New data on mitogenomes of Thienemanniella Kieffer, 1911 (Diptera: Chironomidae, Orthocladiinae).

The mitochondrial genome (mitogenome) has been widely used as a powerful marker in phylogenetic and evolutionary studies of various Dipteran groups. However, only a few mitogenomes from the Thienemanniella genus have been reported till now. Furthermore, there is still indeterminacy in the phylogenetic relationships of the genus Thienemanniella. In this study, mitogenomes of five Thienemanniella species were sequenced and analyzed newly. Combined with the published mitogenome of Thienemanniella nipponica, the obtained results showed that mitogenomes of Thienemanniella were conserved in structure, and all genes were observed to be arranged in the same gene order as the ancestral mitogenome. Nucleotide composition varied significantly among different genes, and the control region displayed the highest A + T content. All protein coding genes are subjected to purification selection, and the fastest evolving gene is ATP8. Maximum likelihood and Bayesian inference analyses showed the phylogeny of Thienemanniella which was supported in five topologies. Our present study provides valuable insight into the phylogenetic relationships of Thienemanniella species.

Chloroplast genome, nuclear ITS regions, mitogenome regions, and Skmer analysis resolved the genetic relationship among Cinnamomum species in Sri Lanka.

Cinnamomum species have gained worldwide attention because of their economic benefits. Among them, C. verum (synonymous with C. zeylanicum Blume), commonly known as Ceylon Cinnamon or True Cinnamon is mainly produced in Sri Lanka. In addition, Sri Lanka is home to seven endemic wild cinnamon species, C. capparu-coronde, C. citriodorum, C. dubium, C. litseifolium, C. ovalifolium, C. rivulorum and C. sinharajaense. Proper identification and genetic characterization are fundamental for the conservation and commercialization of these species. While some species can be identified based on distinct morphological or chemical traits, others cannot be identified easily morphologically or chemically. The DNA barcoding using rbcL, matK, and trnH-psbA regions could not also resolve the identification of Cinnamomum species in Sri Lanka. Therefore, we generated Illumina Hiseq data of about 20x coverage for each identified species and a C. verum sample (India) and assembled the chloroplast genome, nuclear ITS regions, and several mitochondrial genes, and conducted Skmer analysis. Chloroplast genomes of all eight species were assembled using a seed-based method.According to the Bayesian phylogenomic tree constructed with the complete chloroplast genomes, the C. verum (Sri Lanka) is sister to previously sequenced C. verum (NC_035236.1, KY635878.1), C. dubium and C. rivulorum. The C. verum sample from India is sister to C. litseifolium and C. ovalifolium. According to the ITS regions studied, C. verum (Sri Lanka) is sister to C. verum (NC_035236.1), C. dubium and C. rivulorum. Cinnamomum verum (India) shares an identical ITS region with C. ovalifolium, C. litseifolium, C. citriodorum, and C. capparu-coronde. According to the Skmer analysis C. verum (Sri Lanka) is sister to C. dubium and C. rivulorum, whereas C. verum (India) is sister to C. ovalifolium, and C. litseifolium. The chloroplast gene ycf1 was identified as a chloroplast barcode for the identification of Cinnamomum species. We identified an 18 bp indel region in the ycf1 gene, that could differentiate C. verum (India) and C. verum (Sri Lanka) samples tested.

The mitoepigenome responds to stress, suggesting novel mito-nuclear interactions in vertebrates.

The mitochondria are central in the cellular response to changing environmental conditions resulting from disease states, environmental exposures or normal physiological processes. Although the influences of environmental stressors upon the nuclear epigenome are well characterized, the existence and role of the mitochondrial epigenome remains contentious. Here, by quantifying the mitochondrial epigenomic response of pineal gland cells to circadian stress, we confirm the presence of extensive cytosine methylation within the mitochondrial genome. Furthermore, we identify distinct epigenetically plastic regions (mtDMRs) which vary in cytosinic methylation, primarily in a non CpG context, in response to stress and in a sex-specific manner. Motifs enriched in mtDMRs contain recognition sites for nuclear-derived DNA-binding factors (ATF4, HNF4A) important in the cellular metabolic stress response, which we found to be conserved across diverse vertebrate taxa. Together, these findings suggest a new layer of mito-nuclear interaction in which the nuclear metabolic stress response could alter mitochondrial transcriptional dynamics through the binding of nuclear-derived transcription factors in a methylation-dependent context.

The complete mitochondrial genome of Wellcomia compar (Spirurina: Oxyuridae) and its genome characterization and phylogenetic analysis.

Wellcomia compar (Spirurina: Oxyuridae) is a pinworm that infects wild and captive porcupines. Despite clear records of its morphological structure, its genetics, systematics, and biology are poorly understood. This study aimed to determine the complete mitochondrial (mt) genome of W. compar and reconstruct its phylogenetic relationship with other nematodes. We sequenced the complete mt genome of W. comparand conducted phylogenetic analyses using concatenated coding sequences of 12 protein-coding genes (PCGs) by maximum likelihood and Bayesian inference. The complete mt genome is 14,373 bp in size and comprises 36 genes, including 12 protein-coding, two rRNA and 22 tRNA genes. Apart from 28 intergenic regions, one non-coding region and one overlapping region also occur. A comparison of the gene arrangements of Oxyuridomorpha revealed relatively similar features in W. compar and Wellcomia siamensis. Phylogenetic analysis also showed that W. compar and W. siamensis formed a sister group. In Oxyuridomorpha the genetic distance between W. compar and W. siamensis was 0.0805. This study reports, for the first time, the complete W. compar mt genome sequence obtained from Chinese porcupines. It provides genetic markers for investigating the taxonomy, population genetics, and phylogenetics of pinworms from different hosts and has implications for the diagnosis, prevention, and control of parasitic diseases in porcupines and other animals.

Eimeria zuernii (Eimeriidae: Coccidia): mitochondrial genome and genetic diversity in the Chinese yak.

BACKGROUND: Coccidiosis caused by Eimeria zuernii (Eimeriidae: Coccidia) represents a significant economic threat to the bovine industry. Understanding the evolutionary and genetic biology of E. zuernii can assist in new interaction developments for the prevention and control of this protozoosis. METHODS: We defined the evolutionary and genetic characteristics of E. zuernii by sequencing the complete mitogenome and analyzing the genetic diversity and population structure of 51 isolates collected from eight yak breeding parks in China. RESULTS: The 6176-bp mitogenome of E. zuernii was linear and encoded typical mitochondrial contents of apicomplexan parasites, including three protein-coding genes [PCGs; cytochrome c oxidase subunits I and III (cox1 and cox3), and cytochrome b (cytb)], seven fragmented small subunit (SSU) and 12 fragmented large subunit (LSU) rRNAs. Genome-wide comparative and evolutionary analyses showed cytb and cox3 to be the most and least conserved Eimeria PCGs, respectively, and placed E. zuernii more closely related to Eimeria mephitidis than other Eimeria species. Furthermore, cox1-based genetic structure defined 24 haplotypes of E. zuernii with high haplotype diversities and low nucleotide diversities across eight geographic populations, supporting a low genetic structure and rapid evolutionary rate as well as a previous expansion event among E. zuernii populations. CONCLUSIONS: To our knowledge, this is the first study presenting the phylogeny, genetic diversity, and population structure of the yak E. zuernii, and such information, together with its mitogenomic data, should contribute to a better understanding of the genetic and evolutionary biological studies of apicomplexan parasites in bovines.

South Asian maternal and paternal lineages in southern Thailand and the role of sex-biased admixture.

Previous genome-wide studies have reported South Asian (SA) ancestry in several Mainland Southeast Asian (MSEA) populations; however, additional details concerning population history, in particular the role of sex-specific aspects of the SA admixture in MSEA populations can be addressed with uniparental markers. Here, we generated ∼2.3 mB sequences of the male-specific portions of the Y chromosome (MSY) of a Tai-Kadai (TK)-speaking Southern Thai group (SouthernThai_TK), and complete mitochondrial (mtDNA) genomes of the SouthernThai_TK and an Austronesian (AN)-speaking Southern Thai (SouthernThai_AN) group. We identified new mtDNA haplogroups, e.g. Q3, E1a1a1, B4a1a and M7c1c3 that have not previously reported in Thai populations, but are frequent in Island Southeast Asia and Oceania, suggesting interactions between MSEA and these regions. SA prevalent mtDNA haplogroups were observed at frequencies of ~35-45% in the Southern Thai groups; both of them showed more genetic relatedness to Austroasiatic (AA) speaking Mon than to any other group. For MSY, SouthernThai_TK had ~35% SA prevalent haplogroups and exhibited closer genetic affinity to Central Thais. We also analyzed published data from other MSEA populations and observed SA ancestry in some additional MSEA populations that also reflects sex-biased admixture; in general, most AA- and AN-speaking groups in MSEA were closer to SA than to TK groups based on mtDNA, but the opposite pattern was observed for the MSY. Overall, our results of new genetic lineages and sex-biased admixture from SA to MSEA groups attest to the additional value that uniparental markers can add to studies of genome-wide variation.

Identification of new variants in MTRNR1 and MTRNR2 genes using whole mitochondrial genome sequencing in a Taiwanese family with MERRF (myoclonic epilepsy with ragged-red fibers) syndrome.

Mitochondrial encephalomyopathy is a multi-system disorder mostly caused by inborn errors of the oxidative phosphorylation (OXPHOS) system and usually manifested as complex neurological disorder and muscle weakness. Myoclonic epilepsy with ragged-red fibers (MERRF) syndrome is one of the major subtypes of mitochondrial disease associated with the m.8344A>G mutation in mitochondrial tRNALys gene. In addition to the symptoms in central nervous and muscle systems, a portion of the patients may develop hearing loss, which has been linked to the genetic mutations of mitochondrial DNA (mtDNA) especially in the mitochondrial ribosome RNA (rRNA) gene. Despite a great number of studies focusing on the consequences of mtDNA mutations, the mechanism of pathogenesis of these overt diseases has remained unclear, and there is no specific and effective treatment for MERRF syndromes. In this study, we developed a high-quality mtDNA sequencing method by next generation sequencing technology to search for the additional pathogenic variations of mtDNA from skin fibroblasts of four members in a Taiwanese family with MERRF syndrome. Through uncovering the signatures of all mtDNA variants in the MERRF family, we identified novel mtDNA variants in the genes encoding mitochondrial 12S and 16S rRNAs. The finding from this study will give us further insight into the molecular mechanisms driving the phenotypic variability and timing of onset of the MERRF syndrome.

The mitochondrial genome of the mountain wooly tapir, Tapirus pinchaque and a formal test of the effect of altitude on the adaptive evolution of mitochondrial protein coding genes in odd-toed ungulates.

BACKGROUND: The harsh conditions of high-altitude environments are known to drive the evolution of physiological and morphological traits in endothermic animals. These conditions are expected to result in the adaptive evolution of protein coding genes encoded in mitochondrial genomes that are vital for the oxidative phosphorylation pathway. In this study, we formally tested for signatures of adaptive evolution on mitochondrial protein coding genes in Tapirus pinchaque and other odd-toed ungulates inhabiting high-elevation environments. RESULTS: The AT-rich mitochondrial genome of T. pinchaque is 16,750 bp long. A phylomitogenomic analysis supports the monophyly of the genus Tapirus and families in the Perissodactyla. The ratio of non-synonymous to synonymous substitutions demonstrated that all mitochondrial genes undergo purifying selection in T. pinchaque and other odd ungulates living at high elevations. Over this negative background selection, Branch Models suggested that cox3 and nad6 might be undergoing stronger purifying selection than other mitochondrial protein coding genes. Furthermore, Site Models suggested that one and four sites in nad2 and nad5, respectively, could be experiencing positive selection. However, these results were supported by Likelihood Ratio Tests but not Bayesian Empirical Bayes posterior probabilities. Additional analyses (in DataMonkey) indicated a relaxation of selection strength in nad6, evidence of episodic diversifying selection in cob, and revealed episodic positive/diversifying selection signatures for two sites in nad1, and one site each in nad2 and nad4. CONCLUSION: The mitochondrial genome of T. pinchaque is an important genomic resource for conservation of this species and this study contributes to the understanding of adaptive evolution of mitochondrial protein coding genes in odd-toed ungulates inhabiting high-altitude environments.

Comparison of Two Variant Analysis Programs for Next-Generation Sequencing Data of Whole Mitochondrial Genome.

BACKGROUND: With advance of next-generation sequencing (NGS) techniques, the need for mitochondrial DNA analysis is increasing not only in the forensic area, but also in medical fields. METHODS: Two commercial programs, Converge Software (CS) and Torrent Variant Caller for variant calling of NGS data, were compared with a considerable amount of sequence data of 50 samples with a homogeneous ethnicity. RESULTS: About 2,300 variants were identified and the two programs showed about 90% of consistency. CS, a dedicated analysis program for mitochondrial DNA, showed some advantages for forensic use. By additional visual inspection, several causes of discrepancy in variant calling results were identified. Application of different notation rules for mitochondrial sequence and the minor allele frequency close to detection threshold were the two most significant reasons. CONCLUSION: With prospective improvement of each program, researchers and practitioners should be aware of characteristics of the analysis program they use and prepare their own strategies to determine variants.

The genome sequence of the critically endangered Kroombit tinkerfrog ( Taudactylus pleione).

The Kroombit tinkerfrog ( Taudactylus pleione) is a stream-dwelling amphibian of the Myobatrachidae family. It is listed as Critically Endangered and is at high risk of extinction due to chytridiomycosis. Here, we provide the first genome assembly of the evolutionarily distinct Taudactylus genus. We sequenced PacBio HiFi reads to assemble a high-quality long-read genome and identified the mitochondrial genome. We also generated a global transcriptome from a tadpole to improve gene annotation. The genome was 5.52 Gb in length and consisted of 4,196 contigs with a contig N50 of 8.853 Mb and an L50 of 153. This study provides the first genomic resources for the Kroombit tinkerfrog to assist in future phylogenetic, environmental DNA, conservation breeding, and disease susceptibility studies.

C17orf80 binds the mitochondrial genome to promote its replication.

Serving as the power plant and signaling hub of a cell, mitochondria contain their own genome which encodes proteins essential for energy metabolism and forms DNA-protein assemblies called nucleoids. Mitochondrial DNA (mtDNA) exists in multiple copies within each cell ranging from hundreds to tens of thousands. Maintaining mtDNA homeostasis is vital for healthy cells, and its dysregulation causes multiple human diseases. However, the players involved in regulating mtDNA maintenance are largely unknown though the core components of its replication machinery have been characterized. Here, we identify C17orf80, a functionally uncharacterized protein, as a critical player in maintaining mtDNA homeostasis. C17orf80 primarily localizes to mitochondrial nucleoid foci and exhibits robust double-stranded DNA binding activity throughout the mitochondrial genome, thus constituting a bona fide new mitochondrial nucleoid protein. It controls mtDNA levels by promoting mtDNA replication and plays important roles in mitochondrial metabolism and cell proliferation. Our findings provide a potential target for therapeutics of human diseases associated with defective mtDNA control.

A systematic comparison of human mitochondrial genome assembly tools.

BACKGROUND: Mitochondria are the cell organelles that produce most of the chemical energy required to power the cell's biochemical reactions. Despite being a part of a eukaryotic host cell, the mitochondria contain a separate genome whose origin is linked with the endosymbiosis of a prokaryotic cell by the host cell and encode independent genomic information throughout their genomes. Mitochondrial genomes accommodate essential genes and are regularly utilized in biotechnology and phylogenetics. Various assemblers capable of generating complete mitochondrial genomes are being continuously developed. These tools often use whole-genome sequencing data as an input containing reads from the mitochondrial genome. Till now, no published work has explored the systematic comparison of all the available tools for assembling human mitochondrial genomes using short-read sequencing data. This evaluation is required to identify the best tool that can be well-optimized for small-scale projects or even national-level research. RESULTS: In this study, we have tested the mitochondrial genome assemblers for both simulated datasets and whole genome sequencing (WGS) datasets of humans. For the highest computational setting of 16 computational threads with the simulated dataset having 1000X read depth, MitoFlex took the least execution time of 69 s, and IOGA took the longest execution time of 1278 s. NOVOPlasty utilized the least computational memory of approximately 0.098 GB for the same setting, whereas IOGA utilized the highest computational memory of 11.858 GB. In the case of WGS datasets for humans, GetOrganelle and MitoFlex performed the best in capturing the SNPs information with a mean F1-score of 0.919 at the sequencing depth of 10X. MToolBox and NOVOPlasty performed consistently across all sequencing depths with a mean F1 score of 0.897 and 0.890, respectively. CONCLUSIONS: Based on the overall performance metrics and consistency in assembly quality for all sequencing data, MToolBox performed the best. However, NOVOPlasty was the second fastest tool in execution time despite being single-threaded, and it utilized the least computational resources among all the assemblers when tested on simulated datasets. Therefore, NOVOPlasty may be more practical when there is a significant sample size and a lack of computational resources. Besides, as long-read sequencing gains popularity, mitochondrial genome assemblers must be developed to use long-read sequencing data.

Assembly and phylogenetic analysis of the mitochondrial genome of endangered medicinal plant Huperzia crispata.

Huperzia crispata is a traditional Chinese herb plant and has attracted special attention in recent years for its products Hup A can serve as an acetylcholinesterase inhibitor (AChEI). Although the chloroplast (cp) genome of H. crispata has been studied, there are no reports regarding the Huperzia mitochondrial (mt) genome since the previously reported H. squarrosa has been revised as Phlegmariurus squarrosus. The mt genome of H. crispata was sequenced using a combination of long-read nanopore and Illumina sequencing platforms. The entire H. crispata mt genome was assembled in a circular with a length of 412,594 bp and a total of 91 genes, including 45 tRNAs, 6 rRNAs, 37 protein-coding genes (PCGs), and 3 pseudogenes. Notably, the rps8 gene was present in P. squarrosus and a pseudogene rps8 was presented in H. crispata, which was lacking in most of Pteridophyta and Gymnospermae. Intron-encoded maturase (mat-atp9i85 and mat-cobi787) genes were present in H. crispata and P. squarrosus, but lost in other examined lycophytes, ferns, and Gymnospermae plants. Collinearity analysis showed that the mt genome of H. crispata and P. squarrossus is highly conservative compared to other ferns. Relative synonymous codon usage (RSCU) analysis showed that the amino acids most frequently found were phenylalanine (Phe) (4.77%), isoleucine (Ile) (4.71%), lysine (Lys) (4.26%), while arginine (Arg) (0.32%), and histidine (His) (0.42%) were rarely found. Simple sequence repeats (SSR) analysis revealed that a total of 114 SSRs were identified in the mt genome of H. crispata and account for 0.35% of the whole mt genome. Monomer repeats were the majority types of SSRs and represent 91.89% of the total SSRs. In addition, a total of 1948 interspersed repeats (158 forward, 147 palindromic, and 5 reverse repeats) with a length ranging from 30 bp to 14,945 bp were identified in the H. crispata mt genome and the 30-39-bp repeats were the most abundant type. Gene transfer analysis indicated that a total of 12 homologous fragments were discovered between the cp and mt genomes of H. crispata, accounting for 0.93% and 2.48% of the total cp and mt genomes, respectively. The phylogenetic trees revealed that H. crispata was the sister of P. squarrosus. The Ka/Ks analysis results suggested that most PCGs, except atp6 gene, were subject to purification selection during evolution. Our study provides extensive information on the features of the H. crispata mt genome and will help unravel evolutionary relationships, and molecular identification within lycophytes.

Phylogeny and genetic variations of the three genome compartments in haptophytes shed light on the rapid evolution of coccolithophores.

Haptophyte algae, including coccolithophores, play key roles in global carbon cycling and ecosystem. They exhibit exceptional morphological and functional diversity. However, their phylogeny is mostly based on short markers and genome researches are always limited to few species, hindering a better understanding about their evolution and diversification. In this study, by assembling 69 new plastid genomes, 65 new mitochondrial genomes, and 55 nuclear drafts, we systematically analyzed their genome variations and built the most comprehensive phylogenies in haptophytes and Noelaerhabdaceae, with the latter is the family of the model coccolithophore Emiliania huxleyi. The haptophyte genomes vary significantly in size, gene content, and structure. We detected phylogenetic incongruence of Prymnesiales between genome compartments. In Noelaerhabdaceae, by including Reticulofenestra sessilis and a proper outgroup, we found R. sessilis was not the basal taxon of this family. Noelaerhabdaceae strains have very similar genomic features and conserved sequences, but different gene content and dynamic structure. We speculate that was caused by DNA double-strand break repairs. Our results provide valuable genetic resources and new insights into the evolution of haptophytes, especially coccolithophores.

Exploring the mitochondrial genome of Caridina pseudogracilirostris: a comparative analysis within the Atyidae Family.

BACKGROUND: Caridina pseudogracilirostris is a highly adaptive estuarine species found in brackish waters and marshes along the southwestern and southern coastal regions of India. METHODS AND RESULTS: The whole mitochondrial genome of C. pseudogracilirostris is 15,451 bp in length with 59.3% AT content and encodes 37 genes, including 22 tRNAs, 13 protein-coding genes, and two rRNAs, which are arranged in a distinctive pattern similar to most crustaceans. ML and BI methods were used for phylogenetic analysis of C. pseudogracilirostris clustered with other Caridina species, supporting the monophyly of the Caridina genus within the Atyidae family. The fully annotated mitochondrial genome of C. pseudogracilirostris was submitted to GenBank under accession number OQ534868.1. CONCLUSIONS: We are the first to report on the C. pseudogracilirostris whole mitochondrial genome, which provides a valuable resource for future research on genetics, evolution, phylogenetics, etc., among Caridina species and other species. The phylogenetic investigation supports the monophyly of the Caridina genus within the Atyidae family and emphasizes the value of mitochondrial genome data in determining the evolutionary relationships among crustaceans.

Comparative analysis of the mitochondrial genomes of the family Mactridae (Mollusca: Venerida) and their phylogenetic implications.

Taxonomy and phylogenetic relationships within the family Mactridae have remained debatable because of the plasticity of morphological characteristics and the lack of accurate molecular data, thereby resulting in abundant synonyms and taxa rearrangements. Mitochondrial genomes (mitogenomes) have been widely used as powerful tools to reconstruct phylogenies of various groups of mollusks; however, they have not been used for studying the phylogeny of mactrids specifically. In the present study, mitogenomes of seven Mactridae species, namely Mactra chinensis, Mactra cygnus, Mactra quadrangularis, Mactra cumingii, Mactrinula dolabrata, Raeta pulchella, and Raeta sp., were sequenced by Illumina high-throughput sequencing, and a comparative mitochondrial genomic analysis was conducted. The newly sequenced mitogenomes were double-stranded circular molecules, with all functional genes encoded on the heavy strand. All the new mactrid mitogenomes had two rRNA genes (12S and 16S), 13 protein-coding genes (PCGs) (atp6, cox1, cox2, cox3, cytb, nad1, nad2, nad3, nad4, nad4l, nad5, nad6, and atp8), and 22 tRNAs. The mitogenomes showed considerable variation in AT content, GC skew, and AT skew. The results of the phylogenetic analysis confirmed monophyly of the family Mactridae and suggested that genera Mactrinula, Spisula, Rangia, and Mulinia should not be placed under subfamily Mactrinae. Our results supported that potential cryptic species existed in Mactra antiquata. We also proposed subfamily Kymatoxinae should belong to the family Mactridae rather than Anatinellidae and Mactra alta in China should be Mactra cygnus. Additionally, conservation in functional gene arrangement was found in genera Mactra, Raeta, and Lutraria. But gene orders in S. sachalinensis and S. solida were quite different, questioning their congeneric relationship. Our results further suggested that the taxonomy within the family Mactridae requires an integrative revision.

Complete mitochondrial genome of Agrostis stolonifera: insights into structure, Codon usage, repeats, and RNA editing.

BACKGROUND: Plants possess mitochondrial genomes that are large and complex compared to animals. Despite their size, plant mitochondrial genomes do not contain significantly more genes than their animal counterparts. Studies into the sequence and structure of plant mitochondrial genomes heavily imply that the main mechanism driving replication of plant mtDNA, and offer valuable insights into plant evolution, energy production, and environmental adaptation. RESULTS: This study presents the first comprehensive analysis of Agrostis stolonifera's mitochondrial genome, characterized by a branched structure comprising three contiguous chromosomes, totaling 560,800 bp with a GC content of 44.07%. Annotations reveal 33 unique protein-coding genes (PCGs), 19 tRNA genes, and 3 rRNA genes. The predominant codons for alanine and glutamine are GCU and CAA, respectively, while cysteine and phenylalanine exhibit weaker codon usage biases. The mitogenome contains 73, 34, and 23 simple sequence repeats (SSRs) on chromosomes 1, 2, and 3, respectively. Chromosome 1 exhibits the most frequent A-repeat monomeric SSR, whereas chromosome 2 displays the most common U-repeat monomeric SSR. DNA transformation analysis identifies 48 homologous fragments between the mitogenome and chloroplast genome, representing 3.41% of the mitogenome's total length. The PREP suite detects 460 C-U RNA editing events across 33 mitochondrial PCGs, with the highest count in the ccmFn gene and the lowest in the rps7 gene. Phylogenetic analysis confirms A. stolonifera's placement within the Pooideae subfamily, showing a close relationship to Lolium perenne, consistent with the APG IV classification system. Numerous homologous co-linear blocks are observed in A. stolonifera's mitogenomes and those of related species, while certain regions lack homology. CONCLUSIONS: The unique features and complexities of the A. stolonifera mitochondrial genome, along with its similarities and differences to related species, provide valuable insights into plant evolution, energy production, and environmental adaptation. The findings from this study significantly contribute to the growing body of knowledge on plant mitochondrial genomes and their role in plant biology.

Assembly and comparative analysis of the complete mitochondrial genome of Ilex metabaptista (Aquifoliaceae), a Chinese endemic species with a narrow distribution.

BACKGROUND: Ilex metabaptista is a woody tree species with strong waterlogging tolerance and is also admired as a landscape plant with high development prospects and scientific research value. Unfortunately, populations of this species have declined due to habitat loss. Thus, it is a great challenge for us to efficiently protect I. metabaptista resources from extinction. Molecular biology research can provide the scientific basis for the conservation of species. However, the study of I. metabaptista genetics is still in its infancy. To date, no mitochondrial genome (mitogenome) in the genus Ilex has been analysed in detail. RESULTS: The mitogenome of I. metabaptista was assembled based on the reads from Illumina and Nanopore sequencing platforms; it was a typical circular DNA molecule of 529,560 bp with a GC content of 45.61% and contained 67 genes, including 42 protein-coding genes, 22 tRNA genes, and 3 rRNA genes. Repeat sequence analysis and prediction of RNA editing sites revealed a total of 286 dispersed repeats, 140 simple repeats, 18 tandem repeats, and 543 RNA editing sites. Analysis of codon usage showed that codons ending in A/T were preferred. Gene migration was observed to occur between the mitogenome and chloroplast genome via the detection of homologous fragments. In addition, Ka/Ks analysis revealed that most of the protein-coding genes in the mitogenome had undergone negative selection, and only the ccmB gene had undergone potential positive selection in most asterids. Nucleotide polymorphism analysis revealed the variation in each gene, with atp9 being the most notable. Furthermore, comparative analysis showed that the GC contents were conserved, but the sizes and structure of mitogenomes varied greatly among asterids. Phylogenetic analysis based on the mitogenomes reflected the exact evolutionary and taxonomic status of I. metabaptista. CONCLUSION: In this study, we sequenced and annotated the mitogenome of I. metabaptista and compared it with the mitogenomes of other asterids, which provided essential background information for further understanding of the genetics of this plant and helped lay the foundation for future studies on molecular breeding of I. metabaptista.

Structural and gene composition variation of the complete mitochondrial genome of Mammillaria huitzilopochtli (Cactaceae, Caryophyllales), revealed by de novo assembly.

BACKGROUND: Structural descriptions of complete genomes have elucidated evolutionary processes in angiosperms. In Cactaceae (Caryophyllales), a high structural diversity of the chloroplast genome has been identified within and among genera. In this study, we assembled the first mitochondrial genome (mtDNA) for the short-globose cactus Mammillaria huitzilopochtli. For comparative purposes, we used the published genomes of 19 different angiosperms and the gymnosperm Cycas taitungensis as an external group for phylogenetic issues. RESULTS: The mtDNA of M. huitzilopochtli was assembled into one linear chromosome of 2,052,004 bp, in which 65 genes were annotated. These genes account for 57,606 bp including 34 protein-coding genes (PCGs), 27 tRNAs, and three rRNAs. In the non-coding sequences, repeats were abundant, with a total of 4,550 (179,215 bp). In addition, five complete genes (psaC and four tRNAs) of chloroplast origin were documented. Negative selection was estimated for most (23) of the PCGs. The phylogenetic tree showed a topology consistent with previous analyses based on the chloroplast genome. CONCLUSIONS: The number and type of genes contained in the mtDNA of M. huitzilopochtli were similar to those reported in 19 other angiosperm species, regardless of their phylogenetic relationships. Although other Caryophyllids exhibit strong differences in structural arrangement and total size of mtDNA, these differences do not result in an increase in the typical number and types of genes found in M. huitzilopochtli. We concluded that the total size of mtDNA in angiosperms increases by the lengthening of the non-coding sequences rather than a significant gain of coding genes.

The Mitochondrial Genome of the Holoparasitic Plant Thonningia sanguinea Provides Insights into the Evolution of the Multichromosomal Structure.

Multichromosomal mitochondrial genome (mitogenome) structures have repeatedly evolved in many lineages of angiosperms. However, the underlying mechanism remains unclear. The mitogenomes of three genera of Balanophoraceae, namely Lophophytum, Ombrophytum, and Rhopalocnemis, have already been sequenced and assembled, all showing a highly multichromosomal structure, albeit with different genome and chromosome sizes. It is expected that characterization of additional lineages of this family may expand the knowledge of mitogenome diversity and provide insights into the evolution of the plant mitogenome structure and size. Here, we assembled and characterized the mitogenome of Thonningia sanguinea, which, together with Balanophora, forms a clade sister to the clade comprising Lophophytum, Ombrophytum, and Rhopalocnemis. The mitogenome of T. sanguinea possesses a multichromosomal structure of 18 circular chromosomes of 8.7-19.2 kb, with a total size of 246,247 bp. There are very limited shared regions and poor chromosomal correspondence between T. sanguinea and other Balanophoraceae species, suggesting frequent rearrangements and rapid sequence turnover. Numerous medium- and small-sized repeats were identified in the T. sanguinea mitogenome; however, no repeat-mediated recombination was detected, which was verified by Illumina reads mapping and PCR experiments. Intraspecific mitogenome variations in T. sanguinea are mostly insertions and deletions, some of which can lead to degradation of perfect repeats in one or two accessions. Based on the mitogenome features of T. sanguinea, we propose a mechanism to explain the evolution of a multichromosomal mitogenome from a master circle, which involves mutation in organellar DNA replication, recombination and repair genes, decrease of recombination, and repeat degradation.