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Although plastid genome (plastome) structure is highly conserved across most seed plants, investigations during the past two decades have revealed several disparately related lineages that experienced substantial rearrangements. Most plastomes contain a large inverted repeat and two single-copy regions, and a few dispersed repeats; however, the plastomes of some taxa harbour long repeat sequences (>300 bp). These long repeats make it challenging to assemble complete plastomes using short-read data, leading to misassemblies and consensus sequences with spurious rearrangements. Single-molecule, long-read sequencing has the potential to overcome these challenges, yet there is no consensus on the most effective method for accurately assembling plastomes using long-read data. We generated a pipeline, plastid Genome Assembly Using Long-read data (ptGAUL), to address the problem of plastome assembly using long-read data from Oxford Nanopore Technologies (ONT) or Pacific Biosciences platforms. We demonstrated the efficacy of the ptGAUL pipeline using 16 published long-read data sets. We showed that ptGAUL quickly produces accurate and unbiased assemblies using only ~50× coverage of plastome data. Additionally, we deployed ptGAUL to assemble four new Juncus (Juncaceae) plastomes using ONT long reads. Our results revealed many long repeats and rearrangements in Juncus plastomes compared with basal lineages of Poales. The ptGAUL pipeline is available on GitHub: https://github.com/Bean061/ptgaul., (© 2023 John Wiley & Sons Ltd.)

Cyperaceae, the second largest family in the monocot order Poales, comprises >5500 species and includes the genus Eleocharis with ∼ 250 species. A previous study of complete plastomes of two Eleocharis species documented extensive structural heteroplasmy, gene order changes, high frequency of dispersed repeats along with gene losses and duplications. To better understand the phylogenetic distribution of gene and intron content as well as rates and patterns of sequence evolution within and between mitochondrial and plastid genomes of Eleocharis and Cyperaceae, an additional 29 Eleocharis organelle genomes were sequenced and analyzed. Eleocharis experienced extensive gene loss in both genomes while loss of introns was mitochondria-specific. Eleocharis has higher rates of synonymous (dS) and nonsynonymous (dN) substitutions in the plastid and mitochondrion than most sampled angiosperms, and the pattern was distinct from other eudicot lineages with accelerated rates. Several clades showed higher dS and dN in mitochondrial genes than in plastid genes. Furthermore, nucleotide substitution rates of mitochondrial genes were significantly accelerated on the branch leading to Cyperaceae compared to most angiosperms. Mitochondrial genes of Cyperaceae exhibited dramatic loss of RNA editing sites and a negative correlation between RNA editing and dS values was detected among angiosperms. Mutagenic retroprocessing and dysfunction of DNA replication, repair and recombination genes are the most likely cause of striking rate accelerations and loss of edit sites and introns in Eleocharis and Cyperaceae organelle genomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Gene transfers from mitochondria and plastids to the nucleus are an important process in the evolution of the eukaryotic cell. Plastid (pt) gene losses have been documented in multiple angiosperm lineages and are often associated with functional transfers to the nucleus or substitutions by duplicated nuclear genes targeted to both the plastid and mitochondrion. The plastid genome sequence of Euphorbia schimperi was assembled and three major genomic changes were detected, the complete loss of rpl32 and pseudogenization of rps16 and infA. The nuclear transcriptome of E. schimperi was sequenced to investigate the transfer/substitution of the rpl32 and rps16 genes to the nucleus. Transfer of plastid-encoded rpl32 to the nucleus was identified previously in three families of Malpighiales, Rhizophoraceae, Salicaceae and Passifloraceae. An E. schimperi transcript of pt SOD-1-RPL32 confirmed that the transfer in Euphorbiaceae is similar to other Malpighiales indicating that it occurred early in the divergence of the order. Ribosomal protein S16 (rps16) is encoded in the plastome in most angiosperms but not in Salicaceae and Passifloraceae. Substitution of the E. schimperi pt rps16 was likely due to a duplication of nuclear-encoded mitochondrial-targeted rps16 resulting in copies dually targeted to the mitochondrion and plastid. Sequences of RPS16-1 and RPS16-2 in the three families of Malpighiales (Salicaceae, Passifloraceae and Euphorbiaceae) have high sequence identity suggesting that the substitution event dates to the early divergence within Malpighiales.

The plastid genome (plastome ) has proved a valuable source of data for evaluating evolutionary relationships among angiosperms. Through basic and applied approaches, plastid transformation technology offers the potential to understand and improve plant productivity, providing food, fiber, energy, and medicines to meet the needs of a burgeoning global population. The growing genomic resources available to both phylogenetic and biotechnological investigations is allowing novel insights and expanding the scope of plastome research to encompass new species. In this chapter, we present an overview of some of the seminal and contemporary research that has contributed to our current understanding of plastome evolution and attempt to highlight the relationship between evolutionary mechanisms and the tools of plastid genetic engineering.

Diatoms are the largest group of heterokont algae with more than 100,000 species. As one of the single-celled photosynthetic organisms that inhabit marine, aquatic and terrestrial ecosystems, diatoms contribute ~ 45% of global primary production. Despite their ubiquity and environmental significance, very few diatom plastid genomes (plastomes) have been sequenced and studied. This study explored patterns of nucleotide substitution rates of diatom plastids across the entire suite of plastome protein-coding genes for 40 taxa representing the major clades. The highest substitution rate was lineage-specific within the araphid 2 taxon Astrosyne radiata and radial 2 taxon Proboscia sp. Rate heterogeneity was also evident in different functional classes and individual genes. Similar to land plants, proteins genes involved in photosynthetic metabolism have lower synonymous and nonsynonymous substitutions rates than those involved in transcription and translation. Significant positive correlations were identified between substitution rates and measures of genomic rearrangements, including indels and inversions, which is a similar result to what was found in legume plants. This work advances the understanding of the molecular evolution of diatom plastomes and provides a foundation for future studies.

Gene losses in plastid genomes (plastomes) are often accompanied by functional transfer to the nucleus or substitution of an alternative nuclear-encoded gene. Despite the highly conserved gene content in plastomes of photosynthetic land plants, recent gene loss events have been documented in several disparate angiosperm clades. Among these lineages, Passiflora lacks several essential ribosomal genes, rps7, rps16, rpl20, rpl22, and rpl32, the two largest plastid genes, ycf1 and ycf2, and has a highly divergent rpoA. Comparative transcriptome analyses were performed to determine the fate of the missing genes in Passiflora. Putative functional transfers of rps7, rpl22, and rpl32 to nucleus were detected, with the nuclear transfer of rps7, representing a novel event in angiosperms. Plastid-encoded rps7 was transferred into the intron of a nuclear-encoded plastid-targeted thioredoxin m-type gene, acquiring its plastid transit peptide (TP). Plastid rpl20 likely experienced a novel substitution by a duplicated, nuclear-encoded mitochondrial-targeted rpl20 that has a similar gene structure. Additionally, among rosids, evidence for a third independent transfer of rpl22 in Passiflora was detected that gained a TP from a nuclear gene containing an organelle RNA recognition motif. Nuclear transcripts representing rpoA, ycf1, and ycf2 were not detected. Further analyses suggest that the divergent rpoA remains functional and that the gene is under positive or purifying selection in different clades. Comparative analyses indicate that alternative translocon and motor protein complexes may have substituted for the loss of ycf1 and ycf2 in Passiflora., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Plastid genomes (plastomes) of land plants have a conserved quadripartite structure in a gene-dense unit genome consisting of a large inverted repeat that separates two single copy regions. Recently, alternative plastome structures were suggested in Geraniaceae and in some conifers and Medicago the coexistence of inversion isomers has been noted. In this study, plastome sequences of two Cyperaceae, Eleocharis dulcis (water chestnut) and Eleocharis cellulosa (gulf coast spikerush), were completed. Unlike the conserved plastomes in basal groups of Poales, these Eleocharis plastomes have remarkably divergent features, including large plastome sizes, high rates of sequence rearrangements, low GC content and gene density, gene duplications and losses, and increased repetitive DNA sequences. A novel finding among these features was the unprecedented level of heteroplasmy with the presence of multiple plastome structural types within a single individual. Illumina paired-end assemblies combined with PacBio single-molecule real-time sequencing, long-range polymerase chain reaction, and Sanger sequencing data identified at least four different plastome structural types in both Eleocharis species. PacBio long read data suggested that one of the four E. dulcis plastome types predominates., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The genus Trifolium is the largest of the tribe Trifolieae in the subfamily Papilionoideae (Fabaceae). The paucity of mitochondrial genome (mitogenome) sequences has hindered comparative analyses among the three genomic compartments of the plant cell (nucleus, mitochondrion and plastid). We assembled four mitogenomes from the two subgenera ( Chronosemium and Trifolium ) of the genus. The four Trifolium mitogenomes were compact (294,911-348,724 bp in length) and contained limited repetitive (6.6-8.6%) DNA. Comparison of organelle repeat content highlighted the distinct evolutionary trajectory of plastid genomes in a subset of Trifolium species. Intracellular gene transfer (IGT) was analyzed among the three genomic compartments revealing functional transfer of mitochondrial rps1 to nuclear genome along with other IGT events. Phylogenetic analysis based on mitochondrial and nuclear rps1 sequences revealed that the functional transfer in Trifolieae was independent from the event that occurred in robinioid clade that includes genus Lotus . A novel, independent fission event of ccmFn in Trifolium was identified, caused by a 59 bp deletion. Fissions of this gene reported previously in land plants were reassessed and compared with Trifolium .

Plastid genomes (plastomes) of photosynthetic angiosperms are for the most part highly conserved in their organization, mode of inheritance and rates of nucleotide substitution. A small number of distantly related lineages share a syndrome of features that deviate from this general pattern, including extensive genomic rearrangements, accelerated rates of nucleotide substitution, biparental inheritance and plastome-genome incompatibility. Previous studies of plastomes in Passiflora with limited taxon sampling suggested that the genus exhibits this syndrome. To examine this phenomenon further, 15 new plastomes from Passiflora were sequenced and combined with previously published data to examine the phylogenetic relationships, genome organization and evolutionary rates across all five subgenera and the sister genus Adenia. Phylogenomic analyses using 68 protein-coding genes shared by Passiflora generated a fully resolved and strongly supported tree that is congruent with previous phylogenies based on a few plastid and nuclear loci. This phylogeny was used to examine the distribution of plastome rearrangements across Passiflora. Multiple gene and intron losses and inversions were identified in Passiflora with some occurring in parallel and others that extended across the Passifloraceae. Furthermore, extensive expansions and contractions of the inverted repeat (IR) were uncovered and in some cases this resulted in exclusion of all ribosomal RNA genes from the IR. The most highly rearranged lineage was subgenus Decaloba, which experienced extensive IR expansion that incorporated up to 25 protein-coding genes usually located in large single copy region. Nucleotide substitution rate analyses of 68 protein-coding genes across the genus showed lineage- and locus-specific acceleration. Significant increase in dS, dN and dN/dS was detected for clpP across the genus and for ycf4 in certain lineages. Significant increases in dN and dN/dS for ribosomal subunits and plastid-encoded RNA polymerase genes were detected in the branch leading to the expanded IR-clade in subgenus Decaloba. This subgenus displays the syndrome of unusual features, making it an ideal system to investigate the dynamic evolution of angiosperm plastomes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The predominantly South-African plant genus Pelargonium L'Hér. (Geraniaceae) displays remarkable morphological diversity, several basic chromosome numbers as well as high levels of organelle genomic rearrangements, and represents the 7th largest Cape Floristic Region clade. In this study, we reconstructed a phylogenetic tree based on 74 plastome exons and nuclear rDNA ITS regions for 120 species, which represents 43% taxon coverage for Pelargonium. We also performed a dating analysis to examine the timing of the major radiations in the genus. Phylogenetic analyses of nucleotide, amino acid, and ITS alignments confirmed the previously-documented subgeneric split into five main clades ((C1,C2),(B(A1,A2))) although clade only A1 received low bootstrap support. Using calibration evidence from a range of sources the Pelargonium crown age was estimated to be 9.7 My old, much younger than previous estimates for the genus but similar to recent studies of other Cape Floristic lineages that are part of both Fynbos and Succulent Karoo biomes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In plant evolution, intracellular gene transfer (IGT) is a prevalent, ongoing process. While nuclear and mitochondrial genomes are known to integrate foreign DNA via IGT and horizontal gene transfer (HGT), plastid genomes (plastomes) have resisted foreign DNA incorporation and only recently has IGT been uncovered in the plastomes of a few land plants. In this study, we completed plastome sequences for l0 crop species and describe a number of structural features including variation in gene and intron content, inversions, and expansion and contraction of the inverted repeat (IR). We identified a putative in cinnamon ( J. Presl) and other sequenced Lauraceae and an apparent functional transfer of to the nucleus of quinoa ( Willd.). In the orchard tree cashew ( L.), we report the insertion of an ∼6.7-kb fragment of mitochondrial DNA into the plastome IR. BLASTn analyses returned high identity hits to mitogenome sequences including an intact open reading frame. Using three plastome markers for five species of , we generated a phylogeny to investigate the distribution and timing of the insertion. Four species share the insertion, suggesting that this event occurred <20 million yr ago in a single clade in the genus. Our study extends the observation of mitochondrial to plastome IGT to include long-lived tree species. While previous studies have suggested possible mechanisms facilitating IGT to the plastome, more examples of this phenomenon, along with more complete mitogenome sequences, will be required before a common, or variable, mechanism can be elucidated., (Copyright © 2017 Crop Science Society of America.)

Geraniaceae have emerged as a model system for investigating the causes and consequences of variation in plastid and mitochondrial genomes. Incredible structural variation in plastid genomes (plastomes) and highly accelerated evolutionary rates have been reported in selected lineages and functional groups of genes in both plastomes and mitochondrial genomes (mitogenomes), and these phenomena have been implicated in cytonuclear incompatibility. Previous organelle genome studies have included limited sampling of Geranium, the largest genus in the family with over 400 species. This study reports on rates and patterns of nucleotide substitutions in plastomes and mitogenomes of 17 species of Geranium and representatives of other Geraniaceae. As detected across other angiosperms, substitution rates in the plastome are 3.5 times higher than the mitogenome in most Geranium. However, in the branch leading to Geranium brycei/Geranium incanum mitochondrial genes experienced significantly higher dN and dS than plastid genes, a pattern that has only been detected in one other angiosperm. Furthermore, rate accelerations differ in the two organelle genomes with plastomes having increased dN and mitogenomes with increased dS. In the Geranium phaeum/Geranium reflexum clade, duplicate copies of clpP and rpoA genes that experienced asymmetric rate divergence were detected in the single copy region of the plastome. In the case of rpoA, the branch leading to G. phaeum/G. reflexum experienced positive selection or relaxation of purifying selection. Finally, the evolution of acetyl-CoA carboxylase is unusual in Geraniaceae because it is only the second angiosperm family where both prokaryotic and eukaryotic ACCases functionally coexist in the plastid., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

This study represents the most comprehensive plastome-wide comparison of nucleotide substitution rates across the three subfamilies of Fabaceae: Caesalpinioideae, Mimosoideae, and Papilionoideae. Caesalpinioid and mimosoid legumes have large, unrearranged plastomes compared with papilionoids, which exhibit varying levels of rearrangement including the loss of the inverted repeat (IR) in the IR-lacking clade (IRLC). Using 71 genes common to 39 legume taxa representing all the three subfamilies, we show that papilionoids consistently have higher nucleotide substitution rates than caesalpinioids and mimosoids, and rates in the IRLC papilionoids are generally higher than those in the IR-containing papilionoids. Unsurprisingly, this pattern was significantly correlated with growth habit as most papilionoids are herbaceous, whereas caesalpinioids and mimosoids are largely woody. Both nonsynonymous (dN) and synonymous (dS) substitution rates were also correlated with several biological features including plastome size and plastomic rearrangements such as the number of inversions and indels. In agreement with previous reports, we found that genes in the IR exhibit between three and fourfold reductions in the substitution rates relative to genes within the large single-copy or small single-copy regions. Furthermore, former IR genes in IR-lacking taxa exhibit accelerated rates compared with genes contained in the IR.

For species with minor inverted repeat (IR) boundary changes in the plastid genome (plastome), nucleotide substitution rates were previously shown to be lower in the IR than the single copy regions (SC). However, the impact of large-scale IR expansion/contraction on plastid nucleotide substitution rates among closely related species remains unclear. We included plastomes from 22 Pelargonium species, including eight newly sequenced genomes, and used both pairwise and model-based comparisons to investigate the impact of the IR on sequence evolution in plastids. Ten types of plastome organization with different inversions or IR boundary changes were identified in Pelargonium. Inclusion in the IR was not sufficient to explain the variation of nucleotide substitution rates. Instead, the rate heterogeneity in Pelargonium plastomes was a mixture of locus-specific, lineage-specific and IR-dependent effects. Our study of Pelargonium plastomes that vary in IR length and gene content demonstrates that the evolutionary consequences of retaining these repeats are more complicated than previously suggested., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Premise of the Study: There is a misinterpretation in the literature regarding the variable orientation of the small single copy region of plastid genomes (plastomes). The common phenomenon of small and large single copy inversion, hypothesized to occur through intramolecular recombination between inverted repeats (IR) in a circular, single unit-genome, in fact, more likely occurs through recombination-dependent replication (RDR) of linear plastome templates. If RDR can be primed through both intra- and intermolecular recombination, then this mechanism could not only create inversion isomers of so-called single copy regions, but also an array of alternative sequence arrangements., Methods: We used Illumina paired-end and PacBio single-molecule real-time (SMRT) sequences to characterize repeat structure in the plastome of Monsonia emarginata (Geraniaceae). We used OrgConv and inspected nucleotide alignments to infer ancestral nucleotides and identify gene conversion among repeats and mapped long (>1 kb) SMRT reads against the unit-genome assembly to identify alternative sequence arrangements., Results: Although M. emarginata lacks the canonical IR, we found that large repeats (>1 kilobase; kb) represent ∼22% of the plastome nucleotide content. Among the largest repeats (>2 kb), we identified GC-biased gene conversion and mapping filtered, long SMRT reads to the M. emarginata unit-genome assembly revealed alternative, substoichiometric sequence arrangements., Conclusion: We offer a model based on RDR and gene conversion between long repeated sequences in the M. emarginata plastome and provide support that both intra-and intermolecular recombination between large repeats, particularly in repeat-rich plastomes, varies unit-genome structure while homogenizing the nucleotide sequence of repeats., (© 2017 Botanical Society of America.)

Genes for the plastid-encoded RNA polymerase (PEP) persist in the plastid genomes of all photosynthetic angiosperms. However, three unrelated lineages (Annonaceae, Passifloraceae and Geraniaceae) have been identified with unusually divergent open reading frames (ORFs) in the conserved region of rpoA, the gene encoding the PEP α subunit. We used sequence-based approaches to evaluate whether these genes retain function. Both gene sequences and complete plastid genome sequences were assembled and analyzed from each of the three angiosperm families. Multiple lines of evidence indicated that the rpoA sequences are likely functional despite retaining as low as 30% nucleotide sequence identity with rpoA genes from outgroups in the same angiosperm order. The ratio of non-synonymous to synonymous substitutions indicated that these genes are under purifying selection, and bioinformatic prediction of conserved domains indicated that functional domains are preserved. One of the lineages (Pelargonium, Geraniaceae) contains species with multiple rpoA-like ORFs that show evidence of ongoing inter-paralog gene conversion. The plastid genomes containing these divergent rpoA genes have experienced extensive structural rearrangement, including large expansions of the inverted repeat. We propose that illegitimate recombination, not positive selection, has driven the divergence of rpoA.

Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear-plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The Leguminosae has emerged as a model for studying angiosperm plastome evolution because of its striking diversity of structural rearrangements and sequence variation. However, most of what is known about legume plastomes comes from few genera representing a subset of lineages in subfamily Papilionoideae. We investigate plastome evolution in subfamily Mimosoideae based on two newly sequenced plastomes (Inga and Leucaena) and two recently published plastomes (Acacia and Prosopis), and discuss the results in the context of other legume and rosid plastid genomes. Mimosoid plastomes have a typical angiosperm gene content and general organization as well as a generally slow rate of protein coding gene evolution, but they are the largest known among legumes. The increased length results from tandem repeat expansions and an unusual 13 kb IR-SSC boundary shift in Acacia and Inga. Mimosoid plastomes harbor additional interesting features, including loss of clpP intron1 in Inga, accelerated rates of evolution in clpP for Acacia and Inga, and dN/dS ratios consistent with neutral and positive selection for several genes. These new plastomes and results provide important resources for legume comparative genomics, plant breeding, and plastid genetic engineering, while shedding further light on the complexity of plastome evolution in legumes and angiosperms.

Diatoms are mostly photosynthetic eukaryotes within the heterokont lineage. Variable plastid genome sizes and extensive genome rearrangements have been observed across the diatom phylogeny, but little is known about plastid genome evolution within order- or family-level clades. The Thalassiosirales is one of the more comprehensively studied orders in terms of both genetics and morphology. Seven complete diatom plastid genomes are reported here including four Thalassiosirales: Thalassiosira weissflogii, Roundia cardiophora, Cyclotella sp. WC03&#95;2, Cyclotella sp. L04&#95;2, and three additional non-Thalassiosirales species Chaetoceros simplex, Cerataulina daemon, and Rhizosolenia imbricata. The sizes of the seven genomes vary from 116,459 to 129,498 bp, and their genomes are compact and lack introns. The larger size of the plastid genomes of Thalassiosirales compared to other diatoms is due primarily to expansion of the inverted repeat. Gene content within Thalassiosirales is more conserved compared to other diatom lineages. Gene order within Thalassiosirales is highly conserved except for the extensive genome rearrangement in Thalassiosira oceanica. Cyclotella nana, Thalassiosira weissflogii and Roundia cardiophora share an identical gene order, which is inferred to be the ancestral order for the Thalassiosirales, differing from that of the other two Cyclotella species by a single inversion. The genes ilvB and ilvH are missing in all six diatom plastid genomes except for Cerataulina daemon, suggesting an independent gain of these genes in this species. The acpP1 gene is missing in all Thalassiosirales, suggesting that its loss may be a synapomorphy for the order and this gene may have been functionally transferred to the nucleus. Three genes involved in photosynthesis, psaE, psaI, psaM, are missing in Rhizosolenia imbricata, which represents the first documented instance of the loss of photosynthetic genes in diatom plastid genomes.

Wheat is the most important cereal in the world in terms of acreage and productivity. We sequenced and assembled the plastid genome of one Egyptian wheat cultivar using next-generation sequence data. The size of the plastid genome is 133,873 bp, which is 672 bp smaller than the published plastid genome of "Chinese Spring" cultivar, due mainly to the presence of three sequences from the rice plastid genome. The difference in size between the previously published wheat plastid genome and the sequence reported here is due to contamination of the published genome with rice plastid DNA, most of which is present in three sequences of 332, 131 and 131 bp. The corrected plastid genome of wheat has been submitted to GenBank (accession number KJ592713) and can be used in future comparisons., (Copyright © 2014 Académie des sciences. All rights reserved.)

Date palm is a very important crop in western Asia and northern Africa, and it is the oldest domesticated fruit tree with archaeological records dating back 5000 years. The huge economic value of this crop has generated considerable interest in breeding programs to enhance production of dates. One of the major limitations of these efforts is the uncertainty regarding the number of date palm cultivars, which are currently based on fruit shape, size, color, and taste. Whole mitochondrial and plastid genome sequences were utilized to examine single nucleotide polymorphisms (SNPs) of date palms to evaluate the efficacy of this approach for molecular characterization of cultivars. Mitochondrial and plastid genomes of nine Saudi Arabian cultivars were sequenced. For each species about 60 million 100 bp paired-end reads were generated from total genomic DNA using the Illumina HiSeq 2000 platform. For each cultivar, sequences were aligned separately to the published date palm plastid and mitochondrial reference genomes, and SNPs were identified. The results identified cultivar-specific SNPs for eight of the nine cultivars. Two previous SNP analyses of mitochondrial and plastid genomes identified substantial intra-cultivar ( = intra-varietal) polymorphisms in organellar genomes but these studies did not properly take into account the fact that nearly half of the plastid genome has been integrated into the mitochondrial genome. Filtering all sequencing reads that mapped to both organellar genomes nearly eliminated mitochondrial heteroplasmy but all plastid SNPs remained heteroplasmic. This investigation provides valuable insights into how to deal with interorganellar DNA transfer in performing SNP analyses from total genomic DNA. The results confirm recent suggestions that plastid heteroplasmy is much more common than previously thought. Finally, low levels of sequence variation in plastid and mitochondrial genomes argue for using nuclear SNPs for molecular characterization of date palm cultivars.

Photosynthesis by diatoms accounts for roughly one-fifth of global primary production, but despite this, relatively little is known about their plastid genomes. We report the completely sequenced plastid genomes for eight phylogenetically diverse diatoms and show them to be variable in size, gene and foreign sequence content, and gene order. The genomes contain a core set of 122 protein-coding genes, with 15 additional genes exhibiting complex patterns of 1) gene losses at varying phylogenetic scales, 2) functional transfers to the nucleus, 3) gene duplication, divergence, and differential retention of paralogs, and 4) acquisitions of putatively functional recombinase genes from resident plasmids. The newly sequenced genomes also contain several previously unreported genes, highlighting how poorly characterized diatom plastid genomes are overall. Genome size variation reflects major expansions of the inverted repeat region in some cases but, more commonly, large-scale expansions of intergenic regions, many of which contain unique open reading frames of likely foreign origin. Although many gene clusters are conserved across species, rearrangements appear to be frequent in most lineages.

Geraniaceae plastid genomes are highly rearranged, and each of the four genera already sequenced in the family has a distinct genome organization. This study reports plastid genome sequences of six additional species, Francoa sonchifolia, Melianthus villosus, and Viviania marifolia from Geraniales, and Pelargonium alternans, California macrophylla, and Hypseocharis bilobata from Geraniaceae. These genome sequences, combined with previously published species, provide sufficient taxon sampling to reconstruct the ancestral plastid genome organization of Geraniaceae and the rearrangements unique to each genus. The ancestral plastid genome of Geraniaceae has a 4 kb inversion and a reduced, Pelargonium-like small single copy region. Our ancestral genome reconstruction suggests that a few minor rearrangements occurred in the stem branch of Geraniaceae followed by independent rearrangements in each genus. The genomic comparison demonstrates that a series of inverted repeat boundary shifts and inversions played a major role in shaping genome organization in the family. The distribution of repeats is strongly associated with breakpoints in the rearranged genomes, and the proportion and the number of large repeats (>20 bp and >60 bp) are significantly correlated with the degree of genome rearrangements. Increases in the degree of plastid genome rearrangements are correlated with the acceleration in nonsynonymous substitution rates (dN) but not with synonymous substitution rates (dS). Possible mechanisms that might contribute to this correlation, including DNA repair system and selection, are discussed.

The plastid genome (plastome) has proved a valuable source of data for evaluating evolutionary relationships among angiosperms. Through basic and applied approaches, plastid transformation technology offers the potential to understand and improve plant productivity, providing food, fiber, energy and medicines to meet the needs of a burgeoning global population. The growing genomic resources available to both phylogenetic and biotechnological investigations are allowing novel insights and expanding the scope of plastome research to encompass new species. In this chapter we present an overview of some of the seminal and contemporary research that has contributed to our current understanding of plastome evolution and attempt to highlight the relationship between evolutionary mechanisms and tools of plastid genetic engineering.

Functional gene transfer from the plastid to the nucleus is rare among land plants despite evidence that DNA transfer to the nucleus is relatively frequent. During the course of sequencing plastid genomes from representative species from three rosid genera (Castanea, Prunus, Theobroma) and ongoing projects focusing on the Fagaceae and Passifloraceae, we identified putative losses of rpl22 in these two angiosperm families. We further characterized rpl22 from three species of Passiflora and one species of Quercus and identified sequences that likely represent pseudogenes. In Castanea and Quercus, both members of the Fagaceae, we identified a nuclear copy of rpl22, which consisted of two exons separated by an intron. Exon 1 encodes a transit peptide that likely targets the protein product back to the plastid and exon 2 encodes rpl22. We performed phylogenetic analyses of 97 taxa, including 93 angiosperms and four gymnosperm outgroups using alignments of 81 plastid genes to examine the phylogenetic distribution of rpl22 loss and transfer to the nucleus. Our results indicate that within rosids there have been independent transfers of rpl22 to the nucleus in Fabaceae and Fagaceae and a putative third transfer in Passiflora. The high level of sequence divergence between the transit peptides in Fabaceae and Fagaceae strongly suggest that these represent independent transfers. Furthermore, Blast searches did not identify the "donor" genes of the transit peptides, suggesting a de novo origin. We also performed phylogenetic analyses of rpl22 for 87 angiosperms and four gymnosperms, including nuclear-encoded copies for five species of Fabaceae and Fagaceae. The resulting trees indicated that the transfer of rpl22 to the nucleus does not predate the origin of angiosperms as suggested in an earlier study. Using previously published angiosperm divergence time estimates, we suggest that these transfers occurred approximately 56-58, 34-37, and 26-27 Ma for the Fabaceae, Fagaceae, and Passifloraceae, respectively.

Geraniaceae plastid genomes (plastomes) have experienced a remarkable number of genomic changes. The plastomes of Erodium texanum, Geranium palmatum, and Monsonia speciosa were sequenced and compared with other rosids and the previously published Pelargonium hortorum plastome. Geraniaceae plastomes were found to be highly variable in size, gene content and order, repetitive DNA, and codon usage. Several unique plastome rearrangements include the disruption of two highly conserved operons (S10 and rps2-atpA), and the inverted repeat (IR) region in M. speciosa does not contain all genes in the ribosomal RNA operon. The sequence of M. speciosa is unusually small (128,787 bp); among angiosperm plastomes sequenced to date, only those of nonphotosynthetic species and those that have lost one IR copy are smaller. In contrast, the plastome of P. hortorum is the largest, at 217,942 bp. These genomes have experienced numerous gene and intron losses and partial and complete gene duplications. Some of the losses are shared throughout the family (e.g., trnT-GGU and the introns of rps16 and rpl16); however, other losses are homoplasious (e.g., trnG-UCC intron in G. palmatum and M. speciosa). IR length is also highly variable. The IR in P. hortorum was previously shown to be greatly expanded to 76 kb, and the IR is lost in E. texanum and reduced in G. palmatum (11 kb) and M. speciosa (7 kb). Geraniaceae plastomes contain a high frequency of large repeats (>100 bp) relative to other rosids. Within each plastome, repeats are often located at rearrangement end points and many repeats shared among the four Geraniaceae flank rearrangement end points. GC content is elevated in the genomes and also in coding regions relative to other rosids. Codon usage per amino acid and GC content at third position sites are significantly different for Geraniaceae protein-coding sequences relative to other rosids. Our findings suggest that relaxed selection and/or mutational biases lead to increased GC content, and this in turn altered codon usage. We propose that increases in genomic rearrangements, repetitive DNA, nucleotide substitutions, and GC content may be caused by relaxed selection resulting from improper DNA repair.

Plastid genomes of the grasses (Poaceae) are unusual in their organization and rates of sequence evolution. There has been a recent surge in the availability of grass plastid genome sequences, but a comprehensive comparative analysis of genome evolution has not been performed that includes any related families in the Poales. We report on the plastid genome of Typha latifolia, the first non-grass Poales sequenced to date, and we present comparisons of genome organization and sequence evolution within Poales. Our results confirm that grass plastid genomes exhibit acceleration in both genomic rearrangements and nucleotide substitutions. Poaceae have multiple structural rearrangements, including three inversions, three genes losses (accD, ycf1, ycf2), intron losses in two genes (clpP, rpoC1), and expansion of the inverted repeat (IR) into both large and small single-copy regions. These rearrangements are restricted to the Poaceae, and IR expansion into the small single-copy region correlates with the phylogeny of the family. Comparisons of 73 protein-coding genes for 47 angiosperms including nine Poaceae genera confirm that the branch leading to Poaceae has significantly accelerated rates of change relative to other monocots and angiosperms. Furthermore, rates of sequence evolution within grasses are lower, indicating a deceleration during diversification of the family. Overall there is a strong correlation between accelerated rates of genomic rearrangements and nucleotide substitutions in Poaceae, a phenomenon that has been noted recently throughout angiosperms. The cause of the correlation is unknown, but faulty DNA repair has been suggested in other systems including bacterial and animal mitochondrial genomes.

The plastid genome of Trifolium subterraneum is 144,763 bp, about 20 kb longer than those of closely related legumes, which also lost one copy of the large inverted repeat (IR). The genome has undergone extensive genomic reconfiguration, including the loss of six genes (accD, infA, rpl22, rps16, rps18, and ycf1) and two introns (clpP and rps12) and numerous gene order changes, attributable to 14-18 inversions. All endpoints of rearranged gene clusters are flanked by repeated sequences, tRNAs, or pseudogenes. One unusual feature of the Trifolium subterraneum genome is the large number of dispersed repeats, which comprise 19.5% (ca. 28 kb) of the genome (versus about 4% for other angiosperms) and account for part of the increase in genome size. Nine genes (psbT, rbcL, clpP, rps3, rpl23, atpB, psbN, trnI-cau, and ycf3) have also been duplicated either partially or completely. rpl23 is the most highly duplicated gene, with portions of this gene duplicated six times. Comparisons of the Trifolium plastid genome with the Plant Repeat Database and searches for flanking inverted repeats suggest that the high incidence of dispersed repeats and rearrangements is not likely the result of transposition. Trifolium has 19.5 kb of unique DNA distributed among 160 fragments ranging in size from 30 to 494 bp, greatly surpassing the other five sequenced legume plastid genomes in novel DNA content. At least some of this unique DNA may represent horizontal transfer from bacterial genomes. These unusual features provide direction for the development of more complex models of plastid genome evolution.

Angiosperms are the largest and most successful clade of land plants with >250,000 species distributed in nearly every terrestrial habitat. Many phylogenetic studies have been based on DNA sequences of one to several genes, but, despite decades of intensive efforts, relationships among early diverging lineages and several of the major clades remain either incompletely resolved or weakly supported. We performed phylogenetic analyses of 81 plastid genes in 64 sequenced genomes, including 13 new genomes, to estimate relationships among the major angiosperm clades, and the resulting trees are used to examine the evolution of gene and intron content. Phylogenetic trees from multiple methods, including model-based approaches, provide strong support for the position of Amborella as the earliest diverging lineage of flowering plants, followed by Nymphaeales and Austrobaileyales. The plastid genome trees also provide strong support for a sister relationship between eudicots and monocots, and this group is sister to a clade that includes Chloranthales and magnoliids. Resolution of relationships among the major clades of angiosperms provides the necessary framework for addressing numerous evolutionary questions regarding the rapid diversification of angiosperms. Gene and intron content are highly conserved among the early diverging angiosperms and basal eudicots, but 62 independent gene and intron losses are limited to the more derived monocot and eudicot clades. Moreover, a lineage-specific correlation was detected between rates of nucleotide substitutions, indels, and genomic rearrangements.