the subg. Parvisepalum. Furthermore, Paphiopedilum provides evidence for the ongoing degradation of the ndh genes in the photoautotrophic plants. The estimated substitution rates of the protein coding genes show accelerated rates of evolution in clpP, psbH, and psbZ. Genes transferred to the IR region due to the boundary shift also have higher substitution rates. Conclusions We found IR expansion and SSC contraction in the chloroplast genomes of Paphiopedilum with dense sampling, and the genus shows variation in the size, gene order, and gene content of the SSC region. This genus provides an ideal system to investigate the dynamics of plastome evolution.

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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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