Identifying inversions with breakpoints in the Dystrophin gene through long-read sequencing: report of two cases.

BACKGROUND: Duchenne Muscular Dystrophy (DMD) is an X-linked disorder caused by mutations in the DMD gene, with large deletions being the most common type of mutation. Inversions involving the DMD gene are a less frequent cause of the disorder, largely because they often evade detection by standard diagnostic methods such as multiplex ligation probe amplification (MLPA) and whole exome sequencing (WES). CASE PRESENTATION: Our research identified two intrachromosomal inversions involving the dystrophin gene in two unrelated families through Long-read sequencing (LRS). These variants were subsequently confirmed via Sanger sequencing. The first case involved a pericentric inversion extending from DMD intron 47 to Xq27.3. The second case featured a paracentric inversion between DMD intron 42 and Xp21.1, inherited from the mother. In both cases, simple repeat sequences (SRS) were present at the breakpoints of these inversions. CONCLUSIONS: Our findings demonstrate that LRS is an effective tool for detecting atypical mutations. The identification of SRS at the breakpoints in DMD patients enhances our understanding of the mechanisms underlying structural variations, thereby facilitating the exploration of potential treatments.

W2V-repeated index: Prediction of enhancers and their strength based on repeated fragments.

Enhancers are crucial in gene expression regulation, dictating the specificity and timing of transcriptional activity, which highlights the importance of their identification for unravelling the intricacies of genetic regulation. Therefore, it is critical to identify enhancers and their strengths. Repeated sequences in the genome are repeats of the same or symmetrical fragments. There has been a great deal of evidence that repetitive sequences contain enormous amounts of genetic information. Thus, We introduce the W2V-Repeated Index, designed to identify enhancer sequence fragments and evaluates their strength through the analysis of repeated K-mer sequences in enhancer regions. Utilizing the word2vector algorithm for numerical conversion and Manta Ray Foraging Optimization for feature selection, this method effectively captures the frequency and distribution of K-mer sequences. By concentrating on repeated K-mer sequences, it minimizes computational complexity and facilitates the analysis of larger K values. Experiments indicate that our method performs better than all other advanced methods on almost all indicators.

De Novo Hybrid Assembly Unveils Multi-Chromosomal Mitochondrial Genomes in Ludwigia Species, Highlighting Genomic Recombination, Gene Transfer, and RNA Editing Events.

Biological invasions have been identified as the fifth cause of biodiversity loss, and their subsequent dispersal represents a major ecological challenge. The aquatic invasive species Ludwigia grandiflora subsp. hexapetala (Lgh) and Ludwigia peploides subsp. montevidensis (Lpm) are largely distributed in aquatic environments in North America and in Europe. However, they also present worrying terrestrial forms that are able to colonize wet meadows. To comprehend the mechanisms of the terrestrial adaptation of Lgh and Lpm, it is necessary to develop their genomic resources, which are currently poorly documented. We performed de novo assembly of the mitogenomes of Lgh and Lpm through hybrid assemblies, combining short reads (SR) and/or long reads (LR) before annotating both mitogenomes. We successfully assembled the mitogenomes of Lgh and Lpm into two circular molecules each, resulting in a combined total length of 711,578 bp and 722,518 bp, respectively. Notably, both the Lgh and Lpm molecules contained plastome-origin sequences, comprising 7.8% of the mitochondrial genome length. Additionally, we identified recombinations that were mediated by large repeats, suggesting the presence of multiple alternative conformations. In conclusion, our study presents the first high-quality mitogenomes of Lpm and Lgh, which are the only ones in the Myrtales order found as two circular molecules.

Mitochondrial genome complexity in Stemona sessilifolia: nanopore sequencing reveals chloroplast gene transfer and DNA rearrangements.

Mitochondria are semi-autonomous organelles in eukaryotic cells with their own genome. Plant mitogenomes differ from animal mitogenomes in size, structure, and repetitive DNA sequences. Despite larger sizes, plant mitogenomes do not have significantly more genes. They exhibit diverse structures due to variations in size, repetitive DNA, recombination frequencies, low gene densities, and reduced nucleotide substitution rates. In this study, we analyzed the mitochondrial genome of Stemona sessilifolia using Nanopore and Illumina sequencing. De-novo assembly and annotation were conducted using Unicycler, Geseq, tRNAscan-SE and BLASTN, followed by codon usage, repeat sequence, RNA-editing, synteny, and phylogenetic analyses. S. sessilifolia's mitogenome consisted of one linear contig and six circular contigs totaling 724,751 bp. It had 39 protein-coding genes, 27 tRNA genes, and 3 rRNA genes. Transfer of chloroplast sequences accounted for 13.14% of the mitogenome. Various analyses provided insights into genetic characteristics, evolutionary dynamics, and phylogenetic placement. Further investigations can explore transferred genes' functions and RNA-editing's role in mitochondrial gene expression in S. sessilifolia.

The Ga1 locus of the genus Zea is associated with novel genome structures derived from multiple, independent nonhomologous recombination events.

The Ga1 locus controls cross-incompatibility between field corn and popcorn. The Ga1-S haplotype contains 2 types of pectin methylesterase (PME) genes, ZmPme3 and several copies of ZmGa1P that are expressed in silk and pollen, respectively. The ga1 haplotype contains nonfunctional tandem repeat sequences related to ZmPme3 and ZmGa1P. This haplotype can cross-pollinate freely and is widely present in field corn. The primary objective of this study is to characterize the repeat sequences from a diverse collection of maize and teosinte lines and use this information to understand the evolution of the Ga1 locus. First, we characterized the complexity of the Ga1 genome region in high-quality maize genome assemblies that led to their categorization into 5 groups based on the number and type of PME-like sequences found at this region. Second, we studied duplication events that led to the ga1 and Ga1-S repeats using maximum likelihood phylogenetic reconstruction. Divergence estimates of the ga1 haplotype suggest that the duplication events occurred more than 600 KYA whereas those in Ga1-S occurred at 3 time points, i.e. >600, ∼260, and ∼100 KYA. These estimates suggest that the ga1 and Ga1-S tandem duplication events occurred independently. Finally, analysis of ZmPme3 and ZmGa1P homologs in Zea and Tripsacum genomes suggests that ga1 and Ga1-S repeats originated from an ancestral pair of PME genes that duplicated and diverged through 2 evolutionary branches prior to the domestication of maize.

Complete chloroplast genomes of three wild perennial Hordeum species from Central Asia: genome structure, mutation hotspot, phylogenetic relationships, and comparative analysis.

Hordeum L. is widely distributed in mountain or plateau of subtropical and warm temperate regions around the world. Three wild perennial Hordeum species, including H. bogdanii, H. brevisubulatum, and H. violaceum, have been used as forage and for grassland ecological restoration in high-altitude areas in recent years. To date, the degree of interspecies sequence variation in the three Hordeum species within existing gene pools is still not well-defined. Herein, we sequenced and assembled chloroplast (cp) genomes of the three species. The results revealed that the cp genome of H. bogdanii showed certain sequence variations compared with the cp genomes of the other two species (H. brevisubulatum and H. violaceum), and the latter two were characterized by a higher relative affinity. Parity rule 2 plot (PR2) analysis illuminated that most genes of all ten Hordeum species were concentrated in nucleotide T and G. Numerous single nucleotide polymorphism (SNP) and insertion/deletion (In/Del) events were detected in the three Hordeum species. A series of hotspots regions (tRNA-GGU ~ tRNA-GCA, tRNA-UGU ~ ndhJ, psbE ~ rps18, ndhF ~ tRNA-UAG, etc.) were identified by mVISTA procedures, and the five highly polymorphic genes (tRNA-UGC, tRNA-UAA, tRNA-UUU, tRNA-UAC, and ndhA) were proved by the nucleotide diversity (Pi). Although the distribution and existence of cp simple sequence repeats (cpSSRs) were predicted in the three Hordeum cp genomes, no rearrangement was found between them. A similar phenomenon has been found in the cp genome of the other seven Hordeum species, which has been published so far. In addition, evolutionary relationships were reappraised based on the currently reported cp genome of Hordeum L. This study offers a framework for gaining a better understanding of the evolutionary history of Hordeum species through the re-examination of their cp genomes, and by identifying highly polymorphic genes and hotspot regions that could provide important insights into the genetic diversity and differentiation of these species.

Two Short Repeats in the 5' Untranslated Region of Insulin-like Androgenic Gland Factor in Procambarus clarkii (PcIAG) That Regulate PcIAG Expression.

Insulin-like androgenic gland factor (IAG) plays an important role in sex manipulation in decapods. Understanding the molecular regulation mechanism of IAG in Procambarus clarkii (PcIAG) is important for realizing its sex control. In this study, the promoter and gene structure of PcIAG, mRNA, and miRNA expression profiles after interfering with two siRNAs synthesized according to the two short repeats in the 5' untranslated regions (5'UTR) of PcIAG were analyzed, and miRNAs of exosomes were investigated to explore the role of repeated sequences with tandem two short repeats located in the 5'UTR of PcIAG isolated from the androgenic gland (AG) in the regulation of IAG expression. The results showed that the repeated sequences of 5'UTR only occurred completely in the cDNA from AG, and the function of the two repeats was different in regulating the expression of PcIAG, in which the Wnt signaling pathway may be involved. Furthermore, we found that six miRNAs including miR-133, miR-193, miR-34, miR-1, miR-100, and let-7 might be involved in the regulation of the expression of PcIAG, wherein miR-133 might directly be related with the repeated sequences of 5'UTR.

Complex Physical Structure of Complete Mitochondrial Genome of Quercus acutissima (Fagaceae): A Significant Energy Plant.

Quercus acutissima Carruth. is a Chinese important energy plant with high ecological and economic values. While the species chloroplast genome has been reported, its mitochondrial genome (mitogenome) is still unexplored. Here, we assembled and annotated the Q. acutissima mitogenome, and we compared its characteristic differences with several closely related species. The Q. acutissima mitogenome's main structure is branched with three distinguished contigs (linear molecule 1, circular molecule 2, and circular molecule 3) with 448,982 bp total length and 45.72% GC content. The mitogenome contained 51 genes, including 32 protein-coding, 16 tRNA and 3 rRNA genes. We examined codon usage, repeated sequences, genome recombination, chloroplast to mitochondrion DNA transformation, RNA editing, and synteny in the Q. acutissima mitogenome. Phylogenetic trees based on 29 species mitogenomes clarified the species classification. Our results provided comprehensive information of Q. acutissima mitogenome, and they are expected to provide valuable information for Fagaceae evolutionary biology and to promote the species germplasm utilization.

An Expanding Toolkit for Heterochromatin Repair Studies.

Pericentromeric heterochromatin is mostly composed of repetitive DNA sequences prone to aberrant recombination. Cells have developed highly specialized mechanisms to enable 'safe' homologous recombination (HR) repair while preventing aberrant recombination in this domain. Understanding heterochromatin repair responses is essential to understanding the critical mechanisms responsible for genome integrity and tumor suppression. Here, we review the tools, approaches, and methods currently available to investigate double-strand break (DSB) repair in pericentromeric regions, and also suggest how technologies recently developed for euchromatin repair studies can be adapted to characterize responses in heterochromatin. With this ever-growing toolkit, we are witnessing exciting progress in our understanding of how the 'dark matter' of the genome is repaired, greatly improving our understanding of genome stability mechanisms.

Transcription of rRNA in early mouse embryos promotes chromatin reorganization and expression of major satellite repeats.

During the first cell cycles of early development, the chromatin of the embryo is highly reprogrammed while the embryonic genome starts its own transcription. The spatial organization of the genome is an important process that contributes to regulating gene transcription in time and space. It has, however, been poorly studied in the context of early embryos. To study the cause-and-effect link between transcription and spatial organization in embryos, we focused on ribosomal genes, which are silent initially but start to be transcribed in 2-cell mouse embryos. We demonstrated that ribosomal sequences and early unprocessed rRNAs are spatially organized in a very particular manner between 2-cell and 16-cell stage. By using drugs that interfere with ribosomal DNA transcription, we showed that this organization - which is totally different in somatic cells - depends on an active transcription of ribosomal genes and induces a unique chromatin environment that favors transcription of major satellite sequences once the 4-cell stage has been reached.

Group II intron and repeat-rich red algal mitochondrial genomes demonstrate the dynamic recent history of autocatalytic RNAs.

BACKGROUND: Group II introns are mobile genetic elements that can insert at specific target sequences, however, their origins are often challenging to reconstruct because of rapid sequence decay following invasion and spread into different sites. To advance understanding of group II intron spread, we studied the intron-rich mitochondrial genome (mitogenome) in the unicellular red alga, Porphyridium. RESULTS: Analysis of mitogenomes in three closely related species in this genus revealed they were 3-6-fold larger in size (56-132 kbp) than in other red algae, that have genomes of size 21-43 kbp. This discrepancy is explained by two factors, group II intron invasion and expansion of repeated sequences in large intergenic regions. Phylogenetic analysis demonstrates that many mitogenome group II intron families are specific to Porphyridium, whereas others are closely related to sequences in fungi and in the red alga-derived plastids of stramenopiles. Network analysis of intron-encoded proteins (IEPs) shows a clear link between plastid and mitochondrial IEPs in distantly related species, with both groups associated with prokaryotic sequences. CONCLUSION: Our analysis of group II introns in Porphyridium mitogenomes demonstrates the dynamic nature of group II intron evolution, strongly supports the lateral movement of group II introns among diverse eukaryotes, and reveals their ability to proliferate, once integrated in mitochondrial DNA.

Using Genomics to Shape the Definition of the Agglutinin-Like Sequence (ALS) Family in the Saccharomycetales.

The Candida albicans agglutinin-like sequence (ALS) family is studied because of its contribution to cell adhesion, fungal colonization, and polymicrobial biofilm formation. The goal of this work was to derive an accurate census and sequence for ALS genes in pathogenic yeasts and other closely related species, while probing the boundaries of the ALS family within the Order Saccharomycetales. Bioinformatic methods were combined with laboratory experimentation to characterize 47 novel ALS loci from 8 fungal species. AlphaFold predictions suggested the presence of a conserved N-terminal adhesive domain (NT-Als) structure in all Als proteins reported to date, as well as in S. cerevisiae alpha-agglutinin (Sag1). Lodderomyces elongisporus, Meyerozyma guilliermondii, and Scheffersomyces stipitis were notable because each species had genes with C. albicans ALS features, as well as at least one that encoded a Sag1-like protein. Detection of recombination events between the ALS family and gene families encoding other cell-surface proteins such as Iff/Hyr and Flo suggest widespread domain swapping with the potential to create cell-surface diversity among yeast species. Results from the analysis also revealed subtelomeric ALS genes, ALS pseudogenes, and the potential for yeast species to secrete their own soluble adhesion inhibitors. Information presented here supports the inclusion of SAG1 in the ALS family and yields many experimental hypotheses to pursue to further reveal the nature of the ALS family.

Concentration and integrity indexes of urine cell-free DNA as promising biomarkers for early lung cancer diagnosis.

Aim: To explore the role of urine cell-free DNA (ucfDNA) concentration and integrity indexes as potential biomarkers for lung cancer diagnosis. Materials & methods: Quantitative real-time PCR targeting Arthrobacter luteus (ALU) repeats at three size fragments (ALU-60, 115 and 247 bp) was performed in 55 lung cancer patients and 35 healthy individuals. Results: ucfDNA concentration and integrity indexes were significantly higher in lung cancer patients than in healthy controls. The area under the receiver operating characteristic curve for differentiating patients with stage I/II from healthy controls by ALU fragments concentration were 0.856, 0.909 and 0.932, respectively. In addition, the ucfDNA integrity indexes in patients with lymph node metastasis were significantly higher than in patients with non-metastatic. Conclusion: ucfDNA concentration and integrity indexes could serve as promising biomarkers for lung cancer diagnosis.

Plastome comparative genomics in maples resolves the infrageneric backbone relationships.

Maples (Acer) are among the most diverse and ecologically important tree genera of the north-temperate forests. They include species highly valued as ornamentals and as a source of timber and sugar products. Previous phylogenetic studies employing plastid markers have not provided sufficient resolution, particularly at deeper nodes, leaving the backbone of the maple plastid tree essentially unresolved. We provide the plastid genome sequences of 16 species of maples spanning the sectional diversity of the genus and explore the utility of these sequences as a source of information for genetic and phylogenetic studies in this group. We analyzed the distribution of different types of repeated sequences and the pattern of codon usage, and identified variable regions across the plastome. Maximum likelihood and Bayesian analyses using two partitioning strategies were performed with these and previously published sequences. The plastomes ranged in size from 155,212 to 157,023 bp and had structure and gene content except for Acer palmatum (sect. Palmata), which had longer inverted repeats and an additional copy of the rps19 gene. Two genes, rps2 and rpl22, were found to be truncated at different positions and might be non-functional in several species. Most dispersed repeats, SSRs, and overall variation were detected in the non-coding sequences of the LSC and SSC regions. Fifteen loci, most of which have not been used before in the genus, were identified as the most variable and potentially useful as molecular markers for barcoding and genetic studies. Both ML and Bayesian analyses produced similar results irrespective of the partitioning strategy used. The plastome-based tree largely supported the topology inferred in previous studies using cp markers while providing resolution to the backbone relationships but was highly incongruous with a recently published nuclear tree presenting an opportunity for further research to investigate the causes of discordance, and particularly the role of hybridization in the diversification of the genus. Plastome sequences are valuable tools to resolve deep-level relationships within Acer. The variable loci and SSRs identified in this study will facilitate the development of markers for ecological and evolutionary studies in the genus. This study underscores the potential of plastid genome sequences to improve our understanding of the evolution of maples.

Identification of High Molecular Variation Loci in Complete Chloroplast Genomes of Mammillaria (Cactaceae, Caryophyllales).

In plants, partial DNA sequences of chloroplasts have been widely used in evolutionary studies. However, the Cactaceae family (1500-1800 species) lacks molecular markers that allow a phylogenetic resolution between species and genera. In order to identify sequences with high variation levels, we compared previously reported complete chloroplast genomes of seven species of Mammillaria. We identified repeated sequences (RSs) and two types of DNA variation: short sequence repeats (SSRs) and divergent homologous loci. The species with the highest number of RSs was M. solisioides (256), whereas M. pectinifera contained the highest amount of SSRs (84). In contrast, M. zephyranthoides contained the lowest number (35) of both RSs and SSRs. In addition, five of the SSRs were found in the seven species, but only three of them showed variation. A total of 180 homologous loci were identified among the seven species. Out of these, 20 loci showed a molecular variation of 5% to 31%, and 12 had a length within the range of 150 to 1000 bp. We conclude that the high levels of variation at the reported loci represent valuable knowledge that may help to resolve phylogenetic relationships and that may potentially be convenient as molecular markers for population genetics and phylogeographic studies.

Subtelomeric Transcription and its Regulation.

The subtelomeres, highly heterogeneous repeated sequences neighboring telomeres, are transcribed into coding and noncoding RNAs in a variety of organisms. Telomereproximal subtelomeric regions produce non-coding transcripts i.e., ARRET, αARRET, subTERRA, and TERRA, which function in telomere maintenance. The role and molecular mechanisms of the majority of subtelomeric transcripts remain unknown. This review depicts the current knowledge and puts into perspective the results obtained in different models from yeasts to humans.

Chromosome restructuring among hybridizing wild wheats.

The wheat group offers an outstanding system to address the interplay between hybridization, chromosomal evolution and biological diversification. Most diploid wild wheats originated following hybridization between the A-genome lineage and the B-genome lineage some 4 Myr ago, resulting in an admixed D-genome lineage that presented dramatic radiation accompanied by considerable changes in genome size and chromosomal rearrangements. Comparative profiling of low-copy genes, repeated sequences and transposable elements among those divergent species characterized by different karyotypes highlights high genome dynamics and sheds new light on the processes underlying chromosomal evolution in wild wheats. One of the hybrid clades presents upsizing of metacentric chromosomes going along with the proliferation of specific repeats (i.e. 'genomic obesity'), whereas other species show stable genome size associated with increasing chromosomal asymmetry. Genetic and ecological variation in those specialized species suggest that genome restructuring was coupled with adaptive processes to support the evolution of a majority of acrocentric chromosomes. This synthesis of current knowledge on genome restructuring across the diversity of wild wheats paves the way towards surveys based on latest sequencing technologies to characterize valuable resources and address the significance of chromosomal evolution in species with complex genomes.

Complete Sequence, Multichromosomal Architecture and Transcriptome Analysis of the Solanum tuberosum Mitochondrial Genome.

Mitochondrial genomes (mitogenomes) in higher plants can induce cytoplasmic male sterility and be somehow involved in nuclear-cytoplasmic interactions affecting plant growth and agronomic performance. They are larger and more complex than in other eukaryotes, due to their recombinogenic nature. For most plants, the mitochondrial DNA (mtDNA) can be represented as a single circular chromosome, the so-called master molecule, which includes repeated sequences that recombine frequently, generating sub-genomic molecules in various proportions. Based on the relevance of the potato crop worldwide, herewith we report the complete mtDNA sequence of two S. tuberosum cultivars, namely Cicero and Désirée, and a comprehensive study of its expression, based on high-coverage RNA sequencing data. We found that the potato mitogenome has a multi-partite architecture, divided in at least three independent molecules that according to our data should behave as autonomous chromosomes. Inter-cultivar variability was null, while comparative analyses with other species of the Solanaceae family allowed the investigation of the evolutionary history of their mitogenomes. The RNA-seq data revealed peculiarities in transcriptional and post-transcriptional processing of mRNAs. These included co-transcription of genes with open reading frames that are probably expressed, methylation of an rRNA at a position that should impact translation efficiency and extensive RNA editing, with a high proportion of partial editing implying frequent mis-targeting by the editing machinery.

Methods to Study Trinucleotide Repeat Instability Induced by DNA Damage and Repair.

Trinucleotide repeat (TNR) instability (expansion and deletion) is associated with more than 42 human neurodegenerative diseases and cancer and mediated by DNA replication, repair, recombination, and gene transcription. Somatic TNR instability is involved in the progression of TNR expansion diseases and can be modulated by DNA damage repair and gene transcription. Recent studies from our group and others have shown that DNA base damage and its repair play an active role in modulating TNR instability and are responsible for somatic age-dependent CAG repeat expansion in neurons of Huntington's disease mice induced by oxidative DNA damage. However, it remains to be elucidated how DNA damage, non-B form DNA structures, and DNA repair enzymes and cofactors can coordinate to regulate somatic TNR instability. Understanding the molecular mechanisms underlying DNA damage and repair-mediated somatic TNR instability is critically important for identification of new therapeutic targets for treatment and prevention of TNR-related diseases. Here we describe the methods to study the locations and distribution of DNA base lesions and their effects on TNR instability through DNA base excision repair in in vitro reconstituted human systems.

Isolation and Characterization of Interspersed Repeated Sequences in the Common Lizard, Zootoca vivipara, and Their Conservation in Squamata.

The common lizard (Zootoca vivipara) displays characteristic cytogenetic, reproductive, molecular, and biogeographic variability. This species comprises oviparous and viviparous populations with disjunct distribution and sex chromosome polymorphisms, from simple ZZ/ZW to complex Z1Z1Z2Z2/Z1Z2W systems with different morphologies of the W chromosome. In this study, we used the primers SINE A and SINE B and a newly designed primer pair to (1) obtain information on the presence and distribution of transposable elements (TEs) in 8 squamate families and (2) assess the chromosomal location of SINE Squam elements in Z. vivipara. PCR amplification with SINE A and SINE B produced single or multiple products in different Z. vivipara populations, subsequently used to design the SINE-Zv primers. Using the newly designed SINE-Zv primers, we identified 2 sequences of about 700 and 300 bp (SINE-Zv 700 and SINE-Zv 300) in all the investigated populations of Z. vivipara. Fluorescence in situ hybridizations showed a preferential localization of SINE-Zv sequences in the peritelomeric regions of almost all chromosomes, with the exception of the W. Both sequences contained a distinct segment of SINE Squam2. SINE-Zv 700 appeared to be restricted to Z. vivipara, while SINE-Zv 300 contained a partial Gypsy sequence that is highly conserved among Squamata and showed high identity values (72-93%) with several transcripts from different species. Using the same primers, we also highlighted the presence of another highly conserved Gypsy-like fragment in snakes which displayed significant similarity with the stomatin-like protein 2 of colubrids. Our results suggest that SINEs and the Gypsy-like elements are widely distributed among squamates and may have played an active role in their genomic evolution and differentiation.