Rice (Oryza sativa L.) blast disease is one of the most destructive rice diseases in the world. The fungal pathogen, Magnaporthe oryzae, is the causal agent of rice blast disease. Development of resistant cultivars is the most preferred method to achieve sustainable rice production. However, the effectiveness of resistant cultivars is hindered by the genetic plasticity of the pathogen genome. Therefore, information on genetic resistance and virulence stability are vital to increase our understanding of the molecular basis of blast disease resistance. The present study set out to elucidate the resistance pattern and identify potential simple sequence repeat markers linked with rice blast disease. A backcross population (BC2F1), derived from crossing MR264 and Pongsu Seribu 2 (PS2), was developed using marker-assisted backcross breeding. Twelve microsatellite markers carrying the blast resistance gene clearly demonstrated a polymorphic pattern between both parental lines. Among these, two markers, RM206 and RM5961, located on chromosome 11 exhibited the expected 1:1 testcross ratio in the BC2F1 population. The 195 BC2F1 plants inoculated against M. oryzae pathotype P7.2 showed a significantly different distribution in the backcrossed generation and followed Mendelian segregation based on a single-gene model. This indicates that blast resistance in PS2 is governed by a single dominant gene, which is linked to RM206 and RM5961 on chromosome 11. The findings presented in this study could be useful for future blast resistance studies in rice breeding programs.
Ursodeoxycholic acid (UDCA) is used to treat liver diseases and demonstrates cardioprotective effects. Accumulation of the plasma membrane sphingolipid sphingomyelin in the heart can lead to atherosclerosis and coronary artery disease. Sphingomyelinases (SMases) break down sphingomyelin, producing ceramide, and inhibition of SMases activity can promote cell survival. We hypothesized that UDCA regulates activation of ERK and Akt survival signaling pathways and SMases in protecting cardiac cells against hypoxia. Neonatal cardiomyocytes were isolated from 0- to 2-day-old Sprague Dawley rats, and given 100 μM CoCl2, 150 μM H2O2, or placed in a hypoxia chamber for 24 h. The ameliorative effects of 100-μM UDCA treatment for 12 h were then assessed using MTS, QuantiGene Plex (for Smpd1 and Smpd2), and SMase assays, beating rate assessment, and western blotting (for ERK and Akt). Data were analyzed by the paired Student t-tests and one-way analyses of variance. Cell viability decreased significantly after H2O2 (85%), CoCl2 (50%), and hypoxia chamber (52%) treatments compared to the untreated control (100%). UDCA significantly counteracted the effects of chamber- and CoCl2- induced hypoxia on viability and beating rate. However, no significant differences were observed in acid SMase gene and protein expression between the untreated, CoCl2, and UDCA-CoCl2 groups. In contrast, neutral SMase gene and protein expression did significantly differ between the latter two groups. ERK and Akt phosphorylation was higher in hypoxic cardiomyocytes treated with UDCA than those given CoCl2 alone. In conclusion, UDCA regulates the activation of survival signaling proteins and SMases in neonatal rat cardiomyocytes during hypoxia.
Oil palm tissues are rich in polyphenols, polysaccharides and secondary metabolites; these can co-precipitate with RNA, causing problems for downstream applications. We compared two different methods (one conventional and a kit-based method - Easy-Blue(TM) Total RNA Extraction Kit) to isolate total RNA from leaves, roots and shoot apical meristems of tissue culture derived truncated leaf syndrome somaclonal oil palm seedlings. The quality and quantity of total RNA were compared through spectrophotometry and formaldehyde gel electrophoresis. The specificity and applicability of the protocols were evaluated for downstream applications, including cDNA synthesis and RT-PCR analysis. We found that the conventional method gave higher yields of RNA but took longer, and it was contaminated with genomic DNA. This method required extra genomic DNA removal steps that further reduced the RNA yield. The kit-based method, on the other hand, produced good yields as well as well as good quality RNA, within a very short period of time from a small amount of starting material. Moreover, the RNA from the kit-based method was more suitable for synthesizing cDNA and RT-PCR amplification than the conventional method. Therefore, we conclude that the Easy-BlueTM Total RNA Extraction Kit method is suitable and superior for isolation of total RNA from oil palm leaf, root and shoot apical meristem.
This study aims to investigate the effects of tumor necrosis factor alpha (TNF-α) G308A gene polymorphism on essential hypertension (EHT) with or without type 2 diabetes mellitus (T2DM). The project was conducted on buccal epithelial and blood cells for case and control patients, respectively. Epithelial cells were obtained from the inner part of the cheeks. Techniques including DNA extraction, polymerase chain reaction (PCR), and restriction fragment length polymorphism (RFLP) were utilized to assess biomarkers of DNA damage. Our results demonstrated significant differences between wild and mutated genotypes among EHT patients without T2DM. We also found a significant association between wild and mutated allele frequencies in EHT patients (P < 0.05). Clinical characteristics between the groups (EHT with or without T2DM and controls) showed statistically significant association (P < 0.05). Overall, we show that G308A polymorphism of the TNF-αgene may be a significant genetic risk factor for EHT without T2DM patients in Malaysia.
Cerebral ischemia or ischemic stroke is mainly attributed to vascular and circulation disorders. Among protein biomarkers, RNA profiles have also been identified as markers of ischemic stroke. MicroRNA-145 expression is ostensibly recognized as marker and modulator of vascular smooth muscle cell phenotype; however, expression levels in ischemic stroke had not been investigated. Employing real-time quantitative PCR, we examined the expression profile of circulatory microRNA-145 in healthy control subjects (N = 14) and ischemic stroke patients (N = 32). Circulatory microRNA-145 expression was significantly higher in ischemic stroke patients than in control subjects. This demonstrates that hemostatic mechanisms are affected by ischemic stroke. We conclude that circulating microRNA-145 has potential as a biomarker for ischemic stroke.
A limited backcross procedure was utilized to introgress genes associated with grain quality traits from Oryza rufipogon (Accession No. IRGC 105491), a wild rice from Malaysia, to the cultivated rice O. sativa cv. MR219, a popular high yielding Malaysian rice cultivar. A set of 10 BC(2)F(7) progenies were selected based on the field performance and phenotypic appearance in BC(2)F(5) and BC(2)F(6) generations, which initially started with 266 progenies in the BC(2)F(2) generation. These 10 advanced breeding lines are similar to each other but differ in several important grain quality traits, which can be traced to O. rufipogon introgressions. Phenotyping and genotyping of BC(2)F(7) variants were considered for QTL analysis. The introgressed lines did not show any significant changes compared to the recurrent parent MR219 for the traits grain density and milled rice percentage. All 10 progenies showed significantly higher head rice percentages (70-88%) than the recurrent parent MR219. Variants G13 and G15 had higher amylose contents than MR219. All variants were analyzed using polymorphic SSR markers. Of the 34 SSR markers, only 18 showed introgression from O. rufipogon for chromosomes 1, 2, 3, 5, 6, 8, 10, and 11. Graphical genotypes were prepared for each variant, and association between the introgression regions and the traits that increased grain quality was visualized. Based on marker trait association, some of the QTLs are stable across environments and genetic backgrounds and could be used universally.
Type 2 diabetes mellitus (T2DM) is believed to be associated with excessive production of reactive oxygen species. Glutathione S-transferase (GST) polymorphisms result in decreased or absent enzyme activity and altered oxidative stress, and have been associated with cardiovascular disease (CVD). The present study assessed the effect of GST polymorphisms on the risk of developing T2DM in individuals of Malaysian Malay ethnicity. A total of 287 subjects, consisting of 87 T2DM and 64 CVD/T2DM patients, as well as 136 healthy gender- and age-matched controls were genotyped for selected polymorphisms to evaluate associations with T2DM susceptibility. Genomic DNA was extracted using commercially available kits, and GSTM1, GSTT1, and α-globin sequences were amplified by multiplex polymerase chain reaction. Biochemical parameters were measured with a Hitachi autoanalyzer. The Fisher exact test, the chi-square statistic, and means ± standard deviations were calculated using the SPSS software. Overall, we observed no significant differences regarding genotype and allele frequencies between each group (P = 0.224 and 0.199, respectively). However, in the combined analysis of genotypes and blood measurements, fasting plasma glucose, HbA1c, and triglyceride levels, followed by age, body mass index, waist-hip ratio, systolic blood pressure, and history of T2DM significantly differed according to GST polymorphism (P ˂ 0.05). Genetically induced absence of the GSTT1 enzyme is an independent and powerful predictor of premature vascular morbidity and death in individuals with T2DM, and might be triggered by cigarette smoking's oxidative effects. These polymorphisms could be screened in other ethnicities within Malaysia to determine further possible risk factors.
We evaluated the possible influence of glutathione S-transferase mu (GSTM1) and glutathione S-transferase theta (GSTT1) genes on genetic damage due to occupational exposure, which contributes to accelerate ageing. This study was conducted on 120 car auto repair workshop workers exposed to occupational hazards and 120 controls without this kind of exposure. The null and non-null genotypes of GSTM1 and GSTT1 genes were determined by multiplex PCR. Micronucleus frequency, Comet tail length and relative telomere length differences between the null and non-null genotypes of the GSTM1 gene were significantly greater in the exposed group. Lack of GSTT1 did not affect the damage biomarkers significantly (P > 0.05), while lack of GSTM1 was associated with greater susceptibility to genomic damage due to occupational exposure. It was concluded that early ageing is under the influence of these genes and the environmental and socio-demographic factors. Duration of working time was significantly associated with micronucleus frequency, Comet tail length and relative telomere length.
Mackerel (Scombridae; Rastrelliger) are small commercially important pelagic fish found in tropical regions. They serve as a cheap source of animal protein and are commonly used as live bait. By using a truss morphometrics protocol and RAPD analysis, we examined morphological and genetic variation among 77 individual mackerel that were caught using long lines and gillnets at 11 locations along the west coast of Peninsular Malaysia. Nineteen morphometric traits were evaluated and genetic information was estimated using five 10-base RAPD random primers. Total DNA was extracted from muscle tissue. Morphometric discriminant function analysis revealed that two morphologically distinct groups of Rastrelliger kanagurta and a single group of R. brachysoma can be found along the west coast of Peninsular Malaysia. We also found that the head-related characters and those from the anterior part of the body of Rastrelliger spp significantly contribute to stock assessment of this population. RAPD analysis showed a trend similar to that of the morphometric analysis, suggesting a genetic component to the observed phenotypic differentiation. These data will be useful for developing conservation strategies for these species.
Crohn's disease is a chronic, relapsing inflammatory bowel disease; it affects the mucosa and deeper layers of the digestive wall. Two Crohn's disease patients who carried the JW1 variant and two patients who carried the SNP5 variant were investigated for other co-inherited polymorphisms that could influence Crohn's disease development. Based on the sequencing results, a homozygous 5'-UTR-59 G to A variant in exon 1 (SNP6) was observed in a patient who carried SNP5, while a heterozygous SNP6 variant was detected in the other patient who carried SNP5. No other associated mutations or polymorphisms were detected in the two patients who carried the JW1 variant of the CARD15/NOD2 gene.
Hemagglutinin (HA) protein plays an important role in binding the influenza virus to infected cells and therefore mediates infection. Deposited HA sequences of 86 Asian strains of influenza A (H1N1) viruses during the first outbreak were obtained from the NCBI database and compared. Interaction of the HA protein of influenza A (H1N1) virus with the human sialic acid receptor was also studied using bioinformatics. Overall, not more than three single-point amino acid variants/changes were observed in the HA protein region of influenza A (H1N1) virus from Asian countries when a selected group sequence comparison was made. The bioinformatics study showed that the HA protein of influenza A (H1N1) binds to the sialic acid receptor in human airway receptors, possibly key to air-borne infection in humans.
Blood cockles are among the most economically important brackish water invertebrates found in Malaysia. However, our knowledge of blood cockle phylogeny and systematics is rudimentary, especially for the species Tegillarca granosa. It is unclear, for instance, whether the cockles occurring on the west coast of peninsular Malaysia constitute a single species, or multiple, phylogenetically distinct species. We performed the first DNA molecular phylogenetic analysis of T. granosa to distinguish it from other related species found in other parts of the world and to create a DNA database for the species. An approximately 585-nucleotide fragment of the mitochondrial DNA (cytochrome oxidase I, COI) was sequenced for 150 individual cockles, representing 10 populations: three from the north, four from the central part and three from the southern part of peninsular Malaysia. Phylogenetic analyses of the resulting dataset yielded tree topologies that not only showed the relationship between T. granosa and its closest relatives but its position in the evolutionary tree. Three mitochondrial clades were evident, each containing an individual genus. Using the mutation rate of the COI gene, the divergence time between T. granosa and its closest related species was estimated to be 460 thousand years ago. This study provides a phylogenetic framework for this ecologically prominent and commercially important cockle species.
We examined genetic variation in blood cockles in an effort to obtain information useful for the sustainability, management, and the stability of this species as a major commodity in the fisheries sector. Ten populations of cockles were sampled from the north to the south of the west coast of peninsular Malaysia. The cockles were collected in collaboration with the Fisheries Research Institute, Penang. The population genetic analysis of the cockles were studied via RAPD-PCR and mtDNA sequencing. Three hundred individuals were analyzed with RAPD-PCR experiments. High gene diversity over all loci was observed (Shannon index = 0.549 ± 0.056 and Nei's gene diversity = 0.4852 ± 0.0430 among 35 loci). The second method, mtDNA sequencing, was employed to complement the information obtained from RAPD-PCR. The gene selected for mtDNA sequencing was cytochrome c oxidase I (COI). One hundred and fifty individuals were sequenced, yielding a partial gene of 585 bp. Statistical analysis showed homogeneity in general but did reveal some degree of variability between the populations in Johor and the rest of the populations. The Mantel test showed a positive but nonsignificant correlation between geographic and genetic distances (r = 0.2710, P = 0.622), as in the RAPD analysis. We propose that the homogeneity between distant populations is caused by two factors: 1) the translocation of the spats; 2) larvae are carried by current movement from the north of the peninsula to the south. The different genetic composition found in Johor could be due to pollution, mutagenic substances or physical factors such as the depth of the water column. This population genetic study is the first for this species in peninsular Malaysia. The data from this study have important implications for fishery management, conservation of blood cockles and translocation policies for aquaculture and stock enhancement programs.
The mitochondrial DNA (mtDNA) cytochrome oxidase I (COI) gene has been universally and successfully utilized as a barcoding gene, mainly because it can be amplified easily, applied across a wide range of taxa, and results can be obtained cheaply and quickly. However, in rare cases, the gene can fail to distinguish between species, particularly when exposed to highly sensitive methods of data analysis, such as the Bayesian method, or when taxa have undergone introgressive hybridization, over-splitting, or incomplete lineage sorting. Such cases require the use of alternative markers, and nuclear DNA markers are commonly used. In this study, a dendrogram produced by Bayesian analysis of an mtDNA COI dataset was compared with that of a nuclear DNA ATPS-α dataset, in order to evaluate the efficiency of COI in barcoding Malaysian nerites (Neritidae). In the COI dendrogram, most of the species were in individual clusters, except for two species: Nerita chamaeleon and N. histrio. These two species were placed in the same subcluster, whereas in the ATPS-α dendrogram they were in their own subclusters. Analysis of the ATPS-α gene also placed the two genera of nerites (Nerita and Neritina) in separate clusters, whereas COI gene analysis placed both genera in the same cluster. Therefore, in the case of the Neritidae, the ATPS-α gene is a better barcoding gene than the COI gene.
The identification of new biomarkers for early detection of highly recurrent head and neck cancer is urgently needed. MicroRNAs (miRNAs) are small and non-coding RNAs that regulate cancer-related gene expression, such as tumor protein 53 (TP53) gene expression. This study was carried out to analyze TP53 gene expression using real-time PCR and to determine changes in intracellular p53 level by flow cytometry after downregulation of miRNA-181a miRNA inhibitor in the FaDu cell line. TP53 gene expression showed a 3-fold increment and the p53 protein level was also increased in the miRNA-181a-treated cells. In conclusion, miRNA-181a binds to the TP53 gene and inhibits its expression, decreasing the synthesis of p53.
Plants have been used throughout human history for food and medicine. However, many plants are toxic, and cannot easily be morphologically distinguished from non-toxic plants. DNA identification solves this problem and is widely used. Nonetheless, plant DNA barcode identification faces a number of challenges, and many studies have been conducted to find suitable barcodes. The present study was conducted to test the efficiency of commonly used primers, namely ITS2, rpoC1, and trnH-psbA, in order to find the best DNA barcode markers for the identification of medicinal plants in Malaysia. Fresh leaves from 12 medicinal plants that are commonly used by Malay traditional healers were collected from the Tropical Spice Garden, Pulau Pinang, and subjected to polymerase chain reaction amplification using ITS2, rpoC1, and trnH-psbA DNA markers. We found that trnH-psbA is the best DNA marker for the species-level identification of medicinal plants in Malaysia.
The Malaysian giant prawn is among the most commonly cultured species of the genus Macrobrachium. Stocks of giant prawns from four rivers in Peninsular Malaysia have been used for aquaculture over the past 25 years, which has led to repeated harvesting, restocking, and transplantation between rivers. Consequently, a stock improvement program is now important to avoid the depletion of wild stocks and the loss of genetic diversity. However, the success of such an improvement program depends on our knowledge of the genetic variation of these base populations. The aim of the current study was to estimate genetic variation and differentiation of these riverine sources using novel expressed sequence tag-microsatellite (EST-SSR) markers, which not only are informative on genetic diversity but also provide information on immune and metabolic traits. Our findings indicated that the tested stocks have inbreeding depression due to a significant deficiency in heterozygotes, and FIS was estimated as 0.15538 to 0.31938. An F-statistics analysis suggested that the stocks are composed of one large panmictic population. Among the four locations, stocks from Johor, in the southern region of the peninsular, showed higher allelic and genetic diversity than the other stocks. To overcome inbreeding problems, the Johor population could be used as a base population in a stock improvement program by crossing to the other populations. The study demonstrated that EST-SSR markers can be incorporated in future marker assisted breeding to aid the proper management of the stocks by breeders and stakeholders in Malaysia.
Among 120 simple sequence repeat (SSR) markers, 23 polymorphic markers were used to identify the segregation ratio in 320 individuals of an F(2) rice population derived from Pongsu Seribu 2, a resistant variety, and Mahsuri, a susceptible rice cultivar. For phenotypic study, the most virulent blast (Magnaporthe oryzae) pathotype, P7.2, was used in screening of F(2) population in order to understand the inheritance of blast resistance as well as linkage with SSR markers. Only 11 markers showed a good fit to the expected segregation ratio (1:2:1) for the single gene model (d.f. = 1.0, P < 0.05) in chi-square (χ(2)) analyses. In the phenotypic data analysis, the F(2) population segregated in a 3:1 (R:S) ratio for resistant and susceptible plants, respectively. Therefore, resistance to blast pathotype P7.2 in Pongsu Seribu 2 is most likely controlled by a single nuclear gene. The plants from F(2) lines that showed resistance to blast pathotype P7.2 were linked to six alleles of SSR markers, RM168 (116 bp), RM8225 (221 bp), RM1233 (175 bp), RM6836 (240 bp), RM5961 (129 bp), and RM413 (79 bp). These diagnostic markers could be used in marker assisted selection programs to develop a durable blast resistant variety.
Disease susceptibility and genetic variability in 10 eggplant genotypes were studied after inoculating Phomopsis vexans under confined field conditions. Random amplified polymorphic DNA (RAPD) markers were used to assess genetic variation and relationships among eggplant genotypes. The disease index of leaves ranged 0.208-13.79%, while fruit infection ranged 2.15-42.76%. Two varieties, Dohazari G and Laffa S, were found to be susceptible, 6 were moderately resistant, 1 was moderately susceptible, and BAU Begun-1 was resistant to P. vexans. Amplification of genomic DNA by using 3 RAPD primers produced 20 bands: 14 (70%) were polymorphic and 6 (30%) were monomorphic. The highest intra-variety similarity indices values were found in ISD 006, Ishurdi L, Jessore L, and BAU Begun-1 (100%), while the lowest was in Dohazari G (90%). The lowest genetic distance (0.0513) and the highest genetic identity (0.9500) were observed between the ISD 006 and Ishurdi L combinations. A comparatively higher genetic distance (0.3724) and the lowest genetic identity (0.6891) were observed between the ISD 006 and Dohazari G combinations. A dendogram was constructed based on Nei's genetic distance, which produced 2 main clusters of the genotypes - Cluster I: ISD 006, Ishurdi L, Marich begun L, BAU Begun-1, Marich begun S, and Chega and Cluster 2: Laffa S, Dohazari G, Jessore L, and Singhnath. Genetic variation and its relationship with disease susceptibility were assessed using RAPD markers, to develop disease-resistant varieties and improve eggplant crops.
A genome-wide association study showed that the tagging single nucleotide polymorphism (SNP) rs7566605 in the insulin-induced gene 2 (INSIG2) was associated with obesity. Attempts to replicate this result in different populations have produced inconsistent findings. We aimed to study the association between the rs7566605 SNP with obesity and other metabolic parameters in Malaysian Malays. Anthropometric and obesity-related metabolic parameters and DNA samples were collected. We genotyped the rs7566605 polymorphism in 672 subjects using real-time polymerase chain reaction. No significant associations were found between the rs7566605 tagging SNP of INSIG2 with obesity or other metabolic parameters in the Malaysian Malay population. The INSIG2 rs7566605 SNP may not play a role in the development of obesity-related metabolic traits in Malaysian Malays.