Illumina Sequencing Data Research Articles

The male and female genomes of golden pompano (Trachinotus ovatus) provide insights into the sex chromosome evolution and rapid growth

IntroductionGolden pompano (Trachinotus ovatus) is economically significant important for offshore cage aquaculture in China and Southeast Asian countries. Lack of high-quality genomic data and accurate gene annotations greatly restricts its genetic breeding progress. ObjectivesTo decode the mechanisms of sex determination and rapid growth in golden pompano and facilitate the sex- and growth-aimed genetic breeding. MethodsGenome assemblies of male and female golden pompano were generated using Illumina, PacBio, BioNano, genetic maps and Hi-C sequencing data. Genomic comparisons, whole genome re-sequencing of 202 F1 individuals, QTL mapping and gonadal transcriptomes were used to analyze the sex determining region, sex chromosome evolution, SNP loci, and growth candidate genes. Zebrafish model was used to investigate the functions of growth candidate gene. ResultsFemale (644.45 Mb) and male (652.12 Mb) genomes of golden pompano were assembled and annotated at the chromosome level. Both genomes are highly conserved and no new or highly differentiated sex chromosomes occur. A 3.5 Mb sex determining region on LG15 was identified, where Hsd17b1, Micall2 and Lmx1a were putative candidates for sex determination. Three SNP loci significantly linked to growth were pinpointed, and a growth-linked gene gpsstr1 was identified by locus BSNP1369 (G → C, 17489695, Chr23). Loss of sstr1a (homologue of gpsstr1) in zebrafish caused growth retardation. ConclusionThis study provides insights into sex chromosome evolution, sex determination and rapid growth of golden pompano.

Single-Molecule Real-Time Sequencing for Identifying Sexual-Dimorphism-Related Transcriptomes and Genes in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis)

The Chinese soft-shelled turtle (Pelodiscus sinensis), an economically important aquatic species in China, displays considerable sexual dimorphism: the male P. sinensis is larger and, thus, more popular in the market. In this study, we obtained the full-length (FL) transcriptome data of P. sinensis by using Pacific Biosciences (PacBio)’s isoform sequencing and analyzed the transcriptome structure. In total, 1,536,849 high-quality FL transcripts were obtained through single-molecule real-time (SMRT) sequencing, which were then corrected using Illumina sequencing data. Next, 89,666 nonredundant FL transcripts were generated after mapping to the reference genome of P. sinensis; 291 fusion genes and 17,366 novel isoforms were successfully annotated using data from the nonredundant protein sequence database (NR), eukaryotic orthology groups (KOG), the Gene Ontology (GO) project, and the KEGG Orthology (KO) database. Additionally, 19,324 alternative polyadenylation sites, 101,625 alternative splicing events, 12,392 long noncoding RNAs, and 5916 transcription factors were identified. Smad4, Wif1, and 17-β-hsd were identified as female-biased genes, while Nkd2 and Prp18 held a higher expression level in males than females. In summary, we found differences between male and female P. sinensis individuals in AS, lncRNA, genes, and transcripts, which relate to the Wnt pathway, oocyte meiosis, and the TGF-β pathway. Female-biased genes such as Smad4, Wif1, and 17-β-hsd and male-biased genes such as Nkd2 and Prp18 played important roles in the sex determination of P. sinensis. FL transcripts are a precious resource for characterizing the transcriptome of P. sinensis, laying the foundation for further research on the sex-determination mechanisms of P. sinensis.

Chromosome-level genome assembly of Przevalski’s partridge (Alectoris magna)

Przevalski’s partridge (Alectoris magna) is one of the birds in the genus Alectoris endemic to China. The distribution of A. magna was narrow, and it was only found in parts of the Qinghai, Gansu, and Ningxia provinces. A. magna was considered a monotypic species until it was distinguished into two subspecies. However, external morphological characteristics, rather than genetic differences or evolutionary relationships, are now commonly used as evidence of subspecies differentiation. In this study, a chromosome-level reference genome of A. magna has been constructed by combining Illumina, PacBio and Hi-C sequencing data. The 1135.01 Mb A. magna genome was ultimately assembled. The genome showed 96.9% completeness (BUSCO), with a contig N50 length of 23.34 Mb. The contigs were clustered and oriented on 20 chromosomes, covering approximately 99.96% of the genome assembly. Additionally, altogether 19,103 protein-coding genes were predicted, of which 95.10% were functionally annotated. This high-quality genome assembly could serve as a valuable genomic resource for future research on the functional genomics, genetic protection, and interspecific hybridization of A. magna.

Leaf, root, and soil microbiomes of an invasive plant, Ardisia crenata, differ between its native and exotic ranges

IntroductionEcological underpinnings of the invasion success of exotic plants may be found in their interactions with microbes, either through the enemy release hypothesis and the enhanced mutualism hypothesis. Whereas recent high-throughput sequencing techniques have significantly expanded our understanding of plant-associated microbiomes and their functional guilds, few studies to date have used these techniques to compare the microbiome associated with invasive plants between their native and exotic ranges.MethodsWe extracted fungal and bacterial DNA within leaf endosphere, root endosphere and soil of an invasive plant, Ardisia crenata, sampled from their native range Japan and exotic range Florida, USA. Using Illumina sequencing data, we compared microbial community compositions and diversity between the native and exotic ranges, and tested whether abundance of pathogenic or mutualistic microbes differ between the native or exotic ranges in accordance to the enemy release hypothesis or the enhanced mutualism hypothesis.ResultsFungal and bacterial community compositions differed among leaves, roots and soil, and between the native and exotic ranges. Despite a higher microbial diversity in the soil in the exotic range than in the native range, the microbial diversity within leaf and root was lower in the exotic range compared to the native range. In addition, leaves in the native range harbored a greater number of plant pathogenic fungi compared to those in the exotic range.DiscussionThese patterns suggest plant controls over what microbes become associated with leaves and roots. The higher abundance of leaf pathogenic fungi, including the pathogen which is known to cause specific disease in A. crenata in the exotic range than in the native range, support the enemy release hypothesis and highlighted potential importance of examining microbial communities both above- and below-ground.

Complete genomic sequence of Vibrio fluvialis strain IDH5335 isolated from a patient with diarrhea in Kolkata, India.

We isolated a Vibrio fluvialis strain (IDH5335) from a stool sample collected from a patient with diarrhea. In this announcement, we report the complete genomic sequence of this organism, which was obtained by combining Illumina and Oxford Nanopore sequencing data.

Characterization of Pseudomonas sp. En3, an Endophytic Bacterium from Poplar Leaf Endosphere with Plant Growth-Promoting Properties

Growth-promoting endophytic bacteria possess substantial potential for sustainable agriculture. Here, we isolated an endophytic bacterium, Pseudomonas sp. En3, from the leaf endosphere of Populus tomentosa and demonstrated its significant growth-promoting effects on both poplar and tomato seedlings. The phosphorus solubilization and nitrogen fixation abilities of strain En3 were confirmed via growth experiments on NBRIP and Ashby media, respectively. Salkowski staining and HPLC-MS/MS confirmed that En3 generated indole-3-acetic acid (IAA). The infiltration of En3 into leaf tissues of multiple plants did not induce discernible disease symptoms, and a successful replication of En3 was observed in both poplar and tobacco leaves. Combining Illumina and Nanopore sequencing data, we elucidated that En3 possesses a circular chromosome of 5.35 Mb, exhibiting an average G + C content of 60.45%. The multi-locus sequence analysis (MLSA) and genome average nucleotide identity (ANI) supported that En3 is a novel species of Pseudomonas and constitutes a distinct phylogenetic branch with P. rhizosphaerae and P. coleopterorum. En3 genome annotation analysis revealed the presence of genes associated with nitrogen fixation, phosphate solubilization, sulfur metabolism, siderophore biosynthesis, synthesis of IAA, and ethylene and salicylic acid modulation. The findings suggest that Pseudomonas sp. En3 exhibits significant potential as a biofertilizer for crop and tree cultivation.

Construction of heat stress regulation networks based on Illumina and SMRT sequencing data in potato.

Potato (Solanum tuberosum L.) is one of the most important tuber food crops in the world; however, the cultivated potatoes are susceptible to high temperature, by which potato production is adversely affected. Understanding the coping mechanism of potato to heat stress is essential to secure yield and expand adaptability under environmental conditions with rising temperature. However, the lack of heat-related information has significantly limited the identification and application of core genes. To gain deeper insights into heat tolerance genes, next-generation sequencing and single-molecule real-time sequencing were used to learn the transcriptional response of potato to heat stress and 13,159 differentially expressed genes (DEGs) were identified in this study. All DEGs were grouped into 12 clusters using the K-means clustering algorithm. Gene Ontology enrichment analysis revealed that they were involved in temperature signaling, phytohormone, and protein modification. Among them, there were 950 differentially expressed transcription factors (DETFs). According to the network analysis of DETFs at the sixth hour under heat stress, we found some genes that were previously reported to be associated with photoperiodic tuberization, StCO (CONSTANS), tuber formation, StBEL11 (BEL1-LIKE 11), and earliness in potato, StCDF1 (CYCLING DOF FACTOR 1) responding to temperature. Furthermore, we verified the relative expression levels using quantitative real-time polymerase chain reaction, and the results were consistent with the inferences from transcriptomes. In addition, there were 22,125 alternative splicing events and 2,048 long non-coding RNAs. The database and network established in this study will extend our understanding of potato response to heat stress. It ultimately provided valuable resources for molecular analysis of heat stress response in potato and cultivation of potato varieties with heat tolerance.

Complete Mitochondrial Genome Assembly of an Upland Wild Rice Species, Oryza granulata and Comparative Mitochondrial Genomic Analyses of the Genus Oryza.

Wild upland rice species, including Oryza granulata, possess unique characteristics that distinguish them from other Oryza species. For instance, O. granulata characteristically has a GG genome and is accordingly classified as a basal lineage of the genus Oryza. Here, we deployed a versatile hybrid approach by integrating Illumina and PacBio sequencing data to generate a high-quality mitochondrial genome (mitogenome) assembly for O. granulata. The mitogenome of O. granulata was 509,311 base pairs (bp) with sixty-seven genes comprising two circular chromosomes, five ribosomal RNA (rRNA) coding genes, twenty-five transfer RNA (tRNA) coding genes, and thirty-seven genes coding for proteins. We identified a total of 378 simple sequence repeats (SSRs). The genome also contained 643 pairs of dispersed repeats comprising 340 palindromic and 303 forward. In the O. granulata mitogenome, the length of 57 homologous fragments in the chloroplast genome occupied 5.96% of the mitogenome length. Collinearity analysis of three Oryza mitogenomes revealed high structural variability and frequent rearrangements. Phylogenetic analysis showed that, compared to other related genera, O. granulata had the closest genetic relationship with mitogenomes reported for all members of Oryza, and occupies a position at the base of the Oryza phylogeny. Comparative analysis of complete mitochondrial genome assemblies for Oryza species revealed high levels of mitogenomic diversity, providing a foundation for future conservation and utilization of wild rice biodiversity.

High-quality full genome assembly of historic Xylella fastidiosa strains from ICMP collection using a hybrid sequencing approach.

High-quality complete genomes of five Xylella fastidiosa strains were assembled by combining Nanopore and Illumina sequencing data. Among these, International Collection of Micro-organisms from Plants (ICMP) 8731, ICMP 8742 and ICMP 8745 belong to subspecies fastidiosa while ICMP 8739 and ICMP 8740 were determined as subspecies multiplex. The strains were further classified into sequence types.

Multiple configurations of the plastid and mitochondrial genomes of Caragana spinosa.

In this study, we assembled the complete plastome and mitogenome of Caragana spinosa and explored the multiple configurations of the organelle genomes. Caragana spinosa belongs to the Papilionoidea subfamily and has significant pharmaceutical value. To explore the possible interaction between the organelle genomes, we assembled and analyzed the plastome and mitogenome of C. spinosa using the Illumina and Nanopore DNA sequencing data. The plastome of C. spinosa was 129,995bp belonging to the inverted repeat lacking clade (IRLC), which contained 77 protein-coding genes, 29 tRNA genes, and four rRNA genes. The mitogenome was 378,373bp long and encoded 54 unique genes, including 33 protein-coding, three ribosomal RNA (rRNA), and 18 transfer RNA (tRNA) genes. In addition to the single circular conformation, alternative conformations mediated by one and four repetitive sequences in the plastome and mitogenome were identified and validated, respectively. The inverted repeat (PDR12, the 12th dispersed repeat sequence in C. spinosa plastome) of plastome mediating recombinant was conserved in the genus Caragana. Furthermore, we identified 14 homologous fragments by comparing the sequences of mitogenome and plastome, including eight complete tRNA genes. A phylogenetic analysis of protein-coding genes extracted from the plastid and mitochondrial genomes revealed congruent topologies. Analyses of sequence divergence found one intergenic region, trnN-GUU-ycf1, exhibiting a high degree of variation, which can be used to develop novel molecular markers to distinguish the nine Caragana species accurately. This plastome and mitogenome of C. spinosa could provide critical information for the molecular breeding of C. spinosa and be used as a reference genome for other species of Caragana. In this study, we assembled the complete plastome and mitogenome of Caragana spinosa and explored the multiple configurations of the organelle genomes.

Identification of Bona Fide RNA Editing Sites: History, Challenges, and Opportunities.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by the adenosine deaminase acting on the RNA (ADAR) family of enzymes of which there are three members (ADAR1, ADAR2, and ADAR3), is a major gene regulatory mechanism that diversifies the transcriptome. It is widespread in many metazoans, including humans. As inosine is interpreted by cellular machineries mainly as guanosine, A-to-I editing effectively gives A-to-G nucleotide changes. Depending on its location, an editing event can generate new protein isoforms or influence other RNA processing pathways. Researchers have found that ADAR-mediated editing performs diverse functions. For example, it enables living organisms such as cephalopods to adapt rapidly to fluctuating environmental conditions such as water temperature. In development, the loss of ADAR1 is embryonically lethal partly because endogenous double-stranded RNAs (dsRNAs) are no longer marked by inosines, which signal "self", and thus cause the melanoma differentiation-associated protein 5 (MDA5) sensor to trigger a deleterious interferon response. Hence, ADAR1 plays a key role in preventing aberrant activation of the innate immune system. Furthermore, ADAR enzymes have been implicated in myriad human diseases. Intriguingly, some cancer cells are known to exploit ADAR1 activity to dodge immune responses. However, the exact identities of immunogenic RNAs in different biological contexts have remained elusive. Consequently, there is tremendous interest in identifying inosine-containing RNAs in the cell.The identification of A-to-I RNA editing sites is dependent on the sequencing of nucleic acids. Technological and algorithmic advancements over the past decades have revolutionized the way editing events are detected. At the beginning, the discovery of editing sites relies on Sanger sequencing, a first-generation technology. Both RNA, which is reverse transcribed into complementary DNA (cDNA), and genomic DNA (gDNA) from the same source are analyzed. After sequence alignment, one would require an adenosine to be present in the genome but a guanosine to be detected in the RNA sample for a position to be declared as an editing site. However, an issue with Sanger sequencing is its low throughput. Subsequently, Illumina sequencing, a second-generation technology, was invented. By permitting the simultaneous interrogation of millions of molecules, it enables many editing sites to be identified rapidly. However, a key challenge is that the Illumina platform produces short sequencing reads that can be difficult to map accurately. To tackle the challenge, we and others developed computational workflows with a series of filters to discard sites that are likely to be false positives. When Illumina sequencing data sets are properly analyzed, A-to-G variants should emerge as the most dominant mismatch type. Moreover, the quantitative nature of the data allows us to build a comprehensive atlas of editing-level measurements across different biological contexts, providing deep insights into the spatiotemporal dynamics of RNA editing. However, difficulties remain in identifying true A-to-I editing sites in short protein-coding exons or in organisms and diseases where DNA mutations and genomic polymorphisms are prevalent and mostly unknown. Nanopore sequencing, a third-generation technology, promises to address the difficulties, as it allows native RNAs to be sequenced without conversion to cDNA, preserving base modifications that can be directly detected through machine learning. We recently demonstrated that nanopore sequencing could be used to identify A-to-I editing sites in native RNA directly. Although further work is needed to enhance the detection accuracy in single molecules from fewer cells, the nanopore technology holds the potential to revolutionize epitranscriptomic studies.

High-quality wild barley genome assemblies and annotation with Nanopore long reads and Hi-C sequencing data

Wild barley, from “Evolution Canyon (EC)” in Mount Carmel, Israel, are ideal models for cereal chromosome evolution studies. Here, the wild barley EC_S1 is from the south slope with higher daily temperatures and drought, while EC_N1 is from the north slope with a cooler climate and higher relative humidity, which results in a differentiated selection due to contrasting environments. We assembled a 5.03 Gb genome with contig N50 of 3.53 Mb for wild barley EC_S1 and a 5.05 Gb genome with contig N50 of 3.45 Mb for EC_N1 using 145 Gb and 160.0 Gb Illumina sequencing data, 295.6 Gb and 285.35 Gb Nanopore sequencing data and 555.1 Gb and 514.5 Gb Hi-C sequencing data, respectively. BUSCOs and CEGMA evaluation suggested highly complete assemblies. Using full-length transcriptome data, we predicted 39,179 and 38,373 high-confidence genes in EC_S1 and EC_N1, in which 93.6% and 95.2% were functionally annotated, respectively. We annotated repetitive elements and non-coding RNAs. These two wild barley genome assemblies will provide a rich gene pool for domesticated barley.

Chromosome genome assembly and annotation of the spiny red gurnard (Chelidonichthys spinosus)

Chelidonichthys spinosus, a secondary economic fish, is increasingly being exploited and valued in China. However, overfishing has led to it being recognized as one of the most depleted marine species in China. In this study, we generated a chromosome-level genome of C. spinosus using PacBio, Illumina, and Hi-C sequencing data. Ultimately, we assembled a 624.7 Mb genome of C. spinosus, with a contig N50 of 13.77 Mb and scaffold N50 of 28.11 Mb. We further anchored and oriented the assembled sequences onto 24 pseudo-chromosomes using Hi-C techniques. In total, 25,358 protein-coding genes were predicted, of which 24,072 (94.93%) genes were functionally annotated. The dot plot reveals a prominent co-linearity between C. spinosus and Cyclopterus lumpus, indicating a remarkably close phylogenetic relationship between these two species. The assembled genome sequences provide valuable information for elucidating the genetic adaptation and potential molecular basis of C. spinosus. They also have the potential to provide insight into the evolutionary investigation of teleost fish and vertebrates.

Haplotype-resolved chromosomal-level assembly of wasabi (Eutrema japonicum) genome

In Japan, wasabi (Eutrema japonicum) is an important traditional condiment, and is recognized as an endemic species. In the present study, we generated a chromosome-level and haplotype-resolved reference genome for E. japonicum using PacBio CLR (continuous long reads), Illumina, and Hi-C sequencing data. The genome consists of 28 chromosomes that contain 1,512.1 Mb of sequence data, with a scaffold N50 length of 55.67 Mb. We also reported the subgenome and haplotype assignment of the 28 chromosomes by read-mapping and phylogenic analysis. Three validation methods (Benchmarking Universal Single-Copy Orthologs, Merqury, and Inspector) indicated that our obtained genome sequences were a high-quality and high-completeness genome assembly. Comparison of genome assemblies from previously published genomes showed that our obtained genome was of higher quality. Therefore, our genome will serve as a valuable genetic resource for both chemical ecology and evolution research of the genera Eutrema and Brassicaceae, as well as for wasabi breeding.

Genome Sequence Resource of a Colletotrichum graminicola Field Strain from China.

The maize anthracnose stalk rot and leaf blight diseases caused by the fungal pathogen Colletotrichum graminicola is emerging as an important threat to corn production worldwide. In this work, we provide an improved genome assembly of a C. graminicola strain (TZ-3) by using the PacBio Sequel II and Illumina high-throughput sequencing technologies. The genome of TZ-3 consisting of 36 contigs with a length of 59.3 Mb. By correcting and evaluating with the Illumina sequencing data and BUSCO, this genome showed a high assembly quality and integrity. Gene annotation of this genome predicted 11,911 protein-coding genes, among which 983 secreted protein-coding genes and 332 effector genes were predicted. Comparing with previous genomes of C. graminicola strains, TZ-3 genome is more superior in nearly all parameters. The genome assembly and annotation will enhance our knowledge of the pathogen's genetic makeup and molecular mechanisms underlying its pathogenicity, as well as provide valuable insights into the genome variation across different regions.

Structure, sequence and phylogenetic analysis of the complete chloroplast genome of the brown algae Saccharina sp. ye-E (Laminariaceae: Phaeophyceae) from Sakhalin Oblast, Russia

For this investigation, the chloroplast genome of Saccharina sp. ye-E from Russia was uncovered and annotated with Illumina sequencing data to examine the phylogenetic relationship of Saccharina in Laminariaceae from a molecular biology perspective. Analysis of the structural characteristics, simple repeat sequence (SSR) loci, relative species differences, codon preference, and phylogenetic relationships was conducted. The results revealed that the chloroplast genome of Saccharina sp. ye-E had a length of 130,624 bp, containing 139 protein-coding genes (PCGs), 6 ribosomal RNAs (rRNAs) and 57 transfer RNAs (tRNAs) genes, and a total GC content of 31.1%. There were 15 SSR loci in the genome. Effective codon number (ENC) and codon adaptation index (CAI) both indicated the strong codon randomness and codon preference. The phylogenetic tree, based on the complete chloroplast genomes of 10 brown algae, showed that four species of Saccharina genus formed a clade, with Saccharina sp. ye-E and Saccharina latissimi having the most related genetic affinity. It is believed that the determination of the chloroplast genome of Saccharina sp. ye-E will be beneficial for future algal genetics evolution and systematic studies in the Laminariaceae.

Complete circularized genome resources of seven strains of Xylella fastidiosa subsp. fastidiosa using hybrid assembly reveals unknown plasmids.

Xylella fastidiosa (Xf) is a vascular plant pathogenic bacterium native to the Americas that is causing significant epidemics and economic losses in olive and almonds in Europe, where it is a quarantine pathogen. Since its first detection in 2013 in Italy, mandatory surveys across Europe revealed the presence of the bacterium also in France, Spain, and Portugal. Combining ONT and Illumina sequencing data, we assembled high-quality complete genomes of seven Xf subsp. fastidiosa strains isolated from different plants in Spain, the USA, and Mexico. Comparative genomic analyses discovered differences in plasmid content among strains, including plasmids that had been overlooked previously when using Illumina sequencing platform alone. Interestingly, in strain CFBP8073, intercepted in France from plants imported from Mexico, three plasmids were identified, including two (plasmid pXF-P1.CFBP8073 and pXF-P2.CFBP8073) not previously described in X. fastidiosa, and one (pXF5823.CFBP8073) almost identical to a plasmid described in a Xf strain from citrus. Plasmids found in the Spanish strains here were similar to those described previously in other strains from the same subspecies and ST1 isolated in the Balearic Islands and the USA. The genome resources from this work will assist in further studies on the role of plasmids in the epidemiology, ecology, and evolution of this plant pathogen.

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

A chromosome-scale genome assembly of Malus domestica, a multi-stress resistant apple variety

Hanfu apple is the main cultivar grown in the cool areas of Northeast, Northwest, and North China. Here, we proposed a chromosome-level Hanfu genome assembly using PacBio, Illumina and Hi-C sequencing data. The total contig length was 628.99 Mb, with scaffold and contig N50 sizes of 36.18 Mb and 1.25 Mb, respectively. The Hanfu genome had a total of 39,617 genes, of which we predicted the function for 38,816. Evolutionary analysis showed that Hanfu may have undergone a γ-event, a recent whole-genome duplication. A comparative analysis was conducted on the genomes of Hanfu and homozygous triploid HFTH1, which were cultured using the anthers of diploid Hanfu apples. Three variants were identified, including 2,155,184 single nucleotide polymorphisms (SNPs), 413,108 insertions/deletions (indels), and 7,587 structural variants (SVs).This high-quality genome will provide a reference for the genetic improvement of apples and the breeding of more varieties with high resistance and high quality.

Global scenario of the RmtE pan-aminoglycoside-resistance mechanism: emergence of the rmtE4 gene in South America associated with a hospital-related IncL plasmid.

Antimicrobial resistance (AMR) mechanisms, especially those conferring resistance to critically important antibiotics, are a great concern for public health. 16S rRNA methyltransferases (16S-RMTases) abolish the effectiveness of most clinically used aminoglycosides, but some of them are considered sporadic, such as RmtE. The main goals of this work were the genomic analysis of bacteria producing 16S-RMTases from a 'One Health' perspective in Venezuela, and the study of the epidemiological and evolutionary scenario of RmtE variants and their related mobile genetic elements (MGEs) worldwide. A total of 21 samples were collected in 2014 from different animal and environmental sources in the Cumaná region (Venezuela). Highly aminoglycoside-resistant Enterobacteriaceae isolates were selected, identified and screened for 16S-RMTase genes. Illumina and Nanopore whole-genome sequencing data were combined to obtain hybrid assemblies and analyse their sequence type, resistome, plasmidome and pan-genome. Genomic collections of rmtE variants and their associated MGEs were generated to perform epidemiological and phylogenetic analyses. A single 16S-RMTase, the novel RmtE4, was identified in five Klebsiella isolates from wastewater samples of Cumaná. This variant possessed three amino acid modifications with respect to RmtE1-3 (Asn152Asp, Val216Ile and Lys267Ile), representing the most genetic distant among all known and novel variants described in this work, and the second most prevalent. rmtE variants were globally spread, and their geographical distribution was determined by the associated MGEs and the carrying bacterial species. Thus, rmtE4 was found to be confined to Klebsiella isolates from South America, where it was closely related to ISVsa3 and an uncommon IncL plasmid related with hospital environments. This work uncovered the global scenario of RmtE and the existence of RmtE4, which could potentially emerge from South America. Surveillance and control measures should be developed based on these findings in order to prevent the dissemination of this AMR mechanism and preserve public health worldwide.