Large Inverted Repeat Research Articles

Accumulation of Large Lineage-Specific Repeats Coincides with Sequence Acceleration and Structural Rearrangement in Plantago Plastomes.

Repeats can mediate rearrangements and recombination in plant mitochondrial genomes and plastid genomes. While repeat accumulations are linked to heightened evolutionary rates and complex structures in specific lineages, debates persist regarding the extent of their influence on sequence and structural evolution. In this study, 75 Plantago plastomes were analyzed to investigate the relationships between repeats, nucleotide substitution rates, and structural variations. Extensive repeat accumulations were associated with significant rearrangements and inversions in the large inverted repeats (IRs), suggesting that repeats contribute to rearrangement hotspots. Repeats caused infrequent recombination that potentially led to substoichiometric shifting, supported by long-read sequencing. Repeats were implicated in elevating evolutionary rates by facilitating localized hypermutation, likely through DNA damage and repair processes. This study also observed a decrease in nucleotide substitution rates for loci translocating into IRs, supporting the role of biased gene conversion in maintaining lower substitution rates. Combined with known parallel changes in mitogenomes, it is proposed that potential dysfunction in nuclear-encoded genes associated with DNA replication, recombination, and repair may drive the evolution of Plantago organellar genomes. These findings contribute to understanding how repeats impact organellar evolution and stability, particularly in rapidly evolving plant lineages.

Removal of the large inverted repeat from the plastid genome reveals gene dosage effects and leads to increased genome copy number

The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome. Using plastid transformation and subsequent selectable marker gene elimination, we precisely excised the IR, thus generating plants with a substantially reduced plastid genome size. We show that the lack of the IR results in a mildly reduced plastid ribosome number, suggesting a gene dosage benefit from the duplicated presence of the ribosomal RNA operon. Moreover, the IR deletion plants contain an increased number of plastid genomes, suggesting that genome copy number is regulated by measuring total plastid DNA content rather than by counting genomes. Together, our findings (1) demonstrate that the IR can enhance the translation capacity of the plastid, (2) reveal the relationship between genome size and genome copy number, and (3) provide a simplified plastid genome structure that will facilitate future synthetic biology applications.

Comparative analysis of chloroplast genomes of two Chinese local citrus varieties and haplotype analysis with other citrus species

Citrus plants, comprising some of the most widely cultivated fruit crops worldwide, have great research value. We assembled and annotated the chloroplast genomes of two local important citrus varieties of China, ‘Tuju’ and ‘Zangju’, and comparatively analyzed their sequences. The full lengths chloroplast genomes of ‘Tuju’ and ‘Zangju’ were 160,121 and 160,694 bp, respectively. Both featured a quadripartite structure with two copies of a large inverted repeat (IR) separated by small (SSC) and large (LSC) single-copy regions. ‘Tuju’ chloroplast genome contained 134 genes. ‘Zangju’ had one less protein-coding gene (rpl22) than ‘Tuju’. We also found 27 and 21 optimal codons, all of which ended with A or U, for chloroplast protein-coding genes of ‘Tuju’ and ‘Zangju’, respectively. There were 32 haplotypes obtained from 43 citrus chloroplast genomes, some different species or cultivars could share one haplotype. Of them, ‘Tuju’, one individual of Citrus limon, one individual of Citrus maxima and Citrus platymamma shared one haplotype, which implied they had the same maternal origin. The phylogenetic trees supported that all haplotypes could be divided five groups, while haplotype network analysis tended to get three groups. Our study reveals the complex lineage relationship of citrus plants at a certain level, and provides a valuable reference tool for future research on citrus plants.

Co-evolution of large inverted repeats and G-quadruplex DNA in fungal mitochondria may facilitate mitogenome stability: the case of Malassezia

Mitogenomes are essential due to their contribution to cell respiration. Recently they have also been implicated in fungal pathogenicity mechanisms. Members of the basidiomycetous yeast genus Malassezia are an important fungal component of the human skin microbiome, linked to various skin diseases, bloodstream infections, and they are increasingly implicated in gut diseases and certain cancers. In this study, the comparative analysis of Malassezia mitogenomes contributed to phylogenetic tree construction for all species. The mitogenomes presented significant size and gene order diversity which correlates to their phylogeny. Most importantly, they showed the inclusion of large inverted repeats (LIRs) and G-quadruplex (G4) DNA elements, rendering Malassezia mitogenomes a valuable test case for elucidating the evolutionary mechanisms responsible for this genome diversity. Both LIRs and G4s coexist and convergently evolved to provide genome stability through recombination. This mechanism is common in chloroplasts but, hitherto, rarely found in mitogenomes.

Plastid Genome Assembly Using Long-read data.

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.

The first report describes features of the chloroplast genome of Withania frutescens

Genomic studies not only help researcher not only to identify genomic features in organisms, but also facilitate understanding of evolutionary relationships. Species in the Withania genus have medicinal benefits, and one of them is Withania frutescens, which is used to treat various diseases. This report investigates the nucleotides and genic features of chloroplast genome of Withania frutescens and trying to clarify the evolutionary relationship with Withania sp and family Solanaceae. We found that the total size of Withania frutescens chloroplast genome was 153.771 kb (the smallest chloroplast genome in genus Withania). A large single-copy region (91.285 kb), a small single-copy region (18.373 kb) form the genomic region, and are distinct from each other by a large inverted repeat (22.056 kb). 137 chloroplast genes are found including 4 rRNAs, 38 tRNAs and 83 protein-coding genes. The Withania frutescens chloroplast genome as well as four closest relatives was compared for features such as structure, nucleotide composition, simple sequence repeats (SSRs) and codon bias. Compared to other Withania species, Withania frutescens has unique characteristics. It has the smallest chloroplast genome of any Withania species, isoleucine is the major amino acid, and tryptophan is the minor, In addition, there are no ycf3 and ycf4 genes, fourth, there are only fifteen replicative genes, while in most other species there are more. Using fast minimum evolution and neighbor joining, we have reconstructed the trees to confirm the relationship with other Solanacaea species. The Withania frutescens chloroplast genome is submitted under accession no. ON153173.

The Genome of the Mitochondrion-Related Organelle in Cepedea longa, a Large Endosymbiotic Opalinid Inhabiting the Recta of Frogs.

Mitochondrion-related organelles (MROs) are loosely defined as degenerated mitochondria in anaerobic and microaerophilic lineages. Opalinids are commonly regarded as commensals in the guts of cold-blooded amphibians. It may represent an intermediate adaptation stage between the conventional aerobic mitochondria and derived anaerobic MROs. In the present study, we sequenced and analyzed the MRO genome of Cepedea longa. It has a linear MRO genome with large inverted repeat gene regions at both ends. Compared to Blastocystis and Proteromonas lacertae, the MRO genome of C. longa has a higher G + C content and repeat sequences near the central region. Although three Opalinata species have different morphological characteristics, phylogenetic analyses based on eight concatenated nad genes indicate that they are close relatives. The phylogenetic analysis showed that C. longa clustered with P. lacertae with strong support. The 18S rRNA gene-based phylogeny resolved the Opalinea clade as a sister clade to Karotomorpha, which then further grouped with Proteromonas. The paraphyly of Proteromonadea needs to be verified due to the lack of MRO genomes for key species, such as Karotomorpha, Opalina and Protoopalina. Besides, our dataset and analyses offered slight support for the paraphyly of Bigyra.

Chloroplast Genome Annotation Tools: Prolegomena to the Identification of Inverted Repeats.

The development of next-generation sequencing technology and the increasing amount of sequencing data have brought the bioinformatic tools used in genome assembly into focus. The final step of the process is genome annotation, which works on assembled genome sequences to identify the location of genome features. In the case of organelle genomes, specialized annotation tools are used to identify organelle genes and structural features. Numerous annotation tools target chloroplast sequences. Most chloroplast DNA genomes have a quadripartite structure caused by two copies of a large inverted repeat. We investigated the strategies of six annotation tools (Chloë, Chloroplot, GeSeq, ORG.Annotate, PGA, Plann) for identifying inverted repeats and analyzed their success using publicly available complete chloroplast sequences of taxa belonging to the asterid and rosid clades. The annotation tools use two different approaches to identify inverted repeats, using existing general search tools or implementing stand-alone solutions. The chloroplast sequences studied show that there are different types of imperfections in the assembled data and that each tool performs better on some sequences than the others.

Characterization of the Complete Chloroplast Genome of Four Species in Callerya.

Callerya reticulata (Bentham) Schot, Callerya dielsiana (Harms) P.K. Loc ex Z. Wei & Pedley, Callerya nitida var. hirsutissima (Z. Wei) X.Y. Zhu, and Callerya nitida (Bentham) R. Geesink, which belongs to the Leguminosae family, are important medicinal plants in China. The genus Callerya includes 26 species, 18 species are distributed in China, and the vine stems of some species are used as traditional medicinal herbs because they have important pharmacological activity. Due to the high similarity of appearance, it is difficult to identify them in the market by appearance alone. Therefore, circulating of Callerya-related materia medica on the market is confusing, sometimes even leading to drug safety problems. It is urgent to develop molecular methods for their identification. To sequence and analyze the complete chloroplast (cp) genomes of C. reticulata, C. dielsiana, C. nitida var. hirsutissima, and C. nitida and to analyze their cp genome differences as a basis for seeking easier DNA barcoding for their identification. After using Illumina high-throughput sequencing and nanopore sequencing to obtain the genome data, some bioinformatics software was used to assembly and analyze the molecular structure of cp genomes. The complete cp genomes of the four species were circular molecules, which ranged from 130 435 to 132 546 bp, and GC contents ranged from 33.89% to 34.89%. Each of them includes a large single-copy region, a small single-copy region, and without large inverted repeat regions. These results suggested that highly variable regions of the four cp genomes would provide useful plastid markers, which could be used as a potential genomic resource to resolve phylogenetic questions and provide a reference for mining specific DNA barcodes of these species. Our study provided highly effective molecular markers for subsequent phylogenetic analysis, species identification, and biogeographic analysis of Callerya.

Putting small and big pieces together: a genome assembly approach reveals the largest Lamiid plastome in a woody vine.

The plastid genome of flowering plants generally shows conserved structural organization, gene arrangement, and gene content. While structural reorganizations are uncommon, examples have been documented in the literature during the past years. Here we assembled the entire plastome of Bignonia magnifica and compared its structure and gene content with nine other Lamiid plastomes. The plastome of B. magnifica is composed of 183,052 bp and follows the canonical quadripartite structure, synteny, and gene composition of other angiosperms. Exceptionally large inverted repeat (IR) regions are responsible for the uncommon length of the genome. At least four events of IR expansion were observed among the seven Bignoniaceae species compared, suggesting multiple expansions of the IRs over the SC regions in the family. A comparison with 6,231 other complete plastomes of flowering plants available on GenBank revealed that the plastome of B. magnifica is the longest Lamiid plastome described to date. The newly generated plastid genome was used as a source of selected genes. These genes were combined with orthologous regions sampled from other species of Bignoniaceae and all gene alignments concatenated to infer a phylogeny of the family. The tree recovered is consistent with known relationships within the Bignoniaceae.

The mitochondrial and chloroplast genomes of the kelp, Ecklonia radiata

Ecklonia radiata is a dominant kelp across much of the southern hemisphere supporting vast economic, social and ecological values. It is also of emerging interest for aquaculture industries and in blue carbon sequestration. Despite its importance, genomic resources of this key species are scant, yet could provide important knowledge to better manage, cultivate and conserve this species in our changing oceans. In this study we report the complete mitochondrial and draft chloroplast genome for E. radiata. The mitochondrial genome is 37,696 bp in length and contains 66 annotated genes. Two alternative scaffold arrangements were recovered for the chloroplast genome, both of 130,955 bp and differing only in the orientation of a single ~ 43 kbp contig. Annotation of the plastid scaffolds identified 175 genes and two large inverted repeats that are typical of brown algal chloroplast genomes. Comparisons to other kelp genomes indicate that the gene sets reported here are likely complete. The recovery of two chloroplast scaffold assembly arrangements hints at the possibility of heteroplasmy in E. radiata though this requires further investigation. The genomic information reported here will aid in understanding functional response of E. radiata in changing oceans, enhance the development of E. radiata as an aquaculture resource and support efforts to conserve this ecologically important marine species.

Characterization of the complete chloroplast genome of Hippophae salicifolia D. Don (Elaeagnaceae)

Seabuckthorn (Hippophae L.) is a pioneer species widely distributed in Eurasia. We assembled and annotated the chloroplast genome of Hippophae salicifolia from Illumina pair-end data, which was 155,420 bp in length with 36.74% GC content; this plastome featured a quadripartite structure with two copies of a large inverted repeat (IR) of 26,528 bp separated by large single copy (LSC) region of 83,504 bp and small single copy region (SSC) of 18,860 bp. In total, 131 complete genes were annotated, including 38 tRNA, eight rRNA, and 85 protein-coding genes. Phylogenetic analysis recovered H. salicifolia and H. gyantsensis as monophyletic and sister to all other Hippophae species for which complete plastome sequences have been published.

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.

Assembly of the durian chloroplast genome using long PacBio reads

We have assembled the complete sequence of the Durio zibethinus chloroplast genome using long PacBio reads. Durian is a valuable commercial tree that produces durian fruit, which is popular in Southeast Asia. The chloroplast genome assembled into a single 143 kb cyclic contig that contained 111 genes. There were 46 short direct repeats (45 to 586 bp) and five short inverted repeats (63 to 169 bp). The long reads that were used for the assembly span the entire chloroplast with > 10 kb overlaps and multiple long reads join the start of the contig to the end of the contig. The durian chloroplast was found to lack the large inverted repeat that is common in chloroplast genomes. An additional 24 durian varieties were sequenced and compared to the assembly and found to also lack the large inverted repeat. There were nine SNPs among the varieties.

Caught in the Act: Variation in plastid genome inverted repeat expansion within and between populations of Medicago minima.

The inverted repeat (IR) lacking clade (IRLC) is a monophyletic group within the Papilionoideae subfamily of Fabaceae where plastid genomes (plastomes) do not contain the large IR typical of land plants. Recently, an IRLC legume, Medicago minima, was found to have regrown a ~9 kb IR that contained a number of canonical IR genes, and closely related M. lupulina contained an incomplete IR of ~425 bp. Complete plastomes were generated for seven additional species, putative members of the M. minima clade. Polymerase chain reaction was employed to investigate the presence of the IR across M. minima and M. lupulina including individuals of nine and eight Eurasian and North African accessions and 15 and 14 Texas populations, respectively. While no sequence similar to the ~9 kb IR was detected among the seven newly sequenced plastomes, all Eurasian and North African accessions of M. minima contained the IR. Variation in IR extent was detected within and between the Texas populations. Expansions of 13 bp and 11 bp occurred at the boundaries of both IR/small single‐copy regions, and populations had one or the other expansion, but not both. Expansion of the IR was not detected in the accessions from Eurasia and North Africa suggesting recent mutations yielded at least two additional plastid haplotypes in M. minima.

Insights into population structure of East African sweetpotato cultivars from hybrid assembly of chloroplast genomes.

Background: The chloroplast (cp) genome is an important resource for studying plant diversity and phylogeny. Assembly of the cp genomes from next-generation sequencing data is complicated by the presence of two large inverted repeats contained in the cp DNA. Methods: We constructed a complete circular cp genome assembly for the hexaploid sweetpotato using extremely low coverage (<1×) Oxford Nanopore whole-genome sequencing (WGS) data coupled with Illumina sequencing data for polishing. Results: The sweetpotato cp genome of 161,274 bp contains 152 genes, of which there are 96 protein coding genes, 8 rRNA genes and 48 tRNA genes. Using the cp genome assembly as a reference, we constructed complete cp genome assemblies for a further 17 sweetpotato cultivars from East Africa and an I. triloba line using Illumina WGS data. Analysis of the sweetpotato cp genomes demonstrated the presence of two distinct subpopulations in East Africa. Phylogenetic analysis of the cp genomes of the species from the Convolvulaceae Ipomoea section Batatas revealed that the most closely related diploid wild species of the hexaploid sweetpotato is I. trifida. Conclusions: Nanopore long reads are helpful in construction of cp genome assemblies, especially in solving the two long inverted repeats. We are generally able to extract cp sequences from WGS data of sufficiently high coverage for assembly of cp genomes. The cp genomes can be used to investigate the population structure and the phylogenetic relationship for the sweetpotato.

Insights into population structure of East African sweetpotato cultivars from hybrid assembly of chloroplast genomes

Background: The chloroplast (cp) genome is an important resource for studying plant diversity and phylogeny. Assembly of the cp genomes from next-generation sequencing data is complicated by the presence of two large inverted repeats contained in the cp DNA. Methods: We constructed a complete circular cp genome assembly for the hexaploid sweetpotato using extremely low coverage (<1×) Oxford Nanopore whole-genome sequencing (WGS) data coupled with Illumina sequencing data for polishing. Results: The sweetpotato cp genome of 161,274 bp contains 152 genes, of which there are 96 protein coding genes, 8 rRNA genes and 48 tRNA genes. Using the cp genome assembly as a reference, we constructed complete cp genome assemblies for a further 17 sweetpotato cultivars from East Africa and an I. triloba line using Illumina WGS data. Analysis of the sweetpotato cp genomes demonstrated the presence of two distinct subpopulations in East Africa. Phylogenetic analysis of the cp genomes of the species from the Convolvulaceae Ipomoea section Batatas revealed that the most closely related diploid wild species of the hexaploid sweetpotato is I. trifida. Conclusions: Nanopore long reads are helpful in construction of cp genome assemblies, especially in solving the two long inverted repeats. We are generally able to extract cp sequences from WGS data of sufficiently high coverage for assembly of cp genomes. The cp genomes can be used to investigate the population structure and the phylogenetic relationship for the sweetpotato.

The complete chloroplast genome sequence of Chlorella vulgaris and phylogenetic analysis

To understand the process of chloroplast genome evolution, information on repeated sequences, intergenic regions, and pseudogenes in chloroplast DNA is extremely helpful. Chlorella vulgaris is a fast-growing fresh-water microalga cultivated on the industrial scale for applications ranging from food to biofuel production. Structure and expression of the chloroplast genome have been studied in a number of plants. Gene content and the sequence of many genes in chloroplast DNA are relatively conserved among land plants and the Euglenophyta Euglena gracilis. The complete chloroplast genome sequence of C. vulgaris was characterized from Illumina pair-end sequencing. The chloroplast genome of C. vulgaris was 165,412 bp in length and the genome contains no large inverted repeat and has one copy of rRNA gene cluster consisting of 16S, 23S, and 5S rRNA genes. And the genes with two exons containing rrn23 (rRNA), trnl-UAA (tRNA), psba (CDS) and chlL (CDS). The genome contains 114 complete genes, including 78 protein-coding genes (45 protein-coding gene species), 33 tRNA genes (26 tRNA species), and 3 rRNA genes (3 rRNA species). The neighbour-joining phylogenetic analysis showed that C. vulgaris and C. vulgarisNC001865 clustered together as sisters to other Salvia species.

The complete chloroplast genome sequence of Taxus yunnanensis

The first complete chloroplast genome (cpDNA) sequence of Taxus yunnanensis was determined from Illumina HiSeq pair-end sequencing data in this study. The cpDNA is 129,190 bp in length. Like other species of taxus genus, the chloroplast genome of T. yunnanensis has lost one of the large inverted repeats (IRs). The genome contains 116 genes, including 82 protein-coding genes, 4 ribosomal RNA genes, and 30 transfer RNA genes. Further phylogenomic analysis showed that T. yunnanensis closed to T. brevifolia in Lauraceae family.

Mitochondrial genome of Salix cardiophylla and its implications for infrageneric division of the genus of Salix

Salix cardiophylla was a member of the genus of Salix in family Salicaceae with unique morphological traits, and once recognized as a separate genus, Toisusu Kimura. Here, we sequenced and assembled the complete mitochondrial genome of S. cardiophylla, which was 735,173 bp in length, including 56 genes, 28 protein-coding genes, 3 rRNA genes, 25 tRNA genes, and one large inverted repeat regions with length of 13,603 bp. Phylogenetic analysis based on 26 mitochondrial CDS confirmed that S. cardiophylla is a member of Salix, and support its merge into Salix in aspect of our new insights on mitogenome phylogenomics.