Optimal set of microsatellite markers required to detect illegitimate progenies in selected oil palm (Elaeis guineensis Jacq.) breeding crosses.

Oil palm is continually being improved via controlled crossing of selected palms to ensure sustainable yields and productivity. As such, correct parental assignment is important as the presence of illegitimates will compromise the progress of improvement. In the present study, we determined the optimal number of microsatellite (SSR) markers for detection of illegitimates in selected oil palm crosses with high confidence. Determining the optimal number of markers to assign parentage will ensure that the DNA fingerprinting will be cost effective for routine use as a quality control tool in oil palm improvement programs. Here, we evaluated a wide range of crosses that included a cross derived from wild germplasm palm. The results revealed that markers with high PIC are informative and detect most of the alleles present in a cross, including those exhibited by the illegitimates. A larger number of optimum sets of markers are needed to detect all illegitimates for crosses with higher levels of genetic diversity. The optimal number of polymorphic SSR markers determined in the present study can ensure that appropriate quality control is implemented for oil palm improvement programs.

Diverse and dynamic roles of F-box proteins in plant biology.

The SCF complex is a widely studied multi-subunit ring E3 ubiquitin ligase that tags targeted proteins with ubiquitin for protein degradation by the ubiquitin 26S-proteasome system (UPS). The UPS is an important system that generally keeps cellular events tightly regulated by purging misfolded or damaged proteins and selectively degrading important regulatory proteins. The specificity of this post-translational regulation is controlled by F-box proteins (FBPs) via selective recognition of a protein-protein interaction motif at the C-terminal domain. Hence, FBPs are pivotal proteins in determining the plant response in multiple scenarios. It is not surprising that the FBP family is one of the largest protein families in the plant kingdom. In this review, the roles of FBPs, specifically in plants, are compiled to provide insights into their involvement in secondary metabolites, plant stresses, phytohormone signalling, plant developmental processes and miRNA biogenesis.

Regulation of terpenoid biosynthesis by miRNA in Persicaria minor induced by Fusarium oxysporum.

BACKGROUND: Persicaria minor (kesum) is an herbaceous plant with a high level of secondary metabolite compounds, particularly terpenoids. These terpenoid compounds have well-established roles in the pharmaceutical and food industries. Although the terpenoids of P. minor have been studied thoroughly, the involvement of microRNA (miRNA) in terpenoid regulation remains poorly understood and needs to be explored. In this study, P. minor plants were inoculated with the pathogenic fungus Fusarium oxysporum for terpenoid induction. RESULT: SPME GC-MS analysis showed the highest terpenoid accumulation on the 6th day post-inoculation (dpi) compared to the other treatment time points (0 dpi, 3 dpi, and 9 dpi). Among the increased terpenoid compounds, α-cedrene, valencene and ß-bisabolene were prominent. P. minor inoculated for 6 days was selected for miRNA library construction using next generation sequencing. Differential gene expression analysis showed that 58 miRNAs belonging to 30 families had significantly altered regulation. Among these 58 differentially expressed genes (DEGs), 27 [corrected] miRNAs were upregulated, whereas 31 [corrected] miRNAs were downregulated. Two putative novel pre-miRNAs were identified and validated through reverse transcriptase PCR. Prediction of target transcripts potentially involved in the mevalonate pathway (MVA) was carried out by psRobot software, resulting in four miRNAs: pmi-miR530, pmi-miR6173, pmi-miR6300 and a novel miRNA, pmi-Nov_13. In addition, two miRNAs, miR396a and miR398f/g, were predicted to have their target transcripts in the non-mevalonate pathway (MEP). In addition, a novel miRNA, pmi-Nov_12, was identified to have a target gene involved in green leaf volatile (GLV) biosynthesis. RT-qPCR analysis showed that pmi-miR6173, pmi-miR6300 and pmi-nov_13 were downregulated, while miR396a and miR398f/g were upregulated. Pmi-miR530 showed upregulation at 9 dpi, and dynamic expression was observed for pmi-nov_12. Pmi-6300 and pmi-miR396a cleavage sites were detected through degradome sequence analysis. Furthermore, the relationship between miRNA metabolites and mRNA metabolites was validated using correlation analysis. CONCLUSION: Our findings suggest that six studied miRNAs post-transcriptionally regulate terpenoid biosynthesis in P. minor. This regulatory behaviour of miRNAs has potential as a genetic tool to regulate terpenoid biosynthesis in P. minor.

Correction to: Regulation of terpenoid biosynthesis by miRNA in Persicaria minor induced by Fusarium oxysporum.

Following publication of the original article [1], the authors reported a number of errors, which are listed in this Correction article. The corrections are marked in bold.

Recent Development in Omics Studies.

The central dogma of molecular biology (DNA, RNA, protein and metabolite) has engraved our understanding of genetics in all living organisms. While the concept has been embraced for many decades, the development of high-throughput technologies particularly omics (genomics, transcriptomics, proteomics and metabolomics) has revolutionised the field to incorporate big data analysis including bioinformatics and systems biology as well as synthetic biology area. These omics approaches as well as systems and synthetic biology areas are now increasingly popular as seen by the growing numbers of publication throughout the years. Several journals which have published most of these related fields are also listed in this chapter to overview their impact and target journals.

Rapid and targeted introgression of fgr gene through marker-assisted backcrossing in rice (Oryza sativa L.).

While it is crucial for developing countries like Malaysia to achieve self-sufficiency in rice (Oryza sativa L.), it is equally critical to be able to produce high-quality rice, specifically fragrant rice, which demands are often met through importation. The present study was aimed at developing high-yielding fragrant rice, in a timely and cost-effective manner. A marker-assisted backcross (MABC) approach was optimised to introgress the fragrance gene (fgr) into two high-yielding Malaysian varieties, MR84 and MR219, within two years utilising less than 50 molecular markers. Coupled with phenotypic screening, one single foreground marker (fgr-SNP) and 48 background markers were selected and utilised, revealing recovery of at least 90% of recurrent parent genome (RPG) in merely two backcross generations. Collectively, the yield potential of the developed BC2F2 lines (BLs) was higher (P > 0.05) than the donor parent, MRQ74, and similar (P < 0.05) to both the recurrent parents, MR84 and MR219. In addition, some of the developed BLs showed good grain quality, such as having long grain. We believe that this is the first report comprising the validation and utilisation of the single functional marker system (fgr-SNP) in introgressing the fgr gene into different rice varieties.

Green leaf volatiles: biosynthesis, biological functions and their applications in biotechnology.

Plants have evolved numerous constitutive and inducible defence mechanisms to cope with biotic and abiotic stresses. These stresses induce the expression of various genes to activate defence-related pathways that result in the release of defence chemicals. One of these defence mechanisms is the oxylipin pathway, which produces jasmonates, divinylethers and green leaf volatiles (GLVs) through the peroxidation of polyunsaturated fatty acids (PUFAs). GLVs have recently emerged as key players in plant defence, plant-plant interactions and plant-insect interactions. Some GLVs inhibit the growth and propagation of plant pathogens, including bacteria, viruses and fungi. In certain cases, GLVs released from plants under herbivore attack can serve as aerial messengers to neighbouring plants and to attract parasitic or parasitoid enemies of the herbivores. The plants that perceive these volatile signals are primed and can then adapt in preparation for the upcoming challenges. Due to their 'green note' odour, GLVs impart aromas and flavours to many natural foods, such as vegetables and fruits, and therefore, they can be exploited in industrial biotechnology. The aim of this study was to review the progress and recent developments in research on the oxylipin pathway, with a specific focus on the biosynthesis and biological functions of GLVs and their applications in industrial biotechnology.

Biocompatible and mucoadhesive bacterial cellulose-g-poly(acrylic acid) hydrogels for oral protein delivery.

Stimuli-responsive bacterial cellulose-g-poly(acrylic acid) hydrogels were investigated for their potential use as an oral delivery system for proteins. These hydrogels were synthesized using electron beam irradiation without any cross-linking agents, thereby eliminating any potential toxic effects associated with cross-linkers. Bovine serum albumin (BSA), a model protein drug, was loaded into the hydrogels, and the release profile in simulated gastrointestinal fluids was investigated. Cumulative release of less than 10% in simulated gastric fluid (SGF) demonstrated the potential of these hydrogels to protect BSA from the acidic environment of the stomach. Subsequent conformational stability analyses of released BSA by SDS-PAGE, circular dichroism, and an esterase activity assay indicated that the structural integrity and bioactivity of BSA was maintained and preserved by the hydrogels. Furthermore, an increase in BSA penetration across intestinal mucosa tissue was observed in an ex vivo penetration experiment. Our fabricated hydrogels exhibited excellent cytocompatibility and showed no sign of toxicity, indicating the safety of these hydrogels for in vivo applications.

Functional characterization of sesquiterpene synthase from Polygonum minus.

Polygonum minus is an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene from P. minus. P. minus sesquiterpene synthase (PmSTS) has a complete open reading frame (ORF) of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function of PmSTS, we expressed this gene in Arabidopsis thaliana. Two transgenic lines, designated as OE3 and OE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production of ß -sesquiphellandrene.

An F-box Kelch repeat protein, PmFBK2, from Persicaria minor interacts with GID1b to modulate gibberellin signalling.

The F-box protein (FBP) family plays diverse functions in the plant kingdom, with the function of many members still unrevealed. In this study, a specific FBP called PmFBK2, containing Kelch repeats from Persicaria minor, was functionally investigated. Employing the yeast two-hybrid (Y2H) assay, PmFBK2 was found to interact with Skp1-like proteins from P. minor, suggesting its potential to form an E3 ubiquitin ligase, known as the SCF complex. Y2H and co-immunoprecipitation tests revealed that PmFBK2 interacts with full-length PmGID1b. The interaction marks the first documented binding between these two protein types, which have never been reported in other plants before, and they exhibited a negative effect on gibberellin (GA) signal transduction. The overexpression of PmFBK2 in the kmd3 mutant, a homolog from Arabidopsis, demonstrated the ability of PmFBK2 to restore the function of the mutated KMD3 gene. The function restoration was supported by morphophysiological and gene expression analyses, which exhibited patterns similar to the wild type (WT) compared to the kmd3 mutant. Interestingly, the overexpression of PmFBK2 or PmGID1b in Arabidopsis had opposite effects on rosette diameter, seed weight, and plant height. This study provides new insights into the complex GA signalling. It highlights the crucial roles of the interaction between FBP and the GA receptor (GID1b) in regulating GA responses. These findings have implications for developing strategies to enhance plant growth and yield by modulating GA signalling in crops.

Harnessing the potential of non-coding RNA: An insight into its mechanism and interaction in plant biotic stress.

Plants have developed diverse physical and chemical defence mechanisms to ensure their continued growth and well-being in challenging environments. Plants also have evolved intricate molecular mechanisms to regulate their responses to biotic stress. Non-coding RNA (ncRNA) plays a crucial role in this process that affects the expression or suppression of target transcripts. While there have been numerous reviews on the role of molecules in plant biotic stress, few of them specifically focus on how plant ncRNAs enhance resistance through various mechanisms against different pathogens. In this context, we explored the role of ncRNA in exhibiting responses to biotic stress endogenously as well as cross-kingdom regulation of transcript expression. Furthermore, we address the interplay between ncRNAs, which can act as suppressors, precursors, or regulators of other ncRNAs. We also delve into the regulation of ncRNAs in response to attacks from different organisms, such as bacteria, viruses, fungi, nematodes, oomycetes, and insects. Interestingly, we observed that diverse microorganisms interact with distinct ncRNAs. This intricacy leads us to conclude that each ncRNA serves a specific function in response to individual biotic stimuli. This deeper understanding of the molecular mechanisms involving ncRNAs in response to biotic stresses enhances our knowledge and provides valuable insights for future research in the field of ncRNA, ultimately leading to improvements in plant traits.

Transcriptome profiling of genes induced by salicylic acid and methyl jasmonate in Polygonum minus.

The importance of plant secondary metabolites for both mankind and the plant itself has long been established. However, despite extensive research on plant secondary metabolites, plant secondary metabolism and its regulation still remained poorly characterized. In this present study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) transcript profiling was applied to generate the expression profiles of Polygonum minus in response to salicylic acid (SA) and methyl jasmonate (MeJA) elicitations. This study reveals two different sets of genes induced by SA and MeJA, respectively where stress-related genes were proved to lead to the expression of genes involved in plant secondary metabolite biosynthetic pathways. A total of 98 transcript-derived fragments (TDFs) were up-regulated, including 46 from SA-treated and 52 from MeJA-treated samples. The cDNA-AFLP transcripts generated using 64 different Mse1/Taq1 primer combinations showed that treatments with SA and MeJA induced genes mostly involved in scavenging reactive oxygen species, including zeaxanthin epoxidase, cytosolic ascorbate peroxidase 1 and peroxidase. Of these stress-related genes, 15 % of other annotated TDFs are involved mainly in secondary metabolic processes where among these, two genes encoding (+)-delta cadinene synthase and cinnamoyl-CoA reductase were highlighted.

Assessment of antioxidant and cytotoxicity activities of saponin and crude extracts of Chlorophytum borivilianum.

The present paper focused on antioxidant and cytotoxicity assessment of crude and total saponin fraction of Chlorophytum borivilianum as an important medicinal plant. In this study, three different antioxidant activities (2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH), ferrous ion chelating (FIC), and ß -carotene bleaching (BCB) activity) of crude extract and total saponin fraction of C. borivilianum tubers were performed. Crude extract was found to possess higher free radical scavenging activity (ascorbic acid equivalents 2578 ± 111 mg AA/100 g) and bleaching activity (IC50 = 0.7 mg mL(-1)), while total saponin fraction displayed higher ferrous ion chelating (EC50 = 1 mg mL(-1)). Cytotoxicity evaluation of crude extract and total saponin fraction against MCF-7, PC3, and HCT-116 cancer cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) cell viability assay indicated a higher cytotoxicity activity of the crude extract than the total saponin fraction on all cell lines, being most effective and selective on MCF-7 human breast cancer cell line.

Induction and analysis of the alkaloid mitragynine content of a Mitragyna speciosa suspension culture system upon elicitation and precursor feeding.

This study aimed to determine the effects of different concentrations and combinations of the phytohormones 2,4-dichlorophenoxy acetic acid (2,4-D), kinetin, 6-benzylaminopurine (BAP), and 1-naphthaleneacetic acid (NAA) on callus induction and to demonstrate the role of elicitors and exogenous precursors on the production of mitragynine in a Mitragyna speciosa suspension culture. The best callus induction was achieved from petiole explants cultured on WPM that was supplemented with 4 mg L⁻¹ 2,4-D (70.83%). Calli were transferred to liquid media and agitated on rotary shakers to establish Mitragyna speciosa cell suspension cultures. The optimum settled cell volume was achieved in the presence of WPM that contained 3 mg L⁻¹ 2,4-D and 3% sucrose (9.47 ± 0.4667 mL). The treatment of cultures with different concentrations of yeast extract and salicylic acid for different inoculation periods revealed that the highest mitragynine content as determined by HPLC was achieved from the culture treated with 250 mg L⁻¹ yeast extract (9.275 ± 0.082 mg L⁻¹) that was harvested on day 6 of culturing; salicylic acid showed low mitragynine content in all concentrations used. Tryptophan and loganin were used as exogenous precursors; the highest level of mitragynine production was achieved in cultures treated with 3 µM tryptophan and harvested at 6 days (13.226 ± 1.98 mg L⁻¹).

Overview of Repressive miRNA Regulation by Short Tandem Target Mimic (STTM): Applications and Impact on Plant Biology.

The application of miRNA mimic technology for silencing mature miRNA began in 2007. This technique originated from the discovery of the INDUCED BY PHOSPHATE STARVATION 1 (IPS1) gene, which was found to be a competitive mimic that prevents the cleavage of the targeted mRNA by miRNA inhibition at the post-transcriptional level. To date, various studies have been conducted to understand the molecular mimic mechanism and to improve the efficiency of this technology. As a result, several mimic tools have been developed: target mimicry (TM), short tandem target mimic (STTM), and molecular sponges (SPs). STTM is the most-developed tool due to its stability and effectiveness in decoying miRNA. This review discusses the application of STTM technology on the loss-of-function studies of miRNA and members from diverse plant species. A modified STTM approach for studying the function of miRNA with spatial-temporal expression under the control of specific promoters is further explored. STTM technology will enhance our understanding of the miRNA activity in plant-tissue-specific development and stress responses for applications in improving plant traits via miRNA regulation.

Postharvest analysis of lowland transgenic tomato fruits harboring hpRNAi-ACO1 construct.

The plant hormone, ethylene, is an important regulator which involved in regulating fruit ripening and flower senescence. In this study, RNA interference (RNAi) technology was employed to silence the genes involved in ethylene biosynthetic pathway. This was achieved by blocking the expression of specific gene encoding the ACC oxidase. Initially, cDNA corresponding to ACO1 of lowland tomato cultivar (MT1), which has high identity with ACO1 of Solanum lycopersicum in GenBank, was cloned through RT-PCR. Using a partial coding region of ACO1, one hpRNAi transformation vector was constructed and expressed ectopically under the 35S promoter. Results showed that transgenic lines harboring the hpRNA-ACO1 construct had lower ethylene production and a longer shelf life of 32 days as compared to 10 days for wild-type fruits. Changes in cell wall degrading enzyme activities were also investigated in cases where the transgenic fruits exhibited reduced rates of firmness loss, which can be associated with a decrease in pectin methylesterase (PME) and polygalacturonase (PG) activities. However, no significant change was detected in both transgenic and wild-type fruits in terms of ß-galactosidase (ß-Gal) activity and levels of total soluble solid, titratable acid and ascorbic acid.

Effects of 1-methylcyclopropene and modified atmosphere packaging on the antioxidant capacity in pepper "Kulai" during low-temperature storage.

The objective of the present study was to simultaneously evaluate the effect of a postharvest treatment on the pepper's antioxidant content and its ability to retain its economical value during the postharvest period. The fruits were pretreated by modified atmosphere packaging (MAP) with or without treatment with 1-methylcyclopropene (1-MCP) before cold storage at 10°C. Changes in the levels of non-enzymatic antioxidants, including the total phenolic, ascorbic acid levels and the total glutathione level, as well as enzymatic antioxidants, including ascorbate peroxidase (APX), glutathione reductase (GR), and catalase (CAT), were determined. Both treatments successfully extended the shelf life of the fruit for up to 25 days, and a high level of antioxidant capacity was maintained throughout the storage period. However, 1-MCP treatment maintained the high antioxidant capacity for a longer period of time. The 1-MCP-treated peppers maintained high levels of phenolic content, a high reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio, decreased levels of ascorbic acid and CAT activity, and increased levels of APX and GR compared with the peppers that were not treated with 1-MCP. The overall results suggested that a combination of 1-MCP and MAP was the most effective treatment for extending shelf life while retaining the nutritional benefits.

Elaeis oleifera genomic-SSR markers: exploitation in oil palm germplasm diversity and cross-amplification in arecaceae.

Species-specific simple sequence repeat (SSR) markers are favored for genetic studies and marker-assisted selection (MAS) breeding for oil palm genetic improvement. This report characterizes 20 SSR markers from an Elaeis oleifera genomic library (gSSR). Characterization of the repeat type in 2000 sequences revealed a high percentage of di-nucleotides (63.6%), followed by tri-nucleotides (24.2%). Primer pairs were successfully designed for 394 of the E. oleifera gSSRs. Subsequent analysis showed the ability of the 20 selected E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The average Polymorphism Information Content (PIC) value for the SSRs was 0.402, with the tri-repeats showing the highest average PIC (0.626). Low values of observed heterozygosity (H(o)) (0.164) and highly positive fixation indices (F(is)) in the E. oleifera germplasm collection, compared to the E. guineensis, indicated an excess of homozygosity in E. oleifera. The transferability of the markers to closely related palms, Elaeis guineensis, Cocos nucifera and ornamental palms is also reported. Sequencing the amplicons of three selected E. oleifera gSSRs across both species and palm taxa revealed variations in the repeat-units. The study showed the potential of E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The markers are also a valuable genetic resource for studying E. oleifera and other genus in the Arecaceae family.

Simple and rapid molecular techniques for identification of amylose levels in rice varieties.

The polymorphisms of Waxy (Wx) microsatellite and G-T single-nucleotide polymorphism (SNP) in the Wx gene region were analyzed using simplified techniques in fifteen rice varieties. A rapid and reliable electrophoresis method, MetaPhor agarose gel electrophoresis (MAGE), was effectively employed as an alternative to polyacrylamide gel electrophoresis (PAGE) for separating Wx microsatellite alleles. The amplified products containing the Wx microsatellite ranged from 100 to 130 bp in length. Five Wx microsatellite alleles, namely (CT)(10), (CT)(11), (CT)(16), (CT)(17), and (CT)(18) were identified. Of these, (CT)(11) and (CT)(17) were the predominant classes among the tested varieties. All varieties with an apparent amylose content higher than 24% were associated with the shorter repeat alleles; (CT)(10) and (CT)(11), while varieties with 24% or less amylose were associated with the longer repeat alleles. All varieties with intermediate and high amylose content had the sequence AGGTATA at the 5'-leader intron splice site, while varieties with low amylose content had the sequence AGTTATA. The G-T polymorphism was further verified by the PCR-AccI cleaved amplified polymorphic sequence (CAPS) method, in which only genotypes containing the AGGTATA sequence were cleaved by AccI. Hence, varieties with desirable amylose levels can be developed rapidly using the Wx microsatellite and G-T SNP, along with MAGE.

Flavonoid biosynthesis genes putatively identified in the aromatic plant Polygonum minus via Expressed Sequences Tag (EST) analysis.

P. minus is an aromatic plant, the leaf of which is widely used as a food additive and in the perfume industry. The leaf also accumulates secondary metabolites that act as active ingredients such as flavonoid. Due to limited genomic and transcriptomic data, the biosynthetic pathway of flavonoids is currently unclear. Identification of candidate genes involved in the flavonoid biosynthetic pathway will significantly contribute to understanding the biosynthesis of active compounds. We have constructed a standard cDNA library from P. minus leaves, and two normalized full-length enriched cDNA libraries were constructed from stem and root organs in order to create a gene resource for the biosynthesis of secondary metabolites, especially flavonoid biosynthesis. Thus, large-scale sequencing of P. minus cDNA libraries identified 4196 expressed sequences tags (ESTs) which were deposited in dbEST in the National Center of Biotechnology Information (NCBI). From the three constructed cDNA libraries, 11 ESTs encoding seven genes were mapped to the flavonoid biosynthetic pathway. Finally, three flavonoid biosynthetic pathway-related ESTs chalcone synthase, CHS (JG745304), flavonol synthase, FLS (JG705819) and leucoanthocyanidin dioxygenase, LDOX (JG745247) were selected for further examination by quantitative RT-PCR (qRT-PCR) in different P. minus organs. Expression was detected in leaf, stem and root. Gene expression studies have been initiated in order to better understand the underlying physiological processes.