Transposable Element Insertions Research Articles

The rate and spectrum of new mutations in mice inferred by long-read sequencing

All forms of genetic variation originate from new mutations, making it crucial to understand their rates and mechanisms. Here, we use long-read PacBio sequencing to investigate de novo mutations that accumulated in 12 inbred mouse lines derived from three commonly used inbred strains (C3H, C57BL/6, and FVB) maintained for 8-15 generations in a mutation accumulation (MA) experiment. We built chromosome-level genome assemblies based on the MA line founders' genomes, and then employed a combination of read and assembly-based methods to call the complete spectrum of new mutations. On average, there are ~45 mutations per haploid genome per generation, about half of which (54%) are insertions and deletions shorter than 50 bp (indels). The remainder are single nucleotide mutations (SNMs, 44%) and large structural mutations (SMs, 2%). We found that the degree of DNA repetitiveness is positively correlated with SNM and indel rates, and that a substantial fraction of SMs can be explained by homology-dependent mechanisms associated with repeat sequences. Most (90%) indels can be attributed to microsatellite contractions and expansions, and there is a marked bias towards 4 bp indels. Among the different types of SMs, tandem repeat mutations have the highest mutation rate, followed by insertions of transposable elements (TEs). We uncover a rich landscape of active TEs, and notable differences in their spectrum among MA lines and strains, and a high rate of gene retroposition. Our study offers novel insights into mammalian genome evolution, and highlights the importance of repetitive elements in shaping genomic diversity.

Loss-of-Function Mutations in the Fruit Softening Gene POLYGALACTURONASE1 Doubled Fruit Firmness in Strawberry

Abstract Wildtype fruit of cultivated strawberry (Fragaria × ananassa) are typically soft and highly perishable when fully ripe. The development of firm-fruited cultivars by phenotypic selection has greatly increased shelf life, decreased post-harvest perishability, and driven the expansion of strawberry production worldwide. Hypotheses for the firm-fruited phenotype include mutations affecting the expression of genes encoding polygalacturonases that soften fruit by degrading cell wall pectins. Here we show that loss-of-function mutations in the fruit softening gene POLYGALACTURONASE1 (FaPG1; PG1-6A1) double fruit firmness in strawberry. PG1-6A1 was one of three tandemly duplicated polygalacturonase genes found to be in linkage disequilibrium with a quantitative trait locus affecting fruit firmness on chromosome 6A. PG1-6A1 was strongly expressed in soft-fruited (wildtype) homozygotes and weakly expressed in firm-fruited (mutant) homozygotes. Genome-wide association, quantitative trait transcript, DNA sequence, and expression-QTL analyses identified genetic variants in linkage disequilibrium with PG1-6A1 that were positively correlated with fruit firmness and negatively correlated with PG1-6A1 expression. An Enhancer/Suppressor-mutator (En/Spm) transposable element insertion was discovered upstream of PG1-6A1 in mutant homozygotes that we hypothesize transcriptionally downegulates the expression of PG1-6A1. The PG1-6A1 locus was incompletely dominant and explained 26-76% of the genetic variance for fruit firmness among phenotypically diverse individuals. Additional loci are hypothesized to underlie the missing heritability. Highly accurate codominant genotyping assays were developed for modifying fruit firmness by marker-assisted selection of the En/Spm insertion and SNPs associated with the PG1-6A1 locus.

A MYB transcription factor underlying plant height in sorghum qHT7.1 and maize Brachytic 1 loci.

Manipulating plant height is an essential component of crop improvement. Plant height was generally reduced through breeding in wheat, rice, and sorghum to resist lodging and increase grain yield but kept high for bioenergy crops. Here, we positionally cloned a plant height quantitative trait locus (QTL) qHT7.1 as a MYB transcription factor controlling internode elongation, cell proliferation, and cell morphology in sorghum. A 740 bp transposable element insertion in the intronic region caused a partial mis-splicing event, generating a novel transcript that included an additional exon and a premature stop codon, leading to short plant height. The dominant allele had an overall higher expression than the recessive allele across development and internode position, while both alleles' expressions peaked at 46 days after planting and progressively decreased from the top to lower internodes. The orthologue of qHT7.1 was identified to underlie the brachytic1 (br1) locus in maize. A large insertion in exon 3 and a 160 bp insertion at the promoter region were identified in the br1 mutant, while an 18 bp promoter insertion was found to be associated with reduced plant height in a natural recessive allele. CRISPR/Cas9-induced gene knockout of br1 in two maize inbred lines showed significant plant height reduction. These findings revealed functional connections across natural, mutant, and edited alleles of this MYB transcription factor in sorghum and maize. This enriched our understanding of plant height regulation and enhanced our toolbox for fine-tuning plant height for crop improvement.

Polymorphic insertions of DcSto miniature inverted-repeat transposable elements reveal genetic diversity structure within the cultivated carrot.

Miniature inverted-repeat transposable elements (MITEs) are a potent source of polymorphisms in plant genomes. A genotyping system, named DcS-ILP, based on polymorphic insertions of Stowaway MITEs (DcStos) localized in introns and identified in the reference genome DH1, has been developed for carrot. Here, we report an extension of the DcS-ILP genotyping system by incorporation of non-reference insertions identified in resequenced genomes representing the eastern gene pool. We genotyped 52 carrot accessions representing the eastern and western carrot gene pools with 92 markers developed previously (western DcS-ILP panel) together with 84 newly developed markers (eastern DcS-ILP panel). Overall, the DcS-ILP markers revealed a highly structured genetic diversity separating the eastern and the western carrot accessions at K = 2 and differentiating Indian breeding lines from the eastern accessions at K = 3. The eastern DcS-ILP panel proved to be more robust with respect to the eastern carrot gene pool, while it provided little information on the western accessions, as many of the DcSto insertions present in the eastern gene pool were absent in the western gene pool. As the western carrot accessions represent improved cultivars, DcSto insertional polymorphisms allowed detection of a selection-driven bottleneck at the improvement stage. Selection in the course of the improvement stage generally operated on standing variation, as the subset of DcSto insertions present in the western carrot likely originated from transposition events preceding the separation of both gene pools. However, occasional frequency shifts in the opposite direction were also revealed, possibly indicating selection for favorable variants associated with DcSto insertions.

A single dominant GLOBOSA allele accounts for repeated origins of hose-in-hose flowers in Sinningia (Gesneriaceae).

Plants bearing double flowers have long been cultivated as ornamental plants. Hose-in-hose flowers, bearing 2-whorled corolla tubes in whorls 1 and 2, are uncommon but recur in Sinningia (Gesnerioideae, Gesneriaceae). In this study, we selected 15 hose-in-hose cultivars as materials to explore the underlying molecular and genetic mechanisms of this floral architecture. We found that they originated from different hybridization events within the Dircaea clade. Three B-class MADS-box genes were globally expressed in all floral whorls, but only GLOBOSA1 (GLO1) has accumulated a dominant mutation, i.e., the insertion of a hAT-like miniature inverted-repeat transposable element (MITE) into its promoter, that co-segregated with the hose-in-hose phenotype. In addition, all 15 hose-in-hose cultivars contained the same dominant GLO1 allele. Transient gene expression assays confirmed the role of this MITE insertion in up-regulating the promoter activity of GLO1 by providing several cis-regulatory elements. Genetic transformation in heterologous Chirita pumila (Didymocarpoideae, Gesneriaceae) verified that this dominant GLO1 allele is sufficient to confer the hose-in-hose phenotype. We further demonstrated that both the GLO1 allele and the hAT-like MITE descended from wild S. cardinalis with single flowers. This study highlights the significance of wide hybridization in frequent gains of the dominant GLO1 allele and thereafter repeated occurrence of hose-in-hose flowers in Sinningia.

Regulatory Changes in the Fatty Acid Elongase eloF Underlie the Evolution of Sex-specific Pheromone Profiles in Drosophila prolongata.

Pheromones play a key role in regulating sexual behavior throughout the animal kingdom. In Drosophila and other insects, many cuticular hydrocarbons (CHCs) are sexually dimorphic, and some are known to perform pheromonal functions. However, the genetic control of sex-specific CHC production is not understood outside of the model species D. melanogaster. A recent evolutionary change is found in D. prolongata, which, compared to its closest relatives, shows greatly increased sexual dimorphism in both CHCs and the chemosensory system responsible for their perception. A key transition involves a male-specific increase in the proportion of long-chain CHCs. Perfuming D. prolongata females with the male-biased CHCs reduces copulation success, suggesting that these compounds function as sex pheromones. The evolutionary change in CHC profiles correlates with a male-specific increase in the expression of multiple genes involved in CHC biosynthesis, including fatty acid elongases and reductases and other key enzymes. In particular, elongase F, which is responsible for producing female-specific pheromones in D. melanogaster, is strongly upregulated in D. prolongata males compared both to females and to males of the sibling species. Induced mutations in eloF reduce the amount of long-chain CHCs, resulting in a partial feminization of pheromone profiles in D. prolongata males while having minimal effect in females. Transgenic experiments show that sex-biased expression of eloF is caused in part by a putative transposable element insertion in its regulatory region. These results reveal one of the genetic mechanisms responsible for a recent evolutionary change in sexual communication.

Transposable Element (TE) insertion predictions from RNAseq inputs and TE impact on RNA splicing and gene expression in Drosophila brain transcriptomes

Recent studies have suggested that Transposable Elements (TEs) residing in introns frequently splice into and alter primary gene-coding transcripts. To re-examine the exonization frequency of TEs into protein-coding gene transcripts, we re-analyzed a Drosophila neuron circadian rhythm RNAseq dataset and a deep long RNA fly midbrain RNAseq dataset using our Transposon Insertion and Depletion Analyzer (TIDAL) program. Our TIDAL results were able to predict several TE insertions from RNAseq data that were consistent with previous published studies. However, we also uncovered many discrepancies in TE-exonization calls, such as reads that mainly support intron retention of the TE and little support for chimeric mRNA spliced to the TE. We then deployed rigorous genomic DNA-PCR (gDNA-PCR) and RT-PCR procedures on TE-mRNA fusion candidates to see how many of bioinformatics predictions could be validated. By testing a w1118 strain from which the deeper long RNAseq data was derived and comparing to an OreR strain, only 9 of 23 TIDAL candidates (< 40%) could be validated as a novel TE insertion by gDNA-PCR, indicating that deeper study is needed when using RNAseq data as inputs into current TE-insertion prediction programs. Of these validated calls, our RT-PCR results only supported TE-intron retention. Lastly, in the Dscam2 and Bx genes of the w1118 strain that contained intronic TEs, gene expression was 23 times higher than the OreR genes lacking the TEs. This study's validation approach indicates that chimeric TE-mRNAs are infrequent and cautions that more optimization is required in bioinformatics programs to call TE insertions using RNAseq datasets.

Mobilome impacts on physiology in the widely used non-toxic mutant Microcystis aeruginosa PCC 7806 ΔmcyB and toxic wildtype

The Microcystis mobilome is a well-known but understudied component of this bloom-forming cyanobacterium. Through genomic and transcriptomic comparisons, we found five families of transposases that altered the expression of genes in the well-studied toxigenic type-strain, Microcystis aeruginosa PCC 7086, and a non-toxigenic genetic mutant, Microcystis aeruginosa PCC 7806 ΔmcyB. Since its creation in 1997, the ΔmcyB strain has been used in comparative physiology studies against the wildtype strain by research labs throughout the world. Some differences in gene expression between what were thought to be otherwise genetically identical strains have appeared due to insertion events in both intra- and intergenic regions. In our ΔmcyB isolate, a sulfate transporter gene cluster (sbp-cysTWA) showed differential expression from the wildtype, which may have been caused by the insertion of a miniature inverted repeat transposable element (MITE) in the sulfate-binding protein gene (sbp). Differences in growth in sulfate-limited media also were also observed between the two isolates. This paper highlights how Microcystis strains continue to “evolve” in lab conditions and illustrates the importance of insertion sequences / transposable elements in shaping genomic and physiological differences between Microcystis strains thought otherwise identical. This study forces the necessity of knowing the complete genetic background of isolates in comparative physiological experiments, to facilitate the correct conclusions (and caveats) from experiments.

Impact of Chromosomal Fusion and Transposable Elements on the Genomic Evolution and Genetic Diversity of Ilex Species.

The genus Ilex belongs to the sole family and is the single genus within the order Aquifoliales, exhibiting significant phenotypic diversity. However, the genetic differences underlying these phenotypic variations have rarely been studied. In this study, collinearity analyses of three Ilex genomes, Ilex latifolia Thunb., Ilex polyneura (Hand.-Mazz.) S. Y. Hu, and Ilex asprella Champ. ex Benth., indicated a recent fusion event contributing to the reduction of chromosomes in I. asprella. Comparative genome analyses showed slight differences in gene annotation among the three species, implying a minimal disruption of genes following chromosomal fusion in I. asprella. Comprehensive annotation of transposable elements (TEs) revealed that TEs constitute a significant portion of the Ilex genomes, with LTR transposons being predominant. TEs exhibited an inverse relationship with gene density, potentially influencing gene regulation and chromosomal architecture. TE insertions were shown to affect the conformation and binding sites of key genes such as 7-deoxyloganetin glucosyltransferase and transmembrane kinase (TMK) genes, highlighting potential functional impacts. The structural variations caused by TE insertions suggest significant roles in the evolutionary dynamics, leading to either loss or gain of gene function. This study underscores the importance of TEs in shaping the genomic landscape and evolutionary trajectories of Ilex species.

Varying recombination landscapes between individuals are driven by polymorphic transposable elements.

Meiotic recombination is a prominent force shaping genome evolution, and understanding the causes for varying recombination landscapes within and between species has remained a central, though challenging, question. Recombination rates are widely observed to negatively associate with the abundance of transposable elements (TEs), selfish genetic elements that move between genomic locations. While such associations are usually interpreted as recombination influencing the efficacy of selection at removing TEs, accumulating findings suggest that TEs could instead be the cause rather than the consequence. To test this prediction, we formally investigated the influence of polymorphic, putatively active TEs on recombination rates. We developed and benchmarked a novel approach that uses PacBio long-read sequencing to efficiently, accurately, and cost-effectively identify crossovers (COs), a key recombination product, among large numbers of pooled recombinant individuals. By applying this approach to Drosophila strains with distinct TE insertion profiles, we found that polymorphic TEs, especially RNA-based TEs and TEs with local enrichment of repressive marks, reduce the occurrence of COs. Such an effect leads to different CO frequencies between homologous sequences with and without TEs, contributing to varying CO maps between individuals. The suppressive effect of TEs on CO is further supported by two orthogonal approaches-analyzing the distributions of COs in panels of recombinant inbred lines in relation to TE polymorphism and applying marker-assisted estimations of CO frequencies to isogenic strains with and without transgenically inserted TEs. Our investigations reveal how the constantly changing mobilome can actively modify recombination landscapes, shaping genome evolution within and between species.

Gene expression in soft-shell clam (Mya arenaria) transmissible cancer reveals survival mechanisms during host infection and seawater transfer.

Transmissible cancers are unique instances in which cancer cells escape their original host and spread through a population as a clonal lineage, documented in Tasmanian Devils, dogs, and ten bivalve species. For a cancer to repeatedly transmit to new hosts, these lineages must evade strong barriers to transmission, notably the metastasis-like physical transfer to a new host body and rejection by that host's immune system. We quantified gene expression in a transmissible cancer lineage that has spread through the soft-shell clam (Mya arenaria) population to investigate potential drivers of its success as a transmissible cancer lineage, observing extensive differential expression of genes and gene pathways. We observed upregulation of genes involved with genotoxic stress response, ribosome biogenesis and RNA processing, and downregulation of genes involved in tumor suppression, cell adhesion, and immune response. We also observe evidence that widespread genome instability affects the cancer transcriptome via gene fusions, copy number variation, and transposable element insertions. Finally, we incubated cancer cells in seawater, the presumed host-to-host transmission vector, and observed conserved responses to halt metabolism, avoid apoptosis and survive the low-nutrient environment. Interestingly, many of these responses are also present in healthy clam cells, suggesting that bivalve hemocytes may have inherent seawater survival responses that may partially explain why transmissible cancers are so common in bivalves. Overall, this study reveals multiple mechanisms this lineage may have evolved to successfully spread through the soft-shell clam population as a contagious cancer, utilizing pathways known to be conserved in human cancers as well as pathways unique to long-lived transmissible cancers.

Characterization of active transposable elements and their new insertions in tuber propagated greater yam (Dioscorea alata)

BackgroundGreater yam is a key staple crop grown in tropical and subtropical regions, while its asexual propagation mode had led to non-flowering mutations. How transposable elements contribute to its genetic variations is rarely analyzed. We used transcriptome and whole genome sequencing data to identify active transposable elements (TEs) and genetic variation caused by these active TEs. Our aim was to shed light on which TEs would lead to its intraspecies variation.ResultsAnnotation of de novo assembly transcripts indicated that 0.8 − 0.9% of transcripts were TE related, with LTR retrotransposons (LTR-RTs) accounted for 65% TE transcripts. A large portion of these transcripts were non-autonomous TEs, which had incomplete functional domains. The majority of mapped transcripts were distributed in genic deficient regions, with 9% of TEs overlapping with genic regions. Moreover, over 90% TE transcripts exhibited low expression levels and insufficient reads coverage to support full-length structure assembly. Subfamily analysis of Copia and Gypsy, the two LTR-RTs revealed that a small number of subfamilies contained a significantly larger number of members, which play a key role in generating TE transcript. Based on resequencing data, 15,002 L-RT insertion loci were detected for active LTR-RT members. The insertion loci of LTR-RTs were highly divergent among greater yam accessions.ConclusionsThis study showed the ongoing transcription and transpositions of TEs in greater yam, despite low transcription levels and incomplete proteins insufficient for autonomous transposition. While our research did not directly link these TEs to specific yam traits such as tuber yield and propagation mode, it lays a crucial foundation for further research on how these TE insertion polymorphisms (TIPs) might be related to variations in greater yam traits and its tuber propagation mode. Future research may explore the potential roles of TEs in trait variations, such as tuber yield and stress resistance, in greater yam.

Natural Transposable Element Insertions Contribute to Host Fitness in Model Yeasts.

Transposable elements (TEs) are ubiquitous in the eukaryote genomes, but their evolutionary and functional significance remains largely obscure and contentious. Here, we explore the evolution and functional impact of TEs in two model unicellular eukaryotes, the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae, which diverged around 330 to 420 million years ago. We analyze the distribution of LTR retrotransposons (LTR-RTs, the only TE order identified in both species) and their solo-LTR derivatives in 35 strains of S. pombe and 128 strains of S. cerevisiae. We find that natural LTR-RT and solo-LTR insertions exhibit high presence-absence polymorphism among individuals in both species. Population genetics analyses show that solo-LTR insertions experienced functional constraints similar to synonymous sites of host genes in both species, indicating a majority of solo-LTR insertions might have evolved in a neutral manner. When knocking out nine representative solo-LTR insertions separately in the S. pombe strain 972h- and 12 representative solo-LTR insertions separately in the S. cerevisiae strain S288C, we find that one solo-LTR insertion in S. pombe has a significant effect on the fitness and transcriptome of its host. Together, our findings indicate that a fraction of natural TE insertions likely shape their host transcriptomes and thereby contribute to their host fitness, with implications for understanding the functional significance of TEs in eukaryotes.

Genomic analyses of intricate interaction of TE-lncRNA overlapping genes with miRNAs in human diseases.

Transposable elements (TEs) are known to be inserted into genome to create transcript isoforms or to generate long non-coding RNA (lncRNA) sequences. The insertion of TEs generates a gene protein sequence within the genome, but also provides a microRNA (miRNA) regulatory region. To determine the effect of gene sequence changes caused by TE insertion on miRNA binding and to investigate the formation of an overlapping lncRNA that represses it. The distribution of overlapping regions between exons and TE regions with lncRNA was examined using the Bedtools. miRNAs that can bind to those overlapping regions were identified through the miRDB web program. For TE-lncRNA overlapping genes, bioinformatic analysis was conducted using DAVID web database. Differential expression analysis was conducted using data from the GEO dataset and TCGA. Most TEs were distributed more frequently in untranslated regions than open reading frames. There were 30 annotated TE-lncRNA overlapping genes with same strand that could bind to the same miRNA. As a result of identifying the association between these 30 genes and diseases, TGFB2, FCGR2A, DCTN5, and IFI6 were associated with breast cancer, and HMGCS1, FRMD4A, EDNRB, and SNCA were associated with Alzheimer's disease. Analysis of the GEO and TCGA data showed that the relevant expression of miR-891a and miR-28, which bind to the TE overlapping region of DCTN5 and HMGCS1, decreased. This study indicates that the interaction between TE-lncRNA overlapping genes and miRNAs can affect disease progression.

Tolerance thresholds underlie responses to DNA damage during germline development.

Selfish DNA modules like transposable elements (TEs) are particularly active in the germline, the lineage that passes genetic information across generations. New TE insertions can disrupt genes and impair the functionality and viability of germ cells. However, we found that in P-M hybrid dysgenesis in Drosophila, a sterility syndrome triggered by the P-element DNA transposon, germ cells harbor unexpectedly few new TE insertions despite accumulating DNA double-strand breaks (DSBs) and inducing cell cycle arrest. Using an engineered CRISPR-Cas9 system, we show that generating DSBs at silenced P-elements or other noncoding sequences is sufficient to induce germ cell loss independently of gene disruption. Indeed, we demonstrate that both developing and adult mitotic germ cells are sensitive to DSBs in a dosage-dependent manner. Following the mitotic-to-meiotic transition, however, germ cells become more tolerant to DSBs, completing oogenesis regardless of the accumulated genome damage. Our findings establish DNA damage tolerance thresholds as crucial safeguards of genome integrity during germline development.

Actively Expressed Intergenic Genes Generated by Transposable Element Insertions in Gossypium hirsutum Cotton.

The genomes and annotated genes of allotetraploid cotton Gossypium hirsutum have been extensively studied in recent years. However, the expression, regulation, and evolution of intergenic genes (ITGs) have not been completely deciphered. In this study, we identified a novel set of actively expressed ITGs in G. hirsutum cotton, through transcriptome profiling based on deep sequencing data, as well as chromatin immunoprecipitation, followed by sequencing (ChIP-seq) of histone modifications and how the ITGs evolved. Totals of 17,567 and 8249 ITGs were identified in G. hirsutum and Gossypium arboreum, respectively. The expression of ITGs in G. hirsutum was significantly higher than that in G. arboreum. Moreover, longer exons were observed in G. hirsutum ITGs. Notably, 42.3% of the ITGs from G. hirsutum were generated by the long terminal repeat (LTR) insertions, while their proportion in genic genes was 19.9%. The H3K27ac and H3K4me3 modification proportions and intensities of ITGs were equivalent to genic genes. The H3K4me1 modifications were lower in ITGs. Additionally, evolution analyses revealed that the ITGs from G. hirsutum were mainly produced around 6.6 and 1.6 million years ago (Mya), later than the pegged time for genic genes, which is 7.0 Mya. The characterization of ITGs helps to elucidate the evolution of cotton genomes and shed more light on their biological functions in the transcriptional regulation of eukaryotic genes, along with the roles of histone modifications in speciation and diversification.

Metabolomic and Transcriptomic Profiles in Diverse Brassica oleracea Crops Provide Insights into the Genetic Regulation of Glucosinolate Profiles.

Glucosinolates (GSLs) are plant secondary metabolites commonly found in the cruciferous vegetables of the Brassicaceae family, offering health benefits to humans and defense against pathogens and pests to plants. In this study, we investigated 23 GSL compounds' relative abundance in four tissues of five different Brassica oleracea morphotypes. Using the five corresponding high-quality B. oleracea genome assemblies, we identified 183 GSL-related genes and analyzed their expression with mRNA-Seq data. GSL abundance and composition varied strongly, among both tissues and morphotypes, accompanied by different gene expression patterns. Interestingly, broccoli exhibited a nonfunctional AOP2 gene due to a conserved 2OG-FeII_Oxy domain loss, explaining the unique accumulation of two health-promoting GSLs. Additionally, transposable element (TE) insertions were found to affect the gene structure of MAM3 genes. Our findings deepen the understanding of GSL variation and genetic regulation in B. oleracea morphotypes, providing valuable insights for breeding with tailored GSL profiles in these crops.

New insights into the phylogeny of Neogastropoda aided by draft genome sequencing of a volutid snail

AbstractNeogastropoda is a large order of predominantly marine gastropod molluscs, typically predatory or parasitic on other animals. It includes over 16,000 species representing a large post‐Cretaceous radiation, but the internal phylogenetic relationships of contained taxa are far from resolved, with inconsistent results from nuclear genes, mitogenomes, and morphology. One major issue in reconstructing the molecular phylogeny is the lack of high‐quality sequences for early‐diverging families and superfamilies such as Volutidae (Volutoidea). Here, we examine the superfamily‐level phylogenetic relationships in Neogastropoda, aided by newly sequenced draft genome and mitogenome of the volutid snail Fulgoraria chinoi from the deep sea off Japan. The genome of F. chinoi is relatively large at 1.54&amp;amp;#x02009;Gb but exhibits low genome heterozygosity; over 54% of this genome constitutes of repeat contents, and we find evidence for active insertion of transposable elements, particularly LINEs and SINEs. A total of 50,792 protein‐coding genes (PCGs) were predicted from the nuclear genome, and a circular mitogenome was also assembled and annotated. Our phylogenetic analyses using mitogenomes recovered each neogastropod superfamily as monophyletic, though also revealed inconsistent phylogenetic signals within superfamilies. Phylogenetic reconstructions using the PCGs resulted in a robust tree from different models and data matrices, recovering Volutoidea as the earliest diverging superfamily (among those for which comparable data is available) within a monophyletic Neogastropoda. Dated phylogenetic analysis revealed an early Cretaceous radiation of Neogastropoda, congruent with the fossil record. Our study provides a robust internal evolutionary framework for the speciose but genomically undersampled Neogastropoda, and expands the available genomic resources for this order. Genomic data for key missing lineages such as Mitroidea, Olivoidea, and Cancellariidae are much sought in the future for a full understanding of Neogastropoda evolution.

DNA Transposons Favor De Novo Transcript Emergence Through Enrichment of Transcription Factor Binding Motifs.

De novo genes emerge from noncoding regions of genomes via succession of mutations. Among others, such mutations activate transcription and create a new open reading frame (ORF). Although the mechanisms underlying ORF emergence are well documented, relatively little is known about the mechanisms enabling new transcription events. Yet, in many species a continuum between absent and very prominent transcription has been reported for essentially all regions of the genome. In this study, we searched for de novo transcripts by using newly assembled genomes and transcriptomes of seven inbred lines of Drosophila melanogaster, originating from six European and one African population. This setup allowed us to detect sample specific de novo transcripts, and compare them to their homologous nontranscribed regions in other samples, as well as genic and intergenic control sequences. We studied the association with transposable elements (TEs) and the enrichment of transcription factor motifs upstream of de novo emerged transcripts and compared them with regulatory elements. We found that de novo transcripts overlap with TEs more often than expected by chance. The emergence of new transcripts correlates with regions of high guanine-cytosine content and TE expression. Moreover, upstream regions of de novo transcripts are highly enriched with regulatory motifs. Such motifs are more enriched in new transcripts overlapping with TEs, particularly DNA TEs, and are more conserved upstream de novo transcripts than upstream their 'nontranscribed homologs'. Overall, our study demonstrates that TE insertion is important for transcript emergence, partly by introducing new regulatory motifs from DNA TE families.

Transposase-assisted target-site integration for efficient plant genome engineering

The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome-editing approaches to develop improved crops1,2. Often considered to be genome ‘parasites’, transposable elements (TEs) evolved to insert their DNA seamlessly into genomes3–5. Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type6–9. Here we developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the natural ability of the TE to precisely excise and insert into the genome. Inspired by CRISPR-associated transposases that target transposition in a programmable manner in bacteria10–12, we fused the rice Pong transposase protein to the Cas9 or Cas12a programmable nucleases. We demonstrated sequence-specific targeted insertion (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and a gene expression cassette into the genome of the model plant Arabidopsis. We then translated this system into soybean—a major global crop in need of targeted insertion technology. We have engineered a TE ‘parasite’ into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes.