Whole-genome sequencing of four culturable endophytic bacteria from German hardneck garlic cloves (Allium sativum L.).

We present the whole-genome sequence of four bacterial endophytes associated with German hardneck garlic cloves (Allium sativum L.). Among them, Agrobacterium fabrum and Pantoea agglomerans are associated with plant protection, while Rahnella perminowiae and Stenotrophomonas lactitubi are pathogens. These data will facilitate the identification of genes to improve garlic.

Method for detection of pathogenic bacteria from indoor air microbiome samples using high-throughput amplicon sequencing.

The exposure of the air microbiome in indoor air posed a detrimental health effect to the building occupants compared to the outdoor air. Indoor air in hospitals has been identified as a reservoir for various pathogenic microbes. The conventional culture-dependent method has been widely used to access the microbial community in the air. However, it has limited capability in enumerating the complex air microbiome communities, as some of the air microbiomes are uncultivable, slow-growers, and require specific media for cultivation. Here, we utilized a culture-independent method via amplicon sequencing to target the V3 region of 16S rRNA from the pool of total genomic DNA extracted from the dust samples taken from hospital interiors. This method will help occupational health practitioners, researchers, and health authorities to efficiently and comprehensively monitor the presence of harmful air microbiome thus take appropriate action in controlling and minimizing the health risks to the hospital occupants. Key features;•Culture-independent methods offer fast, comprehensive, and unbias profiles of pathogenic and non-pathogenic bacteria from the air microbiomes.•Unlike the culture-dependent method, amplicon sequencing allows bacteria identification to the lowest taxonomy levels.

Nanopore sequencing for identification and characterization of antimicrobial-resistant Escherichia coli and Salmonella spp. from tilapia and shrimp sold at wet markets in Dhaka, Bangladesh.

Wet markets in low-and middle-income countries are often reported to have inadequate sanitation resulting in fecal contamination of sold produce. Consumption of contaminated wet market-sourced foods has been linked to individual illness and disease outbreaks. This pilot study, conducted in two major wet markets in Dhaka city, Bangladesh during a 4-month period in 2021 aimed to assess the occurrence and characteristics of Escherichia coli and non-typhoidal Salmonella spp. (NTS) from tilapia (Oreochromis niloticus) and shrimp (Penaeus monodon). Fifty-four individuals of each species were collected. The identity of the bacterial isolates was confirmed by PCR and their susceptibility toward 15 antimicrobials was tested by disk diffusion. The whole genome of 15 E. coli and nine Salmonella spp. were sequenced using Oxford Nanopore Technology. E. coli was present in 60-74% of tilapia muscle tissue and 41-44% of shrimp muscle tissue. Salmonella spp. was found in skin (29%) and gills (26%) of tilapia, and occasionally in muscle and intestinal samples of shrimp. The E. coli had several Multilocus sequence typing and serotypes and limited antimicrobial resistance (AMR) determinants, such as point mutations on glpT and pmrB. One E. coli (BD17) from tilapia carried resistance genes for beta-lactams, quinolones, and tetracycline. All the E. coli belonged to commensal phylogroups B1 and A and showed no Shiga-toxin and other virulence genes, confirming their commensal non-pathogenic status. Among the Salmonella isolates, five belonged to Kentucky serovar and had similar AMR genes and phenotypic resistance patterns. Three strains of this serovar were ST198, often associated with human disease, carried the same resistance genes, and were genetically related to strains from the region. The two undetermined sequence types of S. Kentucky were distantly related and positioned in a separate phylogenetic clade. Two Brunei serovar isolates, one Augustenborg isolate, and one Hartford isolate showed different resistance profiles. This study revealed high fecal contamination levels in tilapia and shrimp sold at two main wet markets in Dhaka. Together with the occurrence of Salmonella spp., including S. Kentucky ST198, a well-known human pathogen, these results stress the need to improve hygienic practices and sanitation standards at markets to improve food safety and protect consumer health.

A multiplexed RT-PCR assay for nanopore whole genome sequencing of Tilapia lake virus (TiLV).

Tilapia lake virus (TiLV) is a highly contagious viral pathogen that affects tilapia, a globally significant and affordable source of fish protein. To prevent the introduction and spread of TiLV and its impact, there is an urgent need for increased surveillance, improved biosecurity measures, and continuous development of effective diagnostic and rapid sequencing methods. In this study, we have developed a multiplexed RT-PCR assay that can amplify all ten complete genomic segments of TiLV from various sources of isolation. The amplicons generated using this approach were immediately subjected to real-time sequencing on the Nanopore system. By using this approach, we have recovered and assembled 10 TiLV genomes from total RNA extracted from naturally TiLV-infected tilapia fish, concentrated tilapia rearing water, and cell culture. Our phylogenetic analysis, consisting of more than 36 TiLV genomes from both newly sequenced and publicly available TiLV genomes, provides new insights into the high genetic diversity of TiLV. This work is an essential steppingstone towards integrating rapid and real-time Nanopore-based amplicon sequencing into routine genomic surveillance of TiLV, as well as future vaccine development.

Endophytic bacteria associated with wild-type banana seed (Musa balbisiana): whole genome sequencing.

We present the whole-genome sequences of five endophytic bacteria isolated from Musa balbisiana seeds. These strains represent five different genera: Bacillus, Brachybacterium, Enterobacter, Enterococcus, and Pantoea. Among these, three genera (Bacillus, Pantoea, and Enterobacter) were previously recognized for their antagonistic effects against Fusarium wilt, a highly destructive disease that affects banana plants.

Characterization of the complete mitochondrial genome of the critically endangered Southern River Terrapin, Batagur affinis affinis (Reptilia: Geoemydidae).

In this study, we report the nearly complete mitochondrial sequence of Batagur affinis affinis. The assembled mitogenome consists of 13 PCGs, 22 tRNA genes, two rRNAs and one near-complete D-loop region. Of the annotated genes, the ND6 subunit gene and eight tRNA genes were encoded on the L-strand, while the remaining genes were dispersed on the H-strand. Except for CO1, which has a GTG start codon, all protein-coding genes begin with ATG. The mitogenome has been deposited in NCBI GenBank under the accession number OQ409915. Phylogenetic tree analysis based on publicly available mitogenomes indicate the sister grouping of B. affinis affinis with B. kachuga.

Whole-Genome Sequencing of Pseudomonas koreensis Isolated from Diseased Tor tambroides.

Unlike environmental P. koreensis isolated from soil, which has been studied extensively for its role in promoting plant growth, pathogenic P. koreensis isolated from fish has been rarely reported. Therefore, we investigated and isolated the possible pathogen that is responsible for the diseased state of Tor tambroides. Herein, we reported the morphological and biochemical characteristics, as well as whole-genome sequences of a newly identified P. koreensis strain. We assembled a high-quality draft genome of P. koreensis CM-01 with a contig N50 value of 233,601 bp and 99.5% BUSCO completeness. The genome assembly of P. koreensis CM-01 is consists of 6,171,880 bp with a G+C content of 60.5%. Annotation of the genome identified 5538 protein-coding genes, 3 rRNA genes, 54 tRNAs, and no plasmids were found. Besides these, 39 interspersed repeat and 141 tandem repeat sequences, 6 prophages, 51 genomic islands, 94 insertion sequences, 4 clustered regularly interspaced short palindromic repeats, 5 antibiotic-resistant genes, and 150 virulence genes were also predicted in the P. koreensis CM-01 genome. Culture-based approach showed that CM-01 strain exhibited resistance against ampicillin, aztreonam, clindamycin, and cefoxitin with a calculated multiple antibiotic resistance (MAR) index value of 0.4. In addition, the assembled CM-01 genome was successfully annotated against the Cluster of Orthologous Groups of proteins database, Gene Ontology database, and Kyoto Encyclopedia of Genes and Genome pathway database. A comparative analysis of CM-01 with three representative strains of P. koreensis revealed that 92% of orthologous clusters were conserved among these four genomes, and only the CM-01 strain possesses unique elements related to pathogenicity and virulence. This study provides fundamental phenotypic and genomic information for the newly identified P. koreensis strain.

Comprehensive Characterization of a Streptococcus agalactiae Phage Isolated from a Tilapia Farm in Selangor, Malaysia, and Its Potential for Phage Therapy.

The Streptococcus agalactiae outbreak in tilapia has caused huge losses in the aquaculture industry worldwide. In Malaysia, several studies have reported the isolation of S. agalactiae, but no study has reported the isolation of S. agalactiae phages from tilapia or from the culture pond. Here, the isolation of the S. agalactiae phage from infected tilapia is reported and it is named as vB_Sags-UPM1. Transmission electron micrograph (TEM) revealed that this phage showed characteristics of a Siphoviridae and it was able to kill two local S. agalactiae isolates, which were S. agalactiae smyh01 and smyh02. Whole genome sequencing (WGS) of the phage DNA showed that it contained 42,999 base pairs with 36.80% GC content. Bioinformatics analysis predicted that this phage shared an identity with the S. agalactiae S73 chromosome as well as several other strains of S. agalactiae, presumably due to prophages carried by these hosts, and it encodes integrase, which suggests that it was a temperate phage. The endolysin of vB_Sags-UPM1 termed Lys60 showed killing activity on both S. agalactiae strains with varying efficacy. The discovery of the S. agalactiae temperate phage and its antimicrobial genes could open a new window for the development of antimicrobials to treat S. agalactiae infection.

Complete chloroplast genome data of Shorea macrophylla (Engkabang): Structural features, comparative and phylogenetic analysis.

Shorea macrophylla belongs to the Shorea genus under the Dipterocarpaceae family. It is a woody tree that grows in the rainforest in Southeast Asia. The complete chloroplast (cp) genome sequence of S. macrophylla is reported here. The genomic size of S. macrophylla is 150,778 bp and it possesses a circular structure with conserved constitute regions of large single copy (LSC, 83,681 bp) and small single copy (SSC, 19,813 bp) regions, as well as a pair of inverted repeats with a length of 23,642 bp. It has 112 unique genes, including 78 protein-coding genes, 30 tRNA genes, and four rRNA genes. The genome exhibits a similar GC content, gene order, structure, and codon usage when compared to previously reported chloroplast genomes from other plant species. The chloroplast genome of S. macrophylla contained 262 SSRs, the most prevalent of which was A/T, followed by AAT/ATT. Furthermore, the sequences contain 43 long repeat sequences, practically most of them are forward or palindrome type long repeats. The genome structure of S. macrophylla was compared to the genomic structures of closely related species from the same family, and eight mutational hotspots were discovered. The phylogenetic analysis demonstrated a close relationship between Shorea and Parashorea species, indicating that Shorea is not monophyletic. The complete chloroplast genome sequence analysis of S. macrophylla reported in this paper will contribute to further studies in molecular identification, genetic diversity, and phylogenetic research.

Whole-Genome Sequence of Endophytic Bacteria Associated with Poison Ivy Vine (Toxicodendron radicans).

Here, we report the genome assemblies of 11 endophytic bacteria, isolated from poison ivy vine (Toxicodendron radicans). Five species belonging to the genus Pseudomonas, two species of Curtobacterium, one strain of Pantoea agglomerans, and one species from the Bacillus, Cellulomonas, and Enterobacter genera were isolated from the interior tissue of poison ivy.

The complete mitochondrial genomes of Pangasius nasutus and P. conchophilus (Siluriformes: Pangasiidae).

The catfish, Pangasius nasutus and P. conchophilus, are often misidentified between each other due to their similar morphology. Thus, the current study was conducted to differentiate them based on a molecular approach. The complete mitochondrial genomes of P. nasutus and P. conchophilus obtained from the Pahang River (Peninsular Malaysia) were sequenced, assembled, and annotated using next-generation sequencing (NGS). A 16,465 bp and 16,470 bp length mitogenome sequence of P. nasutus and P. conchophilus, respectively, was generated, each containing 13 protein genes, 22 tRNAs, and two rRNAs, typical of most vertebrates. This is the first report of the complete mitochondrial genome sequences of P. nasutus and P. conchophilus. These data are a valuable genetic resource for future studies of these two commercially important species.

A new set of microsatellite markers for Phoxinus lumaireul senso lato, Phoxinus marsilii and Phoxinus krkae for population and molecular taxonomic studies.

Minnows of the genus Phoxinus are common and an often highly abundant fish species in Palearctic freshwater habitats. Phoxinus species have a complex evolutionary history, phylogenetic relationships are not well understood and there are a number of unresolved taxonomic problems. There are currently 23 different mitochondrial genetic lineages identified in the genus Phoxinus, 13 of which are recognized as valid species. The taxonomic status of these lineages requires resolution, including the degree to which they can interbreed. Suitable nuclear molecular markers for studies of population divergence and interbreeding between morphotypes and mitochondrial lineages are lacking for Phoxinus species. Therefore, the authors developed a set of microsatellite markers using genomic information from Phoxinus lumaireul and tested their suitability for this and two related species, Phoxinus krkae and Phoxinus marsilii. Out of 16 microsatellite candidate loci isolated, 12 were found to be in Hardy-Weinberg equilibrium when tested on two P. lumaireul senso lato populations. Seven loci amplified across the three species, enabling the study of intraspecific genetic diversity and population structure within P. marsilii and P. krkae. The markers were able to clearly resolve differences among the three tested species, including the recently described P. krkae, and are therefore suitable for the detection of introgression and hybridization among populations consisting of mixtures of two or more of P. lumaireul s. l., P. marsilii and P. krkae.

Genome-Wide Identification, Characterisation and Phylogenetic Analysis of 52 Striped Catfish (Pangasianodon hypophthalmus) ATP-Binding Cassette (ABC) Transporter Genes.

The Pangasianodon hypophthalmus (striped or tra catfish) is a Pangasiidae family member famous for its high unsaturated fatty acid content flesh. This riverine catfish can breathe in the air unlike the channel catfish. One of the most well-known ecotoxicological protein superfamily, the ATP-binding cassette (ABC) transporters, has been characterised in channel catfish through a genome-wide approach. Therefore, it is interesting to unearth these proteins within the striped catfish genome for a comprehensive comparison across all catfishes available. A total of 52 ABC transporters were discovered from the striped catfish genome. The motif analysis has unconcealed various unshared characteristics of some catfishes. The phylogenetic analysis has evidenced its effectiveness in the successful annotations of these transporter proteins. Duplicated genes such as ABCA1, ABCB3, ABCB6, ABCC5, ABCD3, ABCE1, ABCF2 as well as ABCG2 were uncovered within the striped and channel catfish genomes. This entire set of ABC transporters yields precious genomic data for future ecotoxicological, biochemical and physiological research in striped catfish.

Genome Assembly and Analysis of the Flavonoid and Phenylpropanoid Biosynthetic Pathways in Fingerroot Ginger (Boesenbergia rotunda).

Boesenbergia rotunda (Zingiberaceae), is a high-value culinary and ethno-medicinal plant of Southeast Asia. The rhizomes of this herb have a high flavanone and chalcone content. Here we report the genome analysis of B. rotunda together with a complete genome sequence as a hybrid assembly. B. rotunda has an estimated genome size of 2.4 Gb which is assembled as 27,491 contigs with an N50 size of 12.386 Mb. The highly heterozygous genome encodes 71,072 protein-coding genes and has a 72% repeat content, with class I TEs occupying ~67% of the assembled genome. Fluorescence in situ hybridization of the 18 chromosome pairs at the metaphase showed six sites of 45S rDNA and two sites of 5S rDNA. An SSR analysis identified 238,441 gSSRs and 4604 EST-SSRs with 49 SSR markers common among related species. Genome-wide methylation percentages ranged from 73% CpG, 36% CHG and 34% CHH in the leaf to 53% CpG, 18% CHG and 25% CHH in the embryogenic callus. Panduratin A biosynthetic unigenes were most highly expressed in the watery callus. B rotunda has a relatively large genome with a high heterozygosity and TE content. This assembly and data (PRJNA71294) comprise a source for further research on the functional genomics of B. rotunda, the evolution of the ginger plant family and the potential genetic selection or improvement of gingers.

Characterization of the first mitogenomes of the smallest fish in the world, Paedocypris progenetica, from peat swamp of Peninsular Malaysia, Selangor, and Perak.

The two complete mitochondrial genomes (mitogenomes) of Paedocypris progenetica, the smallest fish in the world which belonged to the Cyprinidae family, were sequenced and assembled. The circular DNA molecules of mitogenomes P1-P. progenetica and S3-P. progenetica were 16,827 and 16,616 bp in length, respectively, and encoded 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and one control region. The gene arrangements of P. progenetica were identical to those of other Paedocypris species. BLAST and phylogenetic analyses revealed variations in the mitogenome sequences of two Paedocypris species from Perak and Selangor. The circular DNA molecule of P. progenetica yield a standard vertebrate gene arrangement and an overall nucleotide composition of A 33.0%, T 27.2%, C 23.5%, and G 15.5%. The overall AT content of this species was consistent with that of other species in other genera. The negative GC-skew and positive ATskew of the control region in P. progenetica indicated rich genetic variability and AT nucleotide bias, respectively. The results of this study provide genomic variation information and enhance the understanding of the mitogenome of P. progenetica. They could later deliver highly valuable new insight into data for phylogenetic analysis and population genetics.

Chromosome-length genome assembly and linkage map of a critically endangered Australian bird: the helmeted honeyeater.

BACKGROUND: The helmeted honeyeater (Lichenostomus melanops cassidix) is a Critically Endangered bird endemic to Victoria, Australia. To aid its conservation, the population is the subject of genetic rescue. To understand, monitor, and modulate the effects of genetic rescue on the helmeted honeyeater genome, a chromosome-length genome and a high-density linkage map are required. RESULTS: We used a combination of Illumina, Oxford Nanopore, and Hi-C sequencing technologies to assemble a chromosome-length genome of the helmeted honeyeater, comprising 906 scaffolds, with length of 1.1 Gb and scaffold N50 of 63.8 Mb. Annotation comprised 57,181 gene models. Using a pedigree of 257 birds and 53,111 single-nucleotide polymorphisms, we obtained high-density linkage and recombination maps for 25 autosomes and Z chromosome. The total sex-averaged linkage map was 1,347 cM long, with the male map being 6.7% longer than the female map. Recombination maps revealed sexually dimorphic recombination rates (overall higher in males), with average recombination rate of 1.8 cM/Mb. Comparative analyses revealed high synteny of the helmeted honeyeater genome with that of 3 passerine species (e.g., 32 Hi-C scaffolds mapped to 30 zebra finch autosomes and Z chromosome). The genome assembly and linkage map suggest that the helmeted honeyeater exhibits a fission of chromosome 1A into 2 chromosomes relative to zebra finch. PSMC analysis showed a ∼15-fold decline in effective population size to ∼60,000 from mid- to late Pleistocene. CONCLUSIONS: The annotated chromosome-length genome and high-density linkage map provide rich resources for evolutionary studies and will be fundamental in guiding conservation efforts for the helmeted honeyeater.

First high-quality genome assembly data of sago palm (Metroxylon sagu Rottboll).

The sago palm (Metroxylon sagu Rottboll) is a tropical halophytic starch-producing, economically important crop palm mainly located in Southeast Asian countries. Recently, a genome survey was conducted on this palm using the Illumina sequencing platform, with a very low (21.5%) BUSCO genome completeness score, and most of them (∼78%) are either fragmented or missing. Thus, in this study, the sago palm genome completeness was further improved with the utilization of the Nanopore sequencing platform that produced longer reads. A hybrid genome assembly was conducted, and the outcome was a much complete sago palm genome with BUSCO completeness achieved at as high as 97.9%, with only ∼2% of them either fragmented or missing. The estimated genome size of the sago palm is 509,812,790 bp in this study. A sum of 33,242 protein-coding genes was revealed from the sago palm genome and around 96.39% of them had been functionally annotated. An investigation on the carbohydrate metabolism KEGG pathways also unearthed that starch synthesis was one of the major sago palm activities. The genome data obtained from this work is indispensable for future molecular evolutionary and genome-wide association studies on the economically important sago palm.

A novel framework for evaluating in situ breeding management strategies in endangered populations.

Conservation breeding management aims to reduce inbreeding and maximize the retention of genetic diversity in endangered populations. However, breeding management of wild populations is still rare, and there is a need for approaches that provide data-driven evidence of the likelihood of success of alternative in situ strategies. Here, we provide an analytical framework that uses in silico simulations to evaluate, for real wild populations, (i) the degree of population-level inbreeding avoidance, (ii) the genetic quality of mating pairs, and (iii) the potential genetic benefits of implementing two breeding management strategies. The proposed strategies aim to improve the genetic quality of breeding pairs by splitting detrimental pairs and allowing the members to re-pair in different ways. We apply the framework to the wild population of the Critically Endangered helmeted honeyeater by combining genomic data and field observations to estimate the inbreeding (i.e., pair-kinship) and genetic quality (i.e., Mate Suitability Index) of all mating pairs for seven consecutive breeding seasons. We found no evidence of population-level inbreeding avoidance and that ~91.6% of breeding pairs were detrimental to the genetic health of the population. Furthermore, the framework revealed that neither proposed management strategy would significantly improve the genetic quality or reduce inbreeding of the mating pairs in this population. Our results demonstrate the usefulness of our analytical framework for testing the efficacy of different in situ breeding management strategies and for making evidence-based management decisions.

Labile sex chromosomes in the Australian freshwater fish family Percichthyidae.

Sex-specific ecology has management implications, but rapid sex-chromosome turnover in fishes hinders sex-marker development for monomorphic species. We used annotated genomes and reduced-representation sequencing data for two Australian percichthyids, Macquarie perch Macquaria australasica and golden perch M. ambigua, and whole genome resequencing for 50 Macquarie perch of each sex, to identify sex-linked loci and develop an affordable sexing assay. In silico pool-seq tests of 1,492,004 Macquarie perch SNPs revealed that a 275-kb scaffold was enriched for gametologous loci. Within this scaffold, 22 loci were sex-linked in a predominantly XY system, with females being homozygous for the X-linked allele at all 22, and males having the Y-linked allele at >7. Seven XY-gametologous loci (all males, but no females, are heterozygous or homozygous for the male-specific allele) were within a 146-bp region. A PCR-RFLP sexing assay targeting one Y-linked SNP, tested in 66 known-sex Macquarie perch and two of each sex of three confamilial species, plus amplicon sequencing of 400 bp encompassing the 146-bp region, revealed that the few sex-linked positions differ between species and between Macquarie perch populations. This indicates sex-chromosome lability in Percichthyidae, supported by nonhomologous scaffolds containing sex-linked loci for Macquarie- and golden perches. The present resources facilitate genomic research in Percichthyidae, including formulation of hypotheses about candidate genes of interest such as transcription factor SOX1b that occurs in the 275-kb scaffold ~38 kb downstream of the 146-bp region containing seven XY-gametologous loci. Sex-linked markers will be useful for determining genetic sex in some populations and studying sex chromosome turnover.

The inconsistent microbiota of Budu, the Malaysian fermented anchovy sauce, revealed through 16S amplicon sequencing.

Budu is a Malaysian fermented anchovy sauce produced by immersing small fishes into a brine solution for 6 to 18 months. Microbial enzymes are known to contribute to fermentation; however, not much is known about the microbial community in Budu. Therefore, a better understanding of the Budu microbiome is necessary to improve the quality, consistency, and safety of the Budu products. In this study, we collected 60 samples from 20 bottles of Budu produced by seven manufacturers. We analyzed their microbiota using V3-V4 16S rRNA amplicon sequencing when we first opened the bottle (month 0), as well as 3 and 7 months post-opening (months 3 and 7). Tetragenococcus was the dominant genus in many samples, reaching a maximum proportion of 98.62%, but was found in low abundance, or absent, in other samples. When Budu samples were not dominated by a dominant taxa, we observed a wider genera diversity such as Staphylococcus, Acinetobacter, Halanaerobium and Bacillus. While the taxonomic composition was relatively stable across sampling periods, samples from two brands showed a sudden increase in relative abundance of the genus Chromobacterium at month 7. Based on prediction of metagenome functions, non-Tetragenococcus-dominated samples were predicted to have enriched functional pathways related to amino acid metabolism and purine metabolism compared to Tetragenococcus-dominated samples; these two pathways are fundamental to fermentation quality and health attributes of fish sauce. Among the non-Tetragenococcus-dominated samples, contributions towards amino acid metabolism and purine metabolism were biased towards the dominant taxa when species evenness is low, while in samples with higher species evenness, the contributions towards the two pathways were predicted to be evenly distributed between taxa. Our results demonstrated that the utility of 16S sequencing to assess batch variation in fermented food production. The distinct microbiota was shown to correlate with characteristic metagenome function including functions potentially related to fermented food nutrition and quality.