Genomic Landscape of Sago Palm (Metroxylon sagu Rottb.)

The sago palm (Metroxylon sagu Rottboll) is a halophytic tree and the ultimate solution to two major global issues: food security and the rapid loss of saline free agricultural lands. This palm can produce high amount of starch, besides being resistant to saline and almost disease free. Unfortunately, this valuable palm is currently underappreciated by the scientific community and the genomic landscape of this palm remained scarce to date. The aim of this study is to sequence and characterise the sago palm chloroplast genome, document the organellar genome copy number changes across various developmental stages, organs, localities and phenotypes as well as sequence the entire nuclear genome of sago palm. The sago palm chloroplast and nuclear genome sizes determined in this study are 157,300 bp and 616,599,600 bp respectively, yielded via next generation sequencing technologies. The organellar genome copy number investigation employing the combinatorial approach of both qPCR and long-PCR assays had unearthed that the mature leaf is generally most enriched with unimpeded plastome and mitogenome among other organs. The Plawei Manit growth stage was discovered to have the highest unimpeded organellar genomes copy numbers (over 180,000) among the five major growth stages. The trunking phenotype was proven to be superior to that of the spiny and non-trunking phenotypes in terms of organellar genomes copy numbers (over 188,000). Interestingly, the organellar genome copy numbers across four main Sarawak localities in Malaysia are indifferent statistically. This study serves as a groundwork for preliminary sago palm genomic landscape to support future haplotype and genome wide association studies. Keywords: Metroxylon sagu, chloroplast genome, nuclear genome, organellar genome, genomic landscap

Salt Tolerance Research in Sago Palm (Metroxylon sagu Rottb.): Past, Present and Future Perspective

The sago palm (Metroxylon sagu Rottb.) is one of the underdogs in the food crop planting industries for its potential which is extremely vast but the community appreciating it is scarce. Its capabilities to thrive well in undesirable environmental conditions, salt tolerance and high starch yield are one of the many advantages it possesses over other food crops like wheat, corn and rice. One important factor to look into for crop plantation is none other than its salt tolerance. The salt tolerance researches on this unique palm have commenced since 1977 and the pace of research was unbelievably slow in progression. Nevertheless, it was not until recently that this palm was being placed in the limelight once more. In this review, we are focusing on salt tolerance research and further detailed on the past, present and future of this research line. It is anticipated that consolidation of talents and resources can come in time and in tandem for the utilization of this cash palm to end world hunger

Organellar genome copy number variations and integrity across different organs, growth stages, phenotypes and main localities of sago palm (Metroxylon sagu Rottboll) in Sarawak, Malaysia

The sago palm (Metroxylon sagu Rottboll) is one valuable food crop that has both high economic and nature value with promising potential to curb future global hunger issues. The organellar genomes, namely mitogenome and plastome, are indispensable in various development processes of plants, orchestrating the survivability and adaptability of the host and fulfilling its physiological needs. The long-PCR and qPCR approaches were used in couple to reveal the organellar genome copy number variations across different organs, growth stages, phenotypes and main localities in Sarawak, Malaysia. In this study, the mature leaf achieved among the highest unimpeded plastome and mitogenome copy number among other organs such as young root, mature root, young shoot, aged leaf and pneumatophore. The Plawei Manit stage scored the highest in terms of unimpeded mitogenome and plastome copy number across other four growth stages. The non-trunking and spiny phenotypes inspected have lower organellar genome copy numbers than the trunking phenotype. All three phenotypes sampled from four different localities in Sarawak, Malaysia showed no significant difference. This study serves as a steppingstone in unravelling the organellar genome copy number variations of sago palm and this knowledge is essential for future genotyping studies

Complete chloroplast genome sequencing of sago palm (Metroxylon sagu Rottb.): Molecular structures, comparative analysis and evolutionary significance

The sago palm (Metroxylon sagu Rottb.) is an underappreciated essential food crop of the century and is deemed to be the ultimate solution to future food security issues globally. To grasp a better comprehension on the molecular basis of sago palm chloroplast, a complete characterization of the 157,300 bp chloroplast genome sequences (encompassing 85,257 bp large single copy region (LSC) and 17,533 bp small single copy region (SSC), sandwiched by a pair of 27,245 bp inverted repeats (IRs)) was performed. The genome houses 113 unique genes: 80 protein-coding genes, 29 tRNA genes and four ribosomal genes. The gene order, AT content and orientation of the M. sagu chloroplast genome closely resembles that of its congeneric species, M. warburgii. The distribution patterns of short and long repeats were also determined along with four highly variable regions, 216 mutation events (164 single nucleotide polymorphisms (SNPs) and 52 insertion/deletions (indels)) between the two Metroxylon genus members. The maximum likelihood phylogenetic tree had unravelled the close relationship of M. sagu, with its closest neighbour M. warburgii, together with other Calameae members like Pigafetta elata, Calamus caryotoides, and Salacca ramosiana. This study had dissected on the entire chloroplast genome of M. sagu and characterized the contents at the molecular level. It is anticipated that the yielded treasurable information will be channelled for species identification and evolutionary analysis within the Metroxylon genus in future

Mutagenesis Analysis of ABCB4 Gene Promoter of Danio rerio

Zebrafish abcb4 gene (ortholog to human ABCB1 gene) serves primarily in multidrug resistance (MDR)mechanism by effluxing chemotherapeutic agents, chemicals, xenobiotics, and numerous anti-cancer drugs out of the cells. This study aims to identify the specific transcription factor binding sites (TFBS) within the promoter region of zebrafish abcb4 gene and determine the functional roles of these factors in abcb4 gene expression regulation via mutagenesis analysis. First, primers were designed to target and amplify the promoter region of zebrafish abcb4 gene through gradient PCR. The zebrafish abcb4 gene promoter was then cloned into pGL3.0 vector and sent for sequencing. The sequencing results revealed high similarity to zebrafish DNA sequence from clone DKEY-24I24 in linkage group 16, indicating a successful cloning of targeted gene. Thereafter, consensus sequence of zebrafish abcb4 gene promoter was generated with the length of 1,392 bp which was close to its expected size during primer design (1,500 bp). Using MATCH tool, 155 TFBSs were found within zebrafish abcb4 gene promoter region. Activator protein 1 (AP-1) TFBS at 1,255 bp was chosen to be mutated through site directed mutagenesis. Mutagenic primers (forward primer: 5’ GGG CAA GGC AGT ATA AAC GTG 3’ and reverse primer: 5’ TTA TGT TTC TAG GGA TTA CGT CAC 3’) were designed to substitute AGT with GGG to remove the AP-1 TFBS. By mutating the zebrafish abcb4 gene promoter, the MDR phenomenon driven by zebrafish abcb4 gene can be elucidated and this might provide clues to the development of tumor and malignancy in human. The results from this study may enrich the knowledge in chemotherapy and cancer treatments

Cucumber Mosaic Virus: Global genome comparison and beyond

Aims: The cucumber mosaic virus (CMV) is categorized under the genus Cucumovirus and family Bromoviridae. This virus is known to infect over 1200 plant species from 100 families, including ornamental and horticultural plants. In this study, we pioneered a global genome comparison to decipher the unknown orchestrators behind the virulence and pathogenicity of CMV via the discovery of important single nucleotide polymorphic markers. Methodology and results: As a result, the genome size was found to be a potential preliminary country-specific marker for South Korea and the GC content can be utilized to preliminarily differentiate Turkey isolates from the others. The motif analysis as well as whole genome and coat protein phylogenetic trees were unable to form country-specific clusters. However, the coat protein haplotype analysis had successfully unconcealed country-specific single nucleotide polymorphic markers for Iran, Turkey and Japan isolates. Moreover, coat protein modelling and gene ontology prediction depicted high conservation across CMV isolates from different countries. Conclusion, significance and impact of study: The country-specific single nucleotide polymorphic markers unearthed in this study may provide significant data towards the profiling of varying virulence and pathogenicity of CMV across the globe in time to combat the yield loss driven by this virus thru the most efficacious biological control measures in the future

Mutagenesis Analysis of ABCB8 Gene Promoter of Danio rerio

The ABCB8 is one of the members under the ABCB subfamily of ATP-Binding Cassette (ABC) transporter which possess the ability in regulating the intracellular iron and heme transport. The loss of function mutation of ABCB8 gene leads to iron and heme accumulation in the cell which is highly toxic to human. However, the information regarding the expression regulation of this gene remains scarce. Hence, the objectives of this project are to determine the transcription factors binding site (TFBS) of ABCB8 and to identify the transcriptional roles of the cis-elements through mutagenesis analysis. To examine this, total genomic DNA was extracted from Danio rerio and the promoter sequence was isolated by using specific pair of primers through polymerase chain reaction (PCR). The sample was sent for DNA sequencing and the result showed 98% similarities to the zebrafish DNA sequence from clone DKEYP-87A6 in linkage group 24. Besides, the TFBS was studied in aspect of TFBS abundance, TFBS composition and TFBS distribution. The two most abundant TFBSs based on liver-specific profile were HNF-3β and C/EBPβ, with 38 and 39 binding sites, respectively. The sequence of ABCB8 promoter gene was mutated through substitution of the AP-1 binding site at location 535 with other nucleotides by using a pair of mutagenic primers (forward primer: 5’-TGGGGGTTTAGATATTGAAAC-3’; reverse primer: 5’-AACTCGC ATACATTTCAGTCATC-3’). This result may benefit the development of new diagnostics and therapeutics for iron-associated disorder

Mutagenesis Analysis of ABCG2 Gene Promoter of Zebrafish (Danio Rerio)

Breast cancer is the commonest cancer among women worldwide and the probability of a woman dying from breast cancer is high (about 1 in 38 of total human population (2.6%)).The main factor for mortality is due to the resistance of this particular disease to chemotherapeutic agents. One of the most well-known proteins to be found to correlate significantly with breast cancer resistance to chemotherapeutic agent is the ATP-binding cassette super-family G member 2 (ABCG2). Knowledge on ABCG2 gene regulation is still lacking in terms of how the increased cytotoxic levels are closely related to induce a hype in gene transcript levels and ultimately cause of the reduction in chemotherapeutic agents. The approach taken in this study is through mutational analysis of selected transcription factor governing the expression of ABCG2. In order to achieve this, a previously cloned ABCG2 promoter which has been isolated (around 1500 bp in size) from Danio rerio and inserted into pGL3.0 plasmid, was subjected to site-directed mutagenesis. Selected transcription factor which is AP-1 was successfully mutated by deletion of 5'- TGACGCG -3' sequence at position 1113 bp from TSS+1 where it would bind in order to define their role in ABCG2 physiological function. Sequencing result after site-directed mutagenesis shows high similarities about 98% with ABCG2 gene of Danio rerio. Upon validation, it was found that the intended AP-1 binding site has been mutated. In future work, the mutated clone here will be subjected to transfection analysis where dual-luciferase assay will be conducted to verify the loss of activity from the ABCG2 promoter upon mutation of the targeted AP-1 site. Hence, the mutagenesis analysis of ABCG2 promoter are able to provide information on the involvement of AP-1 transcription factor in multidrug resistance mechanism of breast cancer and thus will be a potential target for chemotherapeutic agent

Zebrafish (Danio rerio) ecotoxicological ABCB4, ABCC1 and ABCG2a gene promoters depict spatiotemporal xenobiotic multidrug resistance properties against environmental pollutants

Marine organisms are naturally equipped with multixenobiotic resistance mechanisms that are often governed by ATP-binding cassette (ABC) transporter family members. Previous studies focused on the target genes of ABC but little is known about the functionality of their promoter regions. Due to the importance of promoters in ABC transporter gene regulation, we functionally characterized three major xenobiotic transporter promoters of zebrafish, namely ABCB4, ABCC1 and ABCG2a via in silico transcription factor binding analysis and in vivo spatiotemporal expression analysis. The former revealed the major functional contributors (such as AP-1, C/EBP beta, HNF-1 and NF-1 TFBSs) towards promoter activity enhancement across four different tissues (liver, muscle, cell cycle and immune cells) where majority of them discovered were liver-specific whereas the latter unearthed the localization of these promoters at liver and intestinal tracts during late embryogenesis (48, 72 and 96 hpf). This study contributes towards future xenobiotic transporter ecotoxicology studies in zebrafi

Morphometric Analysis and Genetic Relationship of Rasbora spp. in Sarawak, Malaysia

The genus Rasbora is one of the most species-rich genus among the freshwater fishes and cryptic diversity has been a major hindrance in species identification in the past four decades due to their high similarities in terms of morphology. This study aimed to investigate this issue both morphologically and molecularly. In this study, a total of 23 morphometric parameters were used to differentiate the 103 Rasbora fish samples harvested from different regions of Sarawak state of Malaysia via Multivariate Stepwise Discriminant Function Analysis (SDFA). Then, cytochrome oxidase subunit I (COI) gene was utilised to further distinguish 33 of these fishes, followed by sequence and phylogenetic analysis. Our results unravelled pre-anal length as strongest morphometric discriminant (100%) and that all eight Rasbora species tested are monophyletic except for R. sumatrana and R. caudimaculata, revealing possible cryptic Rasbora species. Further investigations are vital to enrich the data from this study for Rasbora cryptic diversity and conservation studies in future