Gene Duplication Research Articles

The three-dimensional genome drives the evolution of asymmetric gene duplicates via enhancer capture-divergence.

Previous evolutionary models of duplicate gene evolution have overlooked the pivotal role of genome architecture. Here, we show that proximity-based regulatory recruitment by distally duplicated genes is an efficient mechanism for modulating tissue-specific production of preexisting proteins. By leveraging genomic asymmetries, we performed a coexpression analysis on Drosophila melanogaster tissue data to show the generality of enhancer capture-divergence (ECD) as a significant evolutionary driver of asymmetric, distally duplicated genes. We use the recently evolved gene HP6/Umbrea as an example of the ECD process. By assaying genome-wide chromosomal conformations in multiple Drosophila species, we show that HP6/Umbrea was inserted near a preexisting, long-distance three-dimensional genomic interaction. We then use this data to identify a newly found enhancer (FLEE1), buried within the coding region of the highly conserved, essential gene MFS18, that likely neofunctionalized HP6/Umbrea. Last, we demonstrate ancestral transcriptional coregulation of HP6/Umbrea's future insertion site, illustrating how enhancer capture provides a highly evolvable, one-step solution to Ohno's dilemma.

Structural variant allelic heterogeneity in MECP2 duplication syndrome provides insight into clinical severity and variability of disease expression

BackgroundMECP2 Duplication Syndrome, also known as X-linked intellectual developmental disorder Lubs type (MRXSL; MIM: 300260), is a neurodevelopmental disorder caused by copy number gains spanning MECP2. Despite varying genomic rearrangement structures, including duplications and triplications, and a wide range of duplication sizes, no clear correlation exists between DNA rearrangement and clinical features. We had previously demonstrated that up to 38% of MRXSL families are characterized by complex genomic rearrangements (CGRs) of intermediate complexity (2 ≤ copy number variant breakpoints < 5), yet the impact of these genomic structures on regulation of gene expression and phenotypic manifestations have not been investigated.MethodsTo study the role of the genomic rearrangement structures on an individual’s clinical phenotypic variability, we employed a comprehensive genomics, transcriptomics, and deep phenotyping analysis approach on 137 individuals affected by MRXSL. Genomic structural information was correlated with transcriptomic and quantitative phenotypic analysis using Human Phenotype Ontology (HPO) semantic similarity scores.ResultsDuplication sizes in the cohort ranging from 64.6 kb to 16.5 Mb were classified into four categories comprising of tandem duplications (48%), terminal duplications (22%), inverted triplications (20%), and other CGRs (10%). Most of the terminal duplication structures consist of translocations (65%) followed by recombinant chromosomes (23%). Notably, 65% of de novo events occurred in the Terminal duplication group in contrast with 17% observed in Tandem duplications. RNA-seq data from lymphoblastoid cell lines indicated that the MECP2 transcript quantity in MECP2 triplications is statistically different from all duplications, but not between other classes of genomic structures. We also observed a significant (p < 0.05) correlation (Pearson R = 0.6, Spearman p = 0.63) between the log-transformed MECP2 RNA levels and MECP2 protein levels, demonstrating that genomic aberrations spanning MECP2 lead to altered MECP2 RNA and MECP2 protein levels. Genotype–phenotype analyses indicated a gradual worsening of phenotypic features, including overall survival, developmental levels, microcephaly, epilepsy, and genitourinary/eye abnormalities in the following order: Tandem duplications, Other complex duplications, Terminal duplications/Translocations, and Triplications encompassing MECP2.ConclusionIn aggregate, this combined analysis uncovers an interplay between MECP2 dosage, genomic rearrangement structure and phenotypic traits. Whereas the level of MECP2 is a key determinant of the phenotype, the DNA rearrangement structure can contribute to clinical severity and disease expression variability. Employing this type of analytical approach will advance our understanding of the impact of genomic rearrangements on genomic disorders and may help guide more targeted therapeutic approaches.

Genome-wide characterization of Solanum tuberosum UGT gene family and functional analysis of StUGT178 in salt tolerance

UDP-glycosyltransferases (UGTs) widely exist in plants and play essential roles in catalyzing the glycosylation reaction associated with metabolic processes. UGT gene family has been identified in many species to date. However, the comprehensive identification and systematic analysis have not been documented yet in the latest potato genome. In this study, a total of 295 UGT members (StUGT) were identified and found to be unevenly distributed on twelve chromosomes of potato. All StUGT genes were classified into 17 groups (A-P, R) and the UGT genes within the same groups have similar structural characterization. Tandem duplication was the major driving force for the StUGT gene expansion. The prediction of cis-acting elements showed that the development process, light, phytohormone, and abiotic stress-responsive elements generally existed in StUGT promoter regions. Analysis of spatial and temporal expression patterns demonstrated that StUGT genes were widely and differentially expressed in various tissues. Additionally, to investigate the salt stress-responsive genes, we analyzed the expression profiles of the StUGT genes under salt treatment. A total of 50 and 20 StUGT genes were continuously up- and down-regulated, respectively, implicating that these genes were involved in the regulation of salt tolerance. Among them, the StUGT178 gene, which was significantly induced by salt stress and contains salt-responsive element, was considered as one of the most relevant candidate genes. Transient transformation of the StUGT178 promoter in tobacco revealed that the transcriptional activation activity of the StUGT178 gene was strengthened under salt treatment. Furthermore, the heterologous expressions of the promoter and coding protein of the StUGT178 gene in Arabidopsis further demonstrated that the StUGT178 gene significantly responds to salt treatment, and enhanced salinity tolerance by regulating antioxidant enzyme activity and H2O2 accumulation. These results provide comprehensive information for a better understanding of the StUGT genes and offer a foundation for uncovering their function associated with salt stress in potato.

A Theory of the Evolutionary Role of Hereditary Tumors (carcino-evo-devo): the History and the Current State. Part 1. From General Principles to Hypothesis, and from Hypothesis to New Concept

The theory of the evolutionary role of hereditary tumors, or carcino-evo-devo theory, may be considered as the next step after A.N. Severtsov theory of phylembryogenesis, the theory of evo-devo, and Susumu Ohno theory of evolution by gene duplication. The carcino-evo-devo theory pretends to be a unifying biological theory, because it unifies within an integrated consideration three main types of biological development – individual, evolutionary and neoplastic development. The carcino-evo-devo theory explains a series of unexplained biological phenomena. In the first place, it explains the mechanisms of progressive evolution and biological complexity increase using the concept of relatively unstable transitory forms and autonomous uncontrolled processes. The theory of the evolutionary role of hereditary tumors has formulated several non-trivial predictions in various fields of biology, which have been confirmed in the lab of the author and in other laboratories. The consequences of the carcino-evo-devo theory have implications in medicine and biotechnology. The first part of the article describes the basic principles from which the main hypothesis followed, the progressive developments of the concept, and the first experimental data in support of non-trivial predictions obtained in the laboratory of the author in the period before 2014, when our monograph “Evolution by Tumor Neofunctionalization” (Kozlov, 2014) has been published.

Two highly selected mutations in the tandemly duplicated CYP6P4a and CYP6P4b genes drive pyrethroid resistance in Anopheles funestus in West Africa

BackgroundGaining a comprehensive understanding of the genetic mechanisms underlying insecticide resistance in malaria vectors is crucial for optimising the effectiveness of insecticide-based vector control methods and developing diagnostic tools for resistance management. Considering the heterogeneity of metabolic resistance in major malaria vectors, the implementation of tailored resistance management strategies is essential for successful vector control. Here, we provide evidence demonstrating that two highly selected mutations in CYP6P4a and CYP6P4b are driving pyrethroid insecticide resistance in the major malaria vector Anopheles funestus, in West Africa.ResultsContinent-wide polymorphism survey revealed escalated signatures of directional selection of both genes between 2014 and 2021. In vitro insecticide metabolism assays with recombinant enzymes from both genes showed that mutant alleles under selection exhibit higher metabolic efficiency than their wild-type counterparts. Using the GAL4-UAS expression system, transgenic Drosophila flies overexpressing mutant alleles exhibited increased resistance to pyrethroids. These findings were consistent with in silico predictions which highlighted changes in enzyme active site architecture that enhance the affinity of mutant alleles for type I and II pyrethroids. Furthermore, we designed two DNA-based assays for the detection of CYP6P4a-M220I and CYP6P4b-D284E mutations, showing their current confinement to West Africa. Genotype/phenotype correlation analyses revealed that these markers are strongly associated with resistance to types I and II pyrethroids and combine to drastically reduce killing effects of pyrethroid bed nets.ConclusionsOverall, this study demonstrated that CYP6P4a and CYP6P4b contribute to pyrethroid resistance in An. funestus and provided two additional insecticide resistance molecular diagnostic tools that would contribute to monitoring and better management of resistance.

Antisense oligonucleotides as a targeted therapeutic approach in model of acute myeloid leukemia.

The genetic and epigenetic alterations observed in acute myeloid leukemia (AML) contribute to its heterogeneity, influencing disease progression response to therapy, and patient outcomes. The use of antisense oligonucleotides (ASOs) technology allows for the design of oligonucleotide inhibitors based on gene sequence information alone, enabling precise targeting of key molecular pathways or specific genes implicated in AML. Midostaurin, a FLT3 specific inhibitor and ASOs targeting particular genes, exons, or mutations was conducted using AML models. This ASOs treatment was designed to bind to exon 7 of the MBNL1 (muscleblind-like) gene. Another target was the FLT3 gene, focusing on two aspects: (a) FLT3-ITD (internal tandem duplication), to inhibit the expression of this aberrant gene form, and (b) the FLT3 in general. Treated and untreated cells were analyzed using quantitative PCR (qPCR), dot blot, and Raman spectroscopy. This study contrasts midostaurin with ASOs that inhibit FLT3 protein production or its isoforms via mRNA degradation. A trend of increased FLT3 expression was observed in midostaurin-treated cells, while ASO-treated cells showed decreased expression, though these changes were not statistically significant. In AML, exon 7 of MBNL1 is involved in several cellular processes and in this study, exon 7 of MBNL1 was targeted for method optimization, with the highest block of the exon 7 gene variant observed 48h post-transfection. Midostaurin, a multitargeted kinase inhibitor, acts against the receptor tyrosine kinase FLT3, a critical molecule in AML pathogenesis. While midostaurin blocks FLT3 signaling pathways, it paradoxically increases FLT3 expression.

Distribution of Polyphosphate Kinase 2 Genes in Bacteria Underscores a Dynamic Evolutionary History.

Polyphosphate kinase 2 (PPK2) enzymes catalyze phosphoryl transfer from polyphosphate to nucleotides and are divided into three classes, each presumed to have different catalytic preferences. With relevance to biotechnology, medicine, and primitive biology, there is significant interest in understanding the evolutionary history of PPK2 enzymes and predicting their functional properties. We reasoned that the distribution and pairing preferences of PPK2 gene classes across the prokaryote tree of life may shed light on these questions. PPK2 was found to be a dynamic gene family, often present in only a subset of species within a clade, even when considering a single genus. Although all possible PPK2 pairs were observed, a ~2-fold enrichment for Class I enzymes in species with multiple PPK2 genes strongly shapes pairing preferences. PPK2 class preference in the absence of PPK1, which synthesizes rather than utilizes polyphosphate, indicates the potential for functional adaptation and/or promiscuity with respect to reaction directionality for all classes, a feature that has previously been associated only with Class I. Patterns of adjacent PPK2 genes revealed signatures of gene duplication, as adjacent genes overwhelmingly belonged to the same class, as well as the potential for an added layer of PPK2 dynamics: hetero-oligomerization of single-domain Class II enzymes to recapitulate the structure of two-domain Class II enzymes. Finally, an updated PPK2 tree constructed from domains instead of genes calls into question established narratives of PPK2 evolution, putting new limits on the extent to which nucleobase promiscuity can be invoked in the early evolution of this family.

Genome-Wide Identification and Evolutionary Analysis of Functional BBM-like Genes in Plant Species

Background/Objectives: BABY BOOM (BBM), a transcription factor from the APETALA2 (AP2) protein family, plays a critical role in somatic embryo induction and apomixis. BBM has now been widely applied to induce apomixis or enhance plant transformation and regeneration efficiency through overexpression or ectopic expression. However, the structural and functional evolutionary history of BBM genes in plants is still not well understood. Methods: The protein sequences of 10 selected plant species were used to locate the branch of BBM-Like by key domain identification and phylogenetic tree construction. The identified BBML genes were used for further conserved motif identification, gene structural analysis, miRNA binding site prediction, cis-acting element prediction, collinear analysis, protein–protein interaction network construction, three-dimensional structure modeling, molecular docking, and expression pattern analysis. Results: A total of 24 BBML proteins were identified from 10 representative plant species. Phylogenetic relationship analysis displayed that BBML proteins from eudicots and monocots were divided into two clusters, with monocots exhibiting a higher number of BBMLs. Gene duplication events indicated that whole genome/segmental duplication were the primary drivers of BBML genes’ evolution in the tested species, with purifying selection playing a key role during evolution processes. Comparative analysis of motif, domains, and gene structures revealed that most BBMLs were highly evolutionarily conserved. The expression patterns of BBML genes revealed significant tissue specificity, particularly in the root and embryo. We also constructed protein–protein interaction networks and molecular docking models to identify functional pathways and key amino acid residues of BBML proteins. The functions of BBMLs may differ between monocots and eudicots, as suggested by the functional enrichment of interacting proteins. Conclusions: Our research delved into the molecular mechanism, evolutionary relationships, functional differentiation, and expression patterns of BBML genes across plants, laying the groundwork for further investigations into the molecular properties and biological roles of BBMLs.

Leveraging Evolutionary Immunology in Interleukin-6 and Interleukin-17 Signaling for Lung Cancer Therapeutics.

Lung cancer is among the most common instances of cancer subtypes and is associated with high mortality rates. Due to the availability of fewer therapies and delayed clinical investigations, the number of cancer incidences is rising dramatically. This is possibly an effect of immune modulations and chemotherapeutic drugs that raises cancer resistance. Among the list, IL-6 and IL-17 are host-derived paradoxical effectors that attune immune responses in malignant lung cells. Their excessive release in the cytokine milieu stabilizes immunosuppressive phenotypes, resulting in cellular perturbations. During tumor development, the significance of these molecules is reflected in their potential to regulate oncogenesis by initiating a myriad of signaling events that influence tumor growth and the metastatic ability of benign cancer cells. Moreover, their transactivation contributes to antiapoptotic mechanisms and favors cancer cell survival via constitutive expression of immunoregulatory molecules. Co-evolution and gene duplication events could be the major drivers behind cytokine evolution, which have prompted generic changes and, hence, the additive effect. The evolutionary model and statistical analysis provide evidence about the cytokines ancestral relationships and site-specific conservation, which is more convincing as both cytokines share cysteine-knot-like structures important in maintaining structural integrity. Funneling through the findings could help find residues that serve a catalytic role in immune functioning. Designing peptides or subunit vaccine formulations against those conserved residues could aid in combating lung cancer pathogenesis.

Clinical and genetic analysis of a case with 2p23.2p22.1 duplication

To report on the phenotype of an adult patient with 2p23.2p22.1 duplication and explore its genotype-phenotype correlation. A pregnant woman who had presented at the Affiliated Drum Tower Hospital of Nanjing University Medical School on January 12, 2024 for a high risk signaled by NIPT was selected as the study subject. Amniotic fluid and peripheral blood samples were collected and subjected to chromosomal microarray analysis (CMA). The phenotype of the patient was observed, the medical history was taken, combined with the result of CMA assay, relevant database was searched for similar cases reported in the literature, and the correlation between genotype and phenotype was analyzed. The CMA result of the patient was arr[GRCh38]2p23.2p22.1(27961669_39280633)×3, which indicated a 11.31 Mb duplication. The woman was found to have short stature, learning disability, visual deficit, sleep disorder and other disorders. The duplication of PPP1CB and SOS1 genes within the 2p23.2p22.1 region can result in Noonan syndrome-like clinical manifestations such as short stature and reduced visual acuity. The duplication of the PPP1CB gene may be associated with the abnormal visual phenotype.

Genome-wide identification, gene cloning, subcellular location and expression analysis of the OPR gene family under salt stress in sweetpotato

BackgroundThe 12-oxo-phytodienoic acid reductase (OPR) enzyme is crucial for the synthesis of jasmonates (JAs), and is involved in the plant stress response. However, the OPR gene family in sweetpotato, an important horticultural crop, remains unidentified.ResultsIn this study, we employed bioinformatics techniques to identify nine IbOPR genes. Phylogenetic analysis revealed that these genes could be divided into Group I and Group II. Synteny analysis indicated that IbOPR evolution was driven by tandem duplication, whole-genome duplication (WGD), and segmental duplication events. The promoter sequences of IbOPRs were found to be associated with stress and hormonal responses. Additionally, we successfully cloned four IbOPRs from "Haida HD7791" and "Haida HD7798" using homologous cloning technology. These sequences were 1203 bp, 1200 bp, 1134 bp, and 1137 bp in length and encoded 400, 399, 377, and 378 amino acids, respectively. The protein sequence similarity between the salt-tolerant variety "Haida HD7791" and the salt-sensitive variety "Haida HD7798" was determined to be 96.75% for IbOPR2, 99.75% for IbOPR3, 92.06% for IbOPR6, and 98.68% for IbOPR7. Phylogenetic analysis categorized IbOPR2 and IbOPR3 proteins into Group II, while IbOPR6 and IbOPR7 proteins belonged to Group I. Subcellular localization experiments showed IbOPR2 protein present in the peroxisome, while IbOPR3, IbOPR6, and IbOPR7 proteins were found in the cytoplasm and nucleus. Salt stress induction experiments demonstrated that IbOPR2, IbOPR3, and IbOPR7 were significantly upregulated only in 'Haida HD7791' after 6 h. In contrast, IbOPR6 was induced in 'Haida HD7798' at 6 h but inhibited in 'Haida HD7791' at later time points (12, 24, 48, and 72 h), highlighting functional differences in salt stress responses.ConclusionsOur findings suggest that IbOPR2 may play a crucial role in sweetpotato's response to salt stress by participating in JAs synthesis. These results provide a foundation for future functional analyses of OPR genes in sweetpotato.

Lineage-specific amino acids define functional attributes of the protomer-protomer interfaces for the Rad51 and Dmc1 recombinases.

Most eukaryotes possess two Rad51/RecA family DNA recombinases that are thought to have arisen from an ancient gene duplication event: Rad51, which is expressed in both mitosis and meiosis; and Dmc1, which is only expressed in meiosis. To explore the evolutionary relationship between these recombinases, here, we present high-resolution CryoEM structures of S. cerevisiae Rad51 filaments and S. cerevisiae Dmc1 filaments bound to ssDNA, which reveal a pair of stacked interfacial aromatic amino acid residues that are nearly universally conserved in Rad51 but are absent from Dmc1. We use a combination of bioinformatics, genetic analysis of natural sequence variation, and deep mutational analysis to probe the functionally tolerated sequence space for these stacked aromatic residues. Our findings demonstrate that the functional landscape of the interfacial aromatic residues within the Rad51 filament is highly constrained. In contrast, the amino acids at the equivalent positions within the Dmc1 filament exhibit a broad functional landscape. This work helps highlight the distinct evolutionary trajectories of these two eukaryotic recombinases, which may have contributed to their functional and mechanistic divergence.

Experimental Evolution of Poxviruses.

Experimental evolution is the process of exposing virus populations to defined selective pressures in a laboratory setting to identify adaptive changes. Coupled with deep sequencing, this experimental approach allows for nucleotide-level resolution of poxvirus adaptive strategies over time. Here, we present a general method of poxvirus experimental evolution, Illumina-based deep sequencing, and bioinformatic analyses to identify structural changes (e.g., gene duplication) as well as local adaptive changes (e.g., small indels and single nucleotide polymorphisms).

Divergence in Regulatory Regions and Gene Duplications May Underlie Chronobiological Adaptation in Desert Tortoises.

Many cellular processes and organismal behaviours are time-dependent, and asynchrony of these phenomena can facilitate speciation through reinforcement mechanisms. The Mojave and Sonoran desert tortoises (Gopherus agassizii and G. morafkai respectively) reside in adjoining deserts with distinct seasonal rainfall patterns and they exhibit asynchronous winter brumation and reproductive behaviours. We used whole genome sequencing of 21 individuals from the two tortoise species and an outgroup to understand genes potentially underlying these characteristics. Genes within the most diverged 1% of the genome (FST ≥ 0.63) with putatively functional variation showed extensive divergence in regulatory elements, particularly promoter regions. Such genes related to UV nucleotide excision repair, mitonuclear and homeostasis functions. Genes mediating chronobiological (cell cycle, circadian and circannual) processes were also among the most highly diverged regions (e.g., XPA and ZFHX3). Putative promoter variants had significant enrichment of genes related to regulatory machinery (ARC-Mediator complex), suggesting that transcriptional cascades driven by regulatory divergence may underlie the behavioural differences between these species, leading to asynchrony-based prezygotic isolation. Further investigation revealed extensive expansion of respiratory and intestinal mucins (MUC5B and MUC5AC) within Gopherus, particularly G. morafkai. This expansion could be a xeric-adaptation to water retention and/or contribute to differential Mycoplasma agassizii infection rates between the two species, as mucins help clear inhaled dust and bacterial. Overall, results highlight the diverse array of genetic changes underlying divergence, adaptation and reinforcement during speciation.

Identification and expression analysis of phosphate transporter (PHT) genesin Brachypodium distachyon in response to phosphorus deficiency.

Phosphorus (P) is a macronutrient that plays a crucial role in critical plant functions. Phosphate transporters (PHTs) ensure the acquisition and translocation of Pi in the plant, thereby playing a key role in maintaining normal plant growth under Pi deficiency conditions. In Brachypodium distachyon, the grass model system, the function of individual PHT genes, remains largely unknown. Here, we identified the complete PHT gene family in B. distachyon, for the first time, and analyzed their expression profiles under Pi deficiency. Overall, 25 PHT genes in B. distachyon (BdPHTs) were identified, which were divided into four clades (PHT1-4). BdPHT genes were found to be unevenly distributed across the five chromosomes. Both segmental and tandem duplication events contributed to PHT gene expansion in B. distachyon which underwent a strong purifying selection. Moreover, exon-intron organization and motif composition were conserved within each PHT group consolidating the classification of the phylogenetic tree. Motif composition differs among the four PHT groups, indicating their functional divergence. Gene expression analysis using real-time quantitative PCR revealed that two BdPHT1 genes (BdPHT1.9 and BdPHT1.10) were upregulated in leaves, and seven (BdPHT1.9, BdPHT1.8, BdPHT1.7, BdPHT1.11, BdPHT1.12, BdPHT1.5, and BdPHT1.13) in roots under P deficiency suggesting their involvement in P uptake and translocation. Therefore, these results lay the foundation for future functional analyses in B. distachyon to improve P deficiency tolerance in B. distachyon and other cereals.

Divergence in MiRNA targeting of AchAco and its role in citrate accumulation in kiwifruit

BackgroundMicroRNA (miRNA) is a crucial post-transcriptional regulatory factor in plant growth and development. Duplicated genes often exhibit functional divergence due to competition for the identical miRNA binding sites. Kiwifruit (Actinidia spp.) is an economically significant horticultural crop renowned for its distinctive flavor, which is largely attributable to elevated citrate levels during fruit development. However, the mechanisms through which miRNA-targeted modules evolve following duplication events and regulate citrate biosynthesis, thereby influencing the unique flavor profile of kiwifruits, remain poorly understood.ResultsIn this study, we examined the expression patterns of miRNAs and interactions with their targets in kiwifruit fruit samples from various pulp tissues and developmental stages. Our analysis identified 46 miRNAs, comprising 44 known miRNAs and two novel/kiwifruit-specific miRNAs, which targeted a total of 1,474 genes. Correlation analysis revealed weak relationships between the expression levels of miRNAs and their target genes. Among these targets, 27 tandemly duplicated genes, and 782 whole genome duplication (WGD) genes exhibited a loss of miRNA binding sites in one of their duplicated copies. Furthermore, weighted gene co-expression network analysis demonstrated that most duplicated genes clustered into distinct gene modules. These findings suggest that the loss of miRNA targets following duplications contributes to expression divergence among gene duplicates, thereby facilitating stable gene expression within the miRNA-targeted network. For instance, the aconitate hydratase genes AchAco4 and AchAco6 were each targeted by different miRNAs, ach-miR-3774 and ach-miR-10371, respectively. Notably, these genes exhibited distinct expression patterns compared to their duplicated counterparts.ConclusionsThis study enhances our understanding of how the miRNA-AchAco module regulates citrate content and provides insights into the molecular network that influences the flavor profile of kiwifruit.Clinical trial numberNot applicable.

SurVIndel2: improving copy number variant calling from next-generation sequencing using hidden split reads

Deletions and tandem duplications (commonly called CNVs) represent the majority of structural variations in a human genome. They can be identified using short reads, but because they frequently occur in repetitive regions, existing methods fail to detect most of them. This is because CNVs in repetitive regions often do not produce the evidence needed by existing short reads-based callers (split reads, discordant pairs or read depth change). Here, we introduce a new CNV short reads-based caller named SurVIndel2. SurVindel2 builds on statistical techniques we previously developed, but also employs a novel type of evidence, hidden split reads, that can uncover many CNVs missed by existing algorithms. We use public benchmarks to show that SurVIndel2 outperforms other popular callers, both on human and non-human datasets. Then, we demonstrate the practical utility of the method by generating a catalogue of CNVs for the 1000 Genomes Project that contains hundreds of thousands of CNVs missing from the most recent public catalogue. We also show that SurVIndel2 is able to complement small indels predicted by Google DeepVariant, and the two software used in tandem produce a remarkably complete catalogue of variants in an individual. Finally, we characterise how the limitations of current sequencing technologies contribute significantly to the missing CNVs.

A pivotal switch in β-domain determines the substrate selectivity of TPS genes involved in Gardenia jasminoides floral scent synthesis.

Plants have evolved a diverse array of secondary metabolites to enhance their adaptability to environmental stresses, with volatile terpenoids being a notable example. Gardenia (Gardenia jasminoides), celebrated for its unique fragrance, is a key natural source of volatile terpenoids. Using our chromosome-level genome and transcriptome data for G. jasminoides, we previously identified six terpene synthases (TPS) involved in the production of its floral scent. Here, we functionally characterized these six key TPS enzymes, aligning their product profiles with volatile organic compound (VOC) analysis. Notably, we identified two highly similar terpene synthases, GjTPS1 and GjTPS2, which share high sequence homology but differ in substrate selectivity. By leveraging AI-based predictions and site-directed mutagenesis, we pinpointed a single amino acid in the β-domain that acts as a "switch," modulating substrate selectivity-an unusual finding, as this residue is outside the active site region. Comparative genomic analyses with Coffea canephora and other eudicots revealed that G. jasminoides TPS genes primarily expanded through dispersed (DSD) and tandem duplications (TD). Our study is the first to reveal a "switch" in a previously deemed "non-functional" region, regulating substrate selectivity in TPS genes. These insights could facilitate breeding elite gardenia varieties and support the rational engineering of terpenoid synthases.

Hydroxylase-oriented mining and functional characterization of camptothecin 10-hydroxylase from Camptotheca acuminata Decne

The regiospecific C-10 hydroxylation of camptothecin (CPT) is challenging from a chemical perspective. This step bridges the natural-sourced CPT to an important industrial material, 10-hydroxycamptothecin (10HCPT). 10HCPT is naturally generated in a medicinal plant, Camptotheca acuminata Decne. Functionally active hydroxylase responsible for regiospecific C-10 hydroxylation of CPT is undoubtedly existed in this plant. Therefore, the multi-omics database of C. acuminata was utilized to perform hydroxylase-oriented mining and screening. Three CYP81 genes were identified and two of them, including CYP81BQ18 and CYP81B256 catalyzed hydroxylation of CPT at the C-10 position. CYP81B256 and CYP81BN24 displayed quercetin C-5’ hydroxylase activity. CYP81B256 exhibited better catalytic performance for CPT than CYP81BQ18 and CPT10H in vitro. Furthermore, CYP81BQ18 and CYP81B256 displayed CPT 10-hydroxylase activity in tobacco. Functional verification in planta suggested that the newly observed CYP81BQ18 and CYP81B256 were involved in 10HCPT biosynthesis in C. acuminata. Subcellular localization analysis revealed that these CYP81 genes were located on the endoplasmic reticulum. Evolutionary analysis indicated that CYP81B256 and CYP81BN24 probably evolved from a same ancestral gene via gene duplication, while CYP81BQ18 evolved independently and acted as a specie-specific hydroxylase. CYP81B256 acted as an evolutionary intermediate gene bridging the alkaloid and flavonoid metabolism in C. acuminata. This research provides valuable insights into the function and evolutionary origin of CYP81s in the regiospecific hydroxylation step for 10HCPT biosynthesis.

BBL enzymes exhibit enantiospecific preferences in the biosynthesis of pyridine alkaloids in Nicotiana tabacum L.

Plant species can accumulate secondary metabolites in optically pure form or, occasionally, as enantiomeric mixtures. Interestingly, enantiomers of the same molecule can confer different biological activities. In tobacco (Nicotiana tabacum L.), the pyridine alkaloids nicotine, nornicotine, anatabine, and anabasine naturally exist as scalemic mixtures of (R)- or (S)-enantiomers, with the (S)-isoforms predominating. The mechanisms by which tobacco alkaloid enantiomers accumulate remain largely unknown. Experiments were carried out involving tobacco genotypes possessing induced deleterious mutations in three genes coding for nicotine demethylase (NND) enzymes and three genes coding for Berberine Bridge Like (BBL) enzymes that act near the end of the nicotine, anatabine, and anabasine biosynthetic pathways. Data indicate that (R)-nicotine is naturally produced at appreciable levels but is observed in only small amounts due to preferential demethylation by NND enzymes. Data further suggest that BBL-a and BBL-b are preferentially involved in the biosynthesis of (S)-alkaloid enantiomers, while BBL-c is preferentially involved in the biosynthesis of (R)-enantiomers. Gene duplication followed by genetic divergence thus played a role in the evolution of scalemic alkaloid accumulation in tobacco. Through a combination of mutation breeding and transgene overexpression, tobacco genotypes were generated in which the predominant alkaloid enantiomers were reversed from the (S)- to the (R)-isoforms. These results shed light on the genetic control of alkaloid accumulation in N. tabacum and on mechanisms of scalemic mixture formation of secondary metabolites in plants.