Isolation, identification and characterisation of Pseudomonas koreensis CM-01 isolated from diseased Malaysian mahseer (Tor tambroides).

Pseudomonas species are one of the most threatening fish pathogens which reside a wide range of environments. In this study, the dominant bacteria were isolated from diseased Malaysian mahseer (Tor tambroides) and tentatively named CM-01. It was identified as Pseudomonas koreensis based on its biochemical, morphological, genetic and physiological information. Its pathogenicity was found to be correlated with twelve virulence genes identified including iron uptake, protease, acylhomoserine lactone synthase gacS/gacA component regulation system, type IV secretion system, hydrogen cyanide production, exolysin, alginate biosynthesis, flagella and pili. The median lethal dose (LD50) for the CM-01 isolate on Malaysian mahseer was documented at 5.01 × 107 CFU/mL. The experimental infection revealed that CM-01 led to significant histological lesions in the fish, ultimately resulting in death. These lesions comprise necrosis, tissue thickening and aggregation. Drug sensitivity tests had shown its susceptibility to beta-lactam combination agents and further suggest its drug of choice. Its growing features had shown its growth at optimal temperature and pH. To the best of our knowledge, this is the first report of P. koreensis linked to diseased T. tambroides. STATEMENT OF RELEVANCE: In this research, a novel strain of Pseudomonas koreensis, CM-01 was isolated from diseased T. tambroides for the first time. The antimicrobial susceptibility, pathogenicity, virulence genes and growth characteristics of CM-01 were studied. These findings established a scientific foundation for the recognition of P. koreensis and the management of fish infections caused by this pathogen.

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.

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.

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.

Transcriptome dataset of sago palm in peat soil.

Sago palm (Metroxylon sagu Rottb.) is an important agricultural starch-producing palm that contributes to Malaysia's economics, especially in the State of Sarawak, Malaysian Borneo. In this palm tree, the central part of the plant storage-starch. Under normal condition, sago palm develop its trunk after 4-5 years being planted. However, sago palms planted on deep-peat soil failed to develop their trunk even after 17 years of being planted. This phenomenon is known as 'non-trunking', which eliminates the economic value of the palms. Numerous research has been done to address the phenomenon, but the molecular mechanisms of sago palm responding toward the responsible stresses are still lacking. Therefore, in this study, leaf samples were collected from trunking (normal) and non-trunking sago palms planted on peat soil for total RNA extraction, followed by next-generation sequencing using the BGISEQ-500 platform. The raw reads were cleaned, and de novo assembled using TRINITY software package. A total of 40.11 Gb bases were sequenced from the sago palm leaf samples. The assembled sequence produced 102,447 unigenes, with N50 score 1809 bp and GC ratio of 44.34%. The alignment of unigenes with seven functional databases (NR, NT, GO, KOG, KEGG, SwissProt and InterPro) resulted in the annotation of 65,523 (63.96%) unigenes. Functional annotation results in the detection of 46,335 coding DNA sequences by Transdecoder. A total of 30,039 simple-sequence repeats distributed on 21,676 unigenes were detected using Primer3 software, and 2355 transcription factor coding unigenes were predicted using getorf and hmmseach software. This work is registered under NCBI BioProject PRJNA781491. The raw RNA sequencing data are available in Sequence Read Archive (SRA) database with accession numbers SRX13165895, SRX13165896, SRX13165897, SRX13165898, SRX13165899, and SRX13165900. Gene expression and annotation information are accessible in public functional genomics data repository Gene Expression Omnibus (GEO) with accession number GSE189085.

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.

The first transcriptome sequencing and data analysis of the Javan mahseer (Tor tambra).

The Javan mahseer (Tor tambra) is one of the most valuable freshwater fish found in Tor species. To date, other than mitogenomic data (BioProject: PRJNA422829), genomic and transcriptomic resources for this species are still lacking which is crucial to understand the molecular mechanisms associated with important traits such as growth, immune response, reproduction and sex determination. For the first time, we sequenced the transcriptome from a whole juvenile fish using Illumina NovaSEQ6000 generating raw paired-end reads. De novo transcriptome assembly generated a draft transcriptome (BUSCO5 completeness of 91.2% [Actinopterygii_odb10 database]) consisting of 259,403 putative transcripts with a total and N50 length of 333,881,215 bp and 2283 bp, respectively. A total count of 77,503 non-redundant protein coding sequences were predicted from the transcripts and used for functional annotation. We mapped the predicted proteins to 304 known KEGG pathways with signal transduction cluster having the highest representation followed by immune system and endocrine system. In addition, transcripts exhibiting significant similarity to previously published growth-and immune-related genes were identified which will facilitate future molecular breeding of Tor tambra.

Improving the phylogenetic resolution of Malaysian and Javan mahseer (Cyprinidae), Tor tambroides and Tor tambra: Whole mitogenomes sequencing, phylogeny and potential mitogenome markers.

The true mahseer (Tor spp.) is one of the highest valued fish in the world due to its high nutritional value and great unique taste. Nevertheless, its morphological characterization and single mitochondrial gene phylogeny in the past had yet to resolve the ambiguity in its taxonomical classification. In this study, we sequenced and assembled 11 complete mahseer mitogenomes collected from Java of Indonesia, Pahang and Terengganu of Peninsular Malaysia as well as Sarawak of East Malaysia. The mitogenome evolutionary relationships among closely related Tor spp. samples were investigated based on maximum likelihood phylogenetic tree construction. Compared to the commonly used COX1 gene fragment, the complete COX1, Cytb, ND2, ND4 and ND5 genes appear to be better phylogenetic markers for genetic differentiation at the population level. In addition, a total of six population-specific mitolineage haplotypes were identified among the mahseer samples analyzed, which this offers hints towards its taxonomical landscape.

Sequencing and characterisation of complete mitogenome DNA for Rasbora sarawakensis (Cypriniformes: Cyprinidae: Rasbora) with phylogenetic consideration.

The Blueline Rasbora (Rasbora sarawakensis) is a small ray-finned fish categorized under the genus Rasbora in the Cyprinidae family. In this study, the complete mitogenome sequence of R. sarawakensis was sequenced using four primers targeting overlapping regions. The mitogenome is 16,709 bp in size, accommodating 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes and a putative control region. Identical gene organisation was detected between this species and other genus counterparts. The heavy strand houses 28 genes while the light strand stores the other nine genes. Most protein-coding genes employ ATG as start codon, excluding COI gene, which utilizes GTG instead. The central conserved sequence blocks (CSB-F, CSB-E and CSB-D), variable sequence blocks (CSB-3, CSB-2 and CSB-1) as well as the terminal associated sequence (TAS) are conserved in the control region. The maximum likelihood phylogenetic tree revealed the divergence of R. sarawakensis from the basal region of the Rasbora clade, where its evolutionary relationships with R. maculatus and R. pauciperforata are poorly resolved as indicated by the low bootstrap values. This work acts as steppingstone towards further molecular evolution and population genetics studies of Rasbora genus in future.

Sequencing and Characterisation of Complete Mitochondrial DNA Genome for Trigonopoma pauciperforatum (Cypriniformes: Cyprinidae: Danioninae) with Phylogenetic Consideration.

The Trigonopoma pauciperforatum or the redstripe rasbora is a cyprinid commonly found in marshes and swampy areas with slight acidic tannin-stained water in the tropics. In this study, the complete mitogenome sequence of T. pauciperforatum was first amplified in two parts using two pairs of overlapping primers and then sequenced. The size of the mitogenome is 16,707 bp, encompassing 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes and a putative control region. Identical gene organisation was detected between this species and other family members. The heavy strand accommodates 28 genes while the light strand houses the remaining nine genes. Most protein-coding genes utilise ATG as start codon except for COI gene which uses GTG instead. The terminal associated sequence (TAS), central conserved sequence block (CSB-F, CSB-D and CSB-E) as well as variable sequence block (CSB-1, CSB-2 and CSB-3) are conserved in the control region. The maximum likelihood phylogenetic tree revealed the divergence of T. pauciperforatum from the basal region of the major clade, where its evolutionary relationships with Boraras maculatus, Rasbora cephalotaenia and R. daniconius are poorly resolved as suggested by the low bootstrap values. This work contributes towards the genetic resource enrichment for peat swamp conservation and comprehensive in-depth comparisons across other phylogenetic researches done on the Rasbora-related genus.

Sequencing and characterization of complete mitogenome DNA of Rasbora tornieri (Cypriniformes: Cyprinidae: Rasbora) and its evolutionary significance.

The yellowtail rasbora (Rasbora tornieri) is a miniature ray-finned fish categorized under the genus Rasbora in the family of Cyprinidae. In this study, a complete mitogenome sequence of R. tornieri was sequenced using four primers targeting two halves of the mitogenome with overlapping flanking regions. The size of mitogenome was 16,573 bp, housing 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes and a putative control region. Identical gene organization was detected between this species and other members of Rasbora genus. The heavy strand encompassed 28 genes while the light strand accommodated the other nine genes. Most protein-coding genes execute ATG as start codon, excluding COI and ND3 genes, which utilized GTG instead. The central conserved sequence blocks (CSB-E, CSB-F and CSB-D), variable sequence blocks (CSB-1, CSB-3 and CSB-2) as well as the terminal associated sequence (TAS) were conserved within the control region. The maximum likelihood phylogenetic family tree revealed the divergence of R. tornieri from the basal region of the Rasbora clade, where its evolutionary relationships with other Rasbora members are poorly resolved as indicated by the low bootstrap values. This work acts as window for further population genetics and molecular evolution studies of Rasbora genus in future.

A survey of recently emerged genome-wide computational enhancer predictor tools.

The race for the discovery of enhancers at a genome-wide scale has been on since the commencement of next generation sequencing decades after the discovery of the first enhancer, SV40. A few enhancer-predicting features such as chromatin feature, histone modifications and sequence feature had been implemented with varying success rates. However, to date, there is no consensus yet on the single enhancer marker that can be employed to ultimately distinguish and uncover enhancers from the enormous genomic regions. Many supervised, unsupervised and semi-supervised computational approaches had emerged to complement and facilitate experimental approaches in enhancer discovery. In this review, we placed our focus on the recently emerged enhancer predictor tools that work on general enhancer features such as sequences, chromatin states and histone modifications, eRNA and of multiple feature approach. Comparisons of their prediction methods and outcomes were done across their functionally similar counterparts. We provide some recommendations and insights for future development of more comprehensive and robust tools.

Distinct developmental expression of two elongase family members in zebrafish.

Despite the known importance of long-chained polyunsaturated fatty acids (LC-PUFA) during development, very little is known about their utilization and biosynthesis during embryogenesis. Combining the advantages of the existence of a complete range of enzymes required for LC-PUFA biosynthesis and the well established developmental biology tools in zebrafish, we examined the expression patterns of three LC-PUFA biosynthesis genes, Elovl2-like elongase (elovl2), Elovl5-like elongase (elovl5) and fatty acyl desaturase (fad) in different zebrafish developmental stages. The presence of all three genes in the brain as early as 24 hours post fertilization (hpf) implies LC-PUFA synthesis activity in the embryonic brain. This expression eventually subsides from 72 hpf onwards, coinciding with the initiation of elovl2 and fad expression in the liver and intestine, 2 organs known to be involved in adult fish LC-PUFA biosynthesis. Collectively, these patterns strongly suggest the necessity for localized production of LC-PUFA in the brain during in early stage embryos prior to the maturation of the liver and intestine. Interestingly, we also showed a specific expression of elovl5 in the proximal convoluted tubule (PCT) of the zebrafish pronephros, suggesting a possible new role for LC-PUFA in kidney development and function.