Insights into The Diversity of Nepenthes L. (Nepenthaceae) Across Peninsular Malaysia, Including The First Sighting of an Undescribed Taxon with Flared Peristomes and Quadruple-Row Ventral Wings

The discoveries of six taxa between 2019 and 2022 have brought the total number of Nepenthes species occurring in Peninsular Malaysia to 18. This article serves as a comprehensive overview, incorporating the latest information and taxonomic insights concerning all species native to the peninsula. The information is derived from published taxonomic records and cumulative field observations conducted since 2013. Notably, it further reports heretofore undocumented discoveries, making it a significant contribution to the current knowledge of tropical pitcher plants. These include a sighting of a population of Nepenthes with unusual traits in the upper montane forest of Banjaran Titiwangsa, which may represent the 19th taxon in Peninsular Malaysia. The plants exhibit conspicuously expanded peristomes and an unusual wing morphology, which altogether provide compelling evidence supporting the recognition of these individuals as a distinct and previously unknown species. Therefore, a provisional taxonomic name—Nepenthes sp. Titiwangsa (pesonawangsa_223) A. Amin—is assigned. The species is inferred to belong to the N. macfarlanei group based on its toothed peristome and the presence of fine hairs below the lid. The partial description, species note, photographs, and a dichotomous key of the proposed new species are provided. In addition, a velvety N. sanguinea population has been observed in northern Banjaran Titiwangsa and treated as a new natural variation (var. velutina) in this articl

In silico analysis of OSRR22 isolated from MR 219 rice and the strategy for developing CRISPR/CAS9 construct for genome editing

The rice response regulator 22 (RR22) has been reported to be negatively regulating salt tolerance in Oryza sativa and involved in the cytokinin signaling pathway; however, it has not been documented in any Malaysian rice and is deemed as an appealing subject for CRIPSR/Cas9 editing. This study analysed the OsRR22 gene from the Malaysian rice cultivar ‘MR 219’ to elucidate its function and determine the gRNA target site, in which the finding served as the most vital part for our current CRISPR/Cas9 genome editing experiment. In brief, the methods employed include total genome isolation, polymerase chain reaction (PCR) amplification, DNA sequencing, genome search, and computational analyses involving an array of in silico tools. The transcript of OsRR22 was 2,019 bp long, composed of six exons and it encoded a highly conserved 696 amino acid residues. Motif analysis revealed the gene product, RR22, contained response regulator (RR) receiver domain (position 27-142), disordered domain (position 154-214), and Myb-like DNA-binding domain (position 214-273). Analysis on the protein-protein interaction (PPI) using STRING revealed RR22 interacted with various proteins including RR24, RR B8A7T0, and a set of HPt domain-containing proteins (B8B4B1, B8AYV8, B8BEM5, B8A9E0 and B8B9H1). Analysis of gene expression profiles via the Rice Expression Database (RED) revealed the OsRR22 (Os06g0183100) gene was highly expressed (FPKM>10) in the leaf and root. In addition, there were 15 genes to be co-expressed (Pearson’s r value > 0.85) with the OsRR22 gene of which high-affinity potassium transporter 9 (HAK9, Os07g0679000) was one of them. Based on the first exon of OsRR22 that encoded a part of the RR receiver domain, a CRISPR-gRNA 20-bp spacer was generated through CCTop. The gRNA spacer was synthesised, annealed, and ligated into the CRSPR/Cas9 pRGEB32 vector. The CRISPR/Cas9 construct—targeting MR 219’s OsRR22 and intended for Agrobacterium-based delivery—was successfully developed. Our study here documents the upstream workflow involved in rice genome editing with an emphasis on the gene characterisation through multiple bioinformatics tools

Genome sequencing and analysis of Trichoderma (Hypocreaceae) isolates exhibiting antagonistic activity against the papaya dieback pathogen, Erwinia mallotivora

Erwinia mallotivora, the causal agent of papaya dieback disease, is a devastating pathogen that has caused a tremendous decrease in Malaysian papaya export and affected papaya crops in neighbouring countries. A few studies on bacterial species capable of suppressing E. mallotivora have been reported, but the availability of antagonistic fungi remains unknown. In this study, mycelial suspensions from five rhizospheric Trichoderma isolates of Malaysian origin were found to exhibit notable antagonisms against E. mallotivora during co-cultivation. We further characterised three isolates, Trichoderma koningiopsis UKM-M-UW RA5, UKM-M-UW RA6, and UKM-M-UW RA3a, that showed significant growth inhibition zones on plate-based inhibition assays. A study of the genomes of the three strains through a combination of Oxford nanopore and Illumina sequencing technologies highlighted potential secondary metabolite pathways that might underpin their antimicrobial properties. Based on these findings, the fungal isolates are proven to be useful as potential biological control agents against E. mallotivora, and the genomic data opens possibilities to further explore the underlying molecular mechanisms behind their antimicrobial activity, with potential synthetic biology applications

Development of CRISPR/Cas9 Construct in Rice (Oryza sativa subsp. indica) Using Golden Gate Cloning Method Towards Drought Tolerance

Rice (Oryza sativa) is a staple food consumed by the majority of the world’s population. Climate change, however, has created a significant threat to our food security as it posed severe effects on rice production. The emergence of genome editing technology has opened a new era in crop improvement. Hence, this study aims to develop the CRISPR/Cas9 construct of drought tolerance for O. sativa subsp. indica cv. IR64 using Golden Gate cloning method. For this purpose, the generation of CRISPR/Cas9 constructs involved several stages, i.e., characterization of SUMO E2-Conjugating Enzyme (OsSCE1) gene, single-guide RNA (sgRNA) design and vector construction. FGENESH, GeneMarkS, InterProScan, and Blast2GO programmes – were used for the OsSCE1 gene characterisation. The putative OsSCE1 gene isolated from IR64 was then verified by sequencing, and the gene was 585 bp long and showed 99% identity with O. sativa on chromosome 10. In silico analysis concluded the gene is involved in abiotic stress regulation. The 20 bp sgRNA was designed manually with the aid of gRNA prediction programmes including CCTop, and Benchling. The virtual vector was validated using the Golden Gate Cloning approach and later confirmed through sequencing. The assembly involved separate vectors containing the OsSCE1 sgRNA sequence, plant selectable marker, and Cas9 cassette to construct standardised elements for hierarchical modular cloning (MoClo). This study demonstrated that our format, as the gene insertion are achievable, resulting in a speedier and more efficient assembly process which may contribute to improve drought tolerance in indica rice. Further study on the Agrobacterium-mediated transformation using the developed construct may be conducted to determine the efficacy of knocking out candidate genes in improving drought tolerance ability O. sativ

Gene co-expression network tools and databases for crop improvement

Transcriptomics has significantly grown as a functional genomics tool for understanding the expression of biological systems. The generated transcriptomics data can be utilised to produce a gene co-expression network that is one of the essential downstream omics data analyses. To date, several gene co-expression network databases that store correlation values, expression profiles, gene names and gene descriptions have been developed. Although these resources remain scattered across the Internet, such databases complement each other and support efficient growth in the functional genomics area. This review presents the features and the most recent gene co-expression network databases in crops and summarises the present status of the tools that are widely used for constructing the gene co-expression network. The highlights of gene co-expression network databases and the tools presented here will pave the way for a robust interpretation of biologically relevant information. With this effort, the researcher would be able to explore and utilise gene co-expression network databases for crops improvement

First Draft Genome Assembly of the Malaysian Stingless Bee, Heterotrigona itama (Apidae, Meliponinae)

The Malaysian stingless bee industry is hugely dependent on wild colonies. Nevertheless, the availability of new queens to establish new colonies is insufficient to meet the growing demand for hives in the industry. Heterotrigona itama is primarily utilized for honey production in the region and the major source of stingless bee colonies comes from the wild. To propagate new colonies domestically, a fundamental understanding of the biology of queen development, especially from the genomics aspect, is necessary. The whole genome was sequenced using a paired-end 150 strategy on the Illumina HiSeq X platform. The shotgun sequencing generated approximately 89 million raw pair-end reads with a total output of 13.37 Gb and a GC content of 37.31%. The genome size of the species was estimated to be approximately 272 Mb. Phylogenetic analysis showed H. itama are much more closely related to the bumble bee (Bombus spp.) than they are to the modern honey bee (Apis spp.). The genome data provided here are expected to contribute to a better understanding of the genetic aspect of queen differentiation as well as of important molecular pathways which are crucial for stingless bee biology, management and conservation

Discovery of pathogenesis related and effector genes of Erwinia mallotivora in Carica papaya (Eksotika I) seedlings via transcriptomic analysis

A comparative transcriptome of Erwinia mallotivora library across early infection time points (6, 24 and 48 h) on papaya seedling Carica papaya (Eksotika I) was performed. A total of 5,680 genes were identified as differentially expressed genes (DEGs). The highest numbers of DEGs in all three E. mallotivora infection time points were accounted in the biosynthesis of secondary metabolites, microbial metabolism in diverse environments and ATP binding cassette transporters based on KEGG-based analysis. The functional annotation of the DEGs via Gene Ontology analysis has revealed a highly complex (more than 2,000 functional terms) yet a specific virulence strategy adapted by E. mallotivora across the infection time points. Our findings have uncovered the key factors and pathogenicity mechanism adopted by E. mallotivora as the infection progresses

Agrobacterium‑mediated in planta transformation of cut coleoptile: a new, simplified, and tissue culture‑independent method to deliver the CRISPR/Cas9 system in rice

Background: Agrobacterium-mediated transformation and particle bombardment are the two common approaches for genome editing in plant species using CRISPR/Cas9 system. Both methods require careful manipulations of undifferentiated cells and tissue culture to regenerate the potentially edited plants. However, tissue culture techniques are laborious and time-consuming. Methods and results: In this study, we have developed a simplified, tissue culture-independent protocol to deliver the CRISPR/Cas9 system through in planta transformation in Malaysian rice (Oryza sativa L. subsp. indica cv. MR219). Sprouting seeds with cut coleoptile were used as the target for the infiltration by Agrobacterium tumefaciens and we achieved 9% transformation efficiency. In brief, the dehusked seeds were surface-sterilised and imbibed, and the coleoptile was cut to expose the apical meristem. Subsequently, the cut coleoptile was inoculated with A. tumefaciens strain EHA105 harbouring CRISPR/Cas9 expression vector. The co-cultivation was conducted for five to six days in a dark room (25°C ±2°C) followed by rooting, acclimatisation, and growing phases. Two-month-old plant leaves were then subjected to a hygromycin selection, and hygromycin-resistant plants were identified as putative transformants. Further validation through the polymerase chain reaction verified the integration of the Cas9 gene in four putative T0 lines. During the fruiting stage, it was confirmed that the Cas9 gene was still present in three randomly selected tillers from two four-month-old transformed plants. Conclusion: This protocol provides a rapid method for editing the rice genome, bypassing the need for tissue culture. This article is the first to report the delivery of the CRISPR/Cas9 system for in planta transformation in rice

Development and assessment of CRISP/Cas9- construct towards drought tolerance in rice (Oryza sativa subsp. indica)

The great majority of the population on earth consume rice (Oryza sativa) as their primary source of carbohydrates, however, seriously threatened by the onset of climate change has posed a significant threat to the cultivation of this staple crop. The revolution of genome editing technology has opened a new era in crop improvement. Hence, this study aims to isolate, characterise, and develop the CRISPR/Cas9 construct for SUMO E2-Conjugating Enzyme (OsSCE1) gene. Development of CRISPR/Cas9 constructs involved several stages, (1) characterisation of OsSCE1 gene, (2) sgRNA design, (3) vector construction, (4) Agrobacterium and in-planta transformation and (5) molecular validations. The OsSCE1 gene was verified by sequencing, showing 99% identity with O. sativa on chromosome 10. The 20 bp sgRNA was designed manually with the help of gRNA prediction programmes including CCTop, and Benchling, and the CRISPR constructs (Level 0, 1 and 2) were confirmed by sequencing. Then, the transgenic rice plants harboring sgRNA: Cas9 targeting IR64 's OsSCE1 gene were generated via Agrobacterium mediated in-planta transformation. Leaves of two-month-old plants were subjected to selection assay (300 mg/L kanamycin) and kanamycin-resistant plants were labelled as putative transformants. Polymerase chain reaction (PCR) amplification of Cas9 and OsSCE1 genes using specific primers verified the integration of the transgenes in 29 T0 lines. The mutations were discovered by examining the targeting location on the genome of the relevant transgenic plants. Sequencing analysis revealed the presence of six transgenic lines (D8, C4, C14, B15, C21, and B18) that exhibited a mutagenesis efficiency of approximately 9% for the OsSCE1 gene. This finding suggests that sgRNA: Cas9-induced gene- targeting could be employed to specifically alter agricultural traits and the CRISPR-Cas9 system has the potential to be a highly effective tool for crop breeding trait enhancements

THE SYSTEMATIC SIGNIFICANCE OF LEAF EPIDERMAL MICROMORPHOLOGY OF TEN NEPENTHES SPECIES (NEPENTHACEAE) FROM PENINSULAR MALAYSIA

GHAZALLI, M. N., TAMIZI, A. A., ESA, M. I. M., BESI, E. E., NIKONG, D., NORDIN, A. R. M. & ZAINI, A. Z. 2019. The systematic significance of leaf epidermal micromorphology of ten Nepenthes species (Nepenthaceae) from Peninsular Malaysia. Reinwardtia 18(2): 81−96. — The pitcher plants of Malaysia belong to the genus Nepenthes and can be found thriving in swampy areas, along the roadside, on hillslopes and in mountainous terrains depending on species and their ecological preferences. In this study, cuticle micromorphology of ten species of Nepenthes (Nepenthaceae) collected from Peninsular Malaysia was intensively studied through scanning electron microscopy (SEM) to characterise and investigate diagnostic characters of cuticle micromorphology that can be useful in Nepenthes classification. A total of eleven characters from the inner and outer cuticles were enumerated in details and these characters have a value either for infrageneric classification or for diagnostic identification of the species. Characters observed and analysed were related to the epidermal cells, subsidiary cells, stomatal complex i.e type of waxes on both epidermal surfaces, abaxial and adaxial cuticular ornamentation, stomata characteristics, stomata formation, stomata frequency, cuticular ornamentation on stomata, shape of the stomata, stomata size, trichome existence and type of trichomes. Nepenthes ampullaria is clearly distinguished from the other species by markedly different types of tufted and multicellular trichomes of the epidermal cells on both leaf epidermal surfaces. For N. alba, its cuticular feature showed groovy cuticular pattern on the abaxial and adaxial surface, hence, can serve as a diagnostic cuticular pattern for this species. From these findings, the species delimitation based on cuticular features show a clear resolution, however some species might be individually distinct based on the combination of characters examined.