Genetic diversity and patterns of population structure of the 94 oil palm lines were investigated using species-specific simple sequence repeat (SSR) markers. We designed primers for 63 SSR loci based on their flanking sequences and conducted amplification in 94 oil palm DNA samples. The amplification result showed that a relatively high level of genetic diversity was observed between oil palm individuals according a set of 21 polymorphic microsatellite loci. The observed heterozygosity (Ho) was 0.3683 and 0.4035, with an average of 0.3859. The Ho value was a reliable determinant of the discriminatory power of the SSR primer combinations. The principal component analysis and unweighted pair-group method with arithmetic averaging cluster analysis showed the 94 oil palm lines were grouped into one cluster. These results demonstrated that the oil palm in Hainan Province of China and the germplasm introduced from Malaysia may be from the same source. The SSR protocol was effective and reliable for assessing the genetic diversity of oil palm. Knowledge of the genetic diversity and population structure will be crucial for establishing appropriate management stocks for this species.
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.
Kemunting (Rhodomyrtus tomentosa) from the Myrtaceae family, is native to Malaysia. It is widely used in traditional medicine to treat various illnesses and possesses significant antibacterial properties. In addition, it has great potential as ornamental in landscape design. Genetic variability studies are important for the rational management and conservation of genetic material. In the present study, inter-simple sequence repeat markers were used to assess the genetic diversity of 18 R. tomentosa populations collected from ten states of Peninsular Malaysia. The 11 primers selected generated 173 bands that ranged in size from 1.6 kb to 130 bp, which corresponded to an average of 15.73 bands per primer. Of these bands, 97.69% (169 in total) were polymorphic. High genetic diversity was documented at the species level (H(T) = 0.2705; I = 0.3973; PPB = 97.69%) but there was a low diversity at population level (H(S) = 0.0073; I = 0 .1085; PPB = 20.14%). The high level of genetic differentiation revealed by G(ST) (73%) and analysis of molecular variance (63%), together with the limited gene flow among population (N(m) = 0.1851), suggests that the populations examined are isolated. Results from an unweighted pair group method with arithmetic mean dendrogram and principal coordinate analysis clearly grouped the populations into two geographic groups. This clear grouping can also be demonstrated by the significant Mantel test (r = 0.581, P = 0.001). We recommend that all the R. tomentosa populations be preserved in conservation program.
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.
Rape seed (Brassica napus L.) is one of the most important oil seed crops in the world. Genetic manipulation of rapeseed requires a suitable tissue culture system and an efficient method for plant regeneration, as well as an efficient transformation procedure. However, development of transgenic B. napus has been problematic, and current studies are limited to cultivated varieties. In this study, we report a protocol for regeneration of transgenic rape after Agrobacterium-mediated transformation of hypocotyls from the spring B. napus 'Precocity' cultivar. We analyzed the effects of plant growth regulators in the medium on regeneration. Additionally, factors affecting the transformation efficiency, including seedling age, Agrobacterium concentration, infection time, and co-cultivation time, were assessed by monitoring GUS expression. Results from these experiments revealed that transformation was optimized when the meristematic parts of the hypocotyls were taken from 8 day-old seedlings, cultured on Murashinge and Skoog basal media containing 0.1 mg/L 1-naphthaleneacetic acid and 2.5 mg/L 6-benzylaminopurine, and incubated in Agrobacterium suspension (OD600 = 0.5) for 3 to 5 min, followed by 2 days of co-cultivation. Integration of T-DNA into the plant genome was confirmed by polymerase chain reaction (PCR), b-glucuronidase histochemical staining, and quantitative real-time PCR. The protocols developed for regeneration, transformation, and rooting described in this study could help to accelerate the development of transgenic spring rape varieties with novel features.
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.
To examine the impact of regional and seasonal variations on the incidence and severity of sheath rot, a major seed-borne disease of rice caused by Sarocladium oryzae, data on incidence and severity were collected from 27 selected fields in the Gazipur, Rangpur, Bogra, Chittagong, Comilla, Gopalgonj, Jessore, Manikgonj, and Bhola districts of Bangladesh in rain-fed and irrigated conditions. Cultural variability of 29 pathogen isolates obtained from 8 different locations was studied on potato dextrose agar (PDA) and genetic variability was determined by DNA fingerprinting using variable number tandem repeat-polymerase chain reaction markers. Overall, disease incidence and severity were higher in irrigated rice. Disease incidence and severity were highest in the Bhola district in rain-fed rice and lowest in irrigated rice. Mycelial growth of 29 representative isolates was found to vary on PDA and the isolates were divided into 6 groups. The range of the overall size of conidia of the selected isolates was 2.40-7.20 x 1.20-2.40 μm. Analysis of the DNA fingerprint types of the 29 isolates of S. oryzae, obtained from the amplification reactions, revealed 10 fingerprinting types (FPTs) that were 80% similar. FPT-1 was the largest group and included 13 isolates (44.8%), while FPT-2 was the third largest group and included 3 isolates. Each of FPT-3, 4, 5, and 6 included only 1 isolate. We observed no relationship between cultural and genetic groupings.
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare, autosomal recessive disorder associated with mutations in the thymidine phosphorylase (TYMP) gene. The main objective of this study was to characterize the genetic profiles of the deceased proband's family members (N = 4) using DNA sequencing and to determine miRNA deregulation in MNGIE using miRNA microarray profiling and bioinformatic analysis. We found that the genetic profile of the younger sister showed similar TYMP gene mutations as that of the proband with the exception of a heterozygous mutation in exon 10. The miRNA microarray revealed 55 significantly up-regulated and 65 significantly down-regulated miRNAs. These miRNAs have been implicated in various mitochondrial dynamics such as energy metabolism, Krebs cycle, mitochondria-associated apoptosis, and mitophagy. In conclusion, we demonstrate that blood miRNAs are deregulated in the pathogenesis of MNGIE and these changes may have therapeutic implications. Further experimental studies will be required to elucidate the functional miRNA-mRNA interactions in MNGIE.
The quality of RNA is crucial when performing microarray experiments. This is particularly important when dealing with preimplantation embryos, from which a minimum yield of RNA of good quality can be produced. We report the optimization of several RNA extraction methods applied to preimplantation embryos at different stages of development. The quality of the samples was confirmed using a microarray and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis. A total of 30 cultured two-cell stage embryos of ICR mice were pooled at the 8-cell, morula, and blastocyst stages. The embryos were divided into two groups comprising DNase-treated and non-DNase-treated RNA samples. Total RNA was extracted using a Pico Pure RNA Isolation Kit following the manufacturer protocol, with some modifications. Lysed samples were bound to a silica-based filter, treated with deoxyribonuclease I (DNase I), and washed several times before elution. RNA concentration and integrity were evaluated using an Agilent 2100 Bioanalyzer and an RNA 6000 Pico Assay kit. Although concentrations of non-DNase-treated RNAs were higher than DNase-treated RNA, DNase-treated RNA gave a higher RNA integrity number compared with non-DNase-treated RNA. Inclusion of DNase treatment in the RNA extraction procedure gave the best quality RNA samples from preimplantation embryos, as validated by microarray and RT-qPCR quality control.
Inheritance of polymorphisms in the interleukin (IL)-10 promoter and IL-12B genes, which influence cytokine production and activities, may define the balance in T helper response in infection and autoimmune diseases. In the present study, we investigated the distribution of the IL-10 promoter and IL-12B gene polymorphisms in a multiethnic Malaysian population. Overall, our findings suggest that the IL-12B and IL-10 -592 genotypes were distributed homogenously across all major ethnic groups, including Malays, Chinese, and Indians, except for polymorphisms at IL-10 -1082. At this gene locus, the ethnic Chinese showed a significantly lower allele frequency of -1082G (2.1%) compared to the Malay (12.2%) and Indian (15.3%) populations. Results for the IL-12B and IL-10 gene polymorphisms were consistent with those reported for the Asian population, but markedly different from those of the African and Caucasian populations. Our findings suggest that there are specific genetic variations between different ethnic groups, which should be examined in all gene population-based association studies.
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.
Grain weight is a major component of rice grain yield and is controlled by quantitative trait loci. Previously, a rice grain weight quantitative trait locus (qGW6) was detected near marker RM587 on chromosome 6 in a backcross population (BC2F2) derived from a cross between Oryza rufipogon IRGC105491 and O. sativa cv. MR219. Using a BC2F5 population, qGW6 was validated and mapped to a region of 4.8 cM (1.2 Mb) in the interval between RM508 and RM588. Fine mapping using a series of BC4F3 near isogenic lines further narrowed the interval containing qGW6 to 88 kb between markers RM19268 and RM19271.1. According to the Duncan multiple range test, 8 BC4F4 near isogenic lines had significantly higher 100-grain weight (4.8 to 7.5% over MR219) than their recurrent parent, MR219 (P < 0.05). According to the rice genome automated annotation database, there are 20 predicted genes in the 88-kb target region, and 9 of them have known functions. Among the genes with known functions in the target region, in silico gene expression analysis showed that 9 were differentially expressed during the seed development stage(s) from gene expression series GSE6893; however, only 3 of them have known functions. These candidates provide targets for further characterization of qGW6, which will assist in understanding the genetic control of grain weight in rice.
Commercial flocks infected by Eimeria species parasites, including Eimeria maxima, have an increased risk of developing clinical or subclinical coccidiosis; an intestinal enteritis associated with increased mortality rates in poultry. Currently, infection control is largely based on chemotherapy or live vaccines; however, drug resistance is common and vaccines are relatively expensive. The development of new cost-effective intervention measures will benefit from unraveling the complex genetic mechanisms that underlie host-parasite interactions, including the identification and characterization of genes encoding proteins such as phosphatidylinositol 4-phosphate 5-kinase (PIP5K). We previously identified a PIP5K coding sequence within the E. maxima genome. In this study, we analyzed two bacterial artificial chromosome clones presenting a ~145-kb E. maxima (Weybridge strain) genomic region spanning the PIP5K gene locus. Sequence analysis revealed that ~95% of the simple sequence repeats detected were located within regions comparable to the previously described feature-rich segments of the Eimeria tenella genome. Comparative sequence analysis with the orthologous E. maxima (Houghton strain) region revealed a moderate level of conserved synteny. Unique segmental organizations and telomere-like repeats were also observed in both genomes. A number of incomplete transposable elements were detected and further scrutiny of these elements in both orthologous segments revealed interesting nesting events, which may play a role in facilitating genome plasticity in E. maxima. The current analysis provides more detailed information about the genome organization of E. maxima and may help to reveal genotypic differences that are important for expression of traits related to pathogenicity and virulence.
Growth factors are polypeptides that are critical for the initiation, progression, and metastasis of cancer. Most tumor cells are capable of synthesizing particular growth factors leading to constitutive pathway activation in these cells through autocrine signaling. Epidermal growth factor (EGF) is a potent mitogenic peptide that exerts direct effects on the proliferation and differentiation of tumor cells in carcinogenesis. By contrast, vascular endothelial growth factor (VEGF) is vital for the invasion and metastasis of neoplasms through the formation of new blood vessels from mature endothelial cells. In this study, we investigated the association between functional polymorphisms of both the EGF and VEGF genes and colorectal cancer (CRC) susceptibility. A total of 130 CRC patients and 212 healthy controls were recruited for this case-control study. Genotyping of genetic variants was conducted via real-time polymerase chain reaction (PCR) amplification with allele-specific TaqMan probes. None of the genotypes of the EGF +61 A>G and VEGF +936 C>T variants was significantly associated with CRC susceptibility among the Malaysian subjects evaluated (P > 0.05). The observed frequency distributions of the EGF +61 A>G polymorphism genotypes showed ethnic heterogeneity, which was not the case for the VEGF +936 C>T genotypes. In conclusion, no positive correlation between these functional polymorphisms and CRC risk was found in this Malaysian population. Studies of the EGF and VEGF genes and CRC susceptibility are scarce, and the results reported thus far differ from one population to another. Hence, more replication studies are warranted before any firm conclusions can be made.
Calamus palustris Griff. is an economically important dioecious rattan species in Southeast Asia. However, dioecy and onset of flowering at 3-4 years old render uncertainties in desired female:male seedling ratios to establish a productive seed orchard for this rattan species. We constructed a subtractive library for male floral tissue to understand the genetic mechanism for gender determination in C. palustris. The subtractive library produced 1536 clones with 1419 clones of high quality. Reverse Northern screening showed 313 clones with differential expression, and sequence analyses clustered them into 205 unigenes, including 32 contigs and 173 singletons. The subtractive library was further validated with reverse transcription-quantitative polymerase chain reaction analysis. Homology identification classified the unigenes into 12 putative functional proteins with 83% unigenes showing significant match to proteins in databases. Functional annotations of these unigenes revealed genes involved in male flower development, including MADS-box genes, pollen-related genes, phytohormones for flower development, and male flower organ development. Our results showed that the male floral genes may play a vital role in sex determination in C. palustris. The identified genes can be exploited to understand the molecular basis of sex determination in C. palustris.
The mosquito Aedes albopictus is indigenous to Southeast Asian and is a vector for arbovirus diseases. Studies examining the population genetics structure of A. albopictus have been conducted worldwide; however, there are no documented reports on the population genetic structure of A. albopictus in Malaysia, particularly in Penang. We examined the population genetics of A. albopictus based on a 445-base pair segment of the mitochondrial DNA cytochrome oxidase 1 gene among 77 individuals from 9 localities representing 4 regions (Seberang Perai Utara, Seberang Perai Tengah, Northeast, and Southwest) of Penang. A total of 37 haplotypes were detected, including 28 unique haplotypes. The other 9 haplotypes were shared among various populations. These shared haplotypes reflect the weak population genetic structure of A. albopictus. The phylogenetic tree showed a low bootstrap value with no genetic structure, which was supported by minimum spanning network analysis. Analysis of mismatch distribution showed poor fit of equilibrium distribution. The genetic distance showed low genetic variation, while pairwise FST values showed no significant difference between all regions in Penang except for some localities. High haplotype diversity and low nucleotide diversity was observed for cytochrome oxidase 1 mtDNA. We conclude that there is no population genetic structure of A. albopictus mosquitoes in the Penang area.
For centuries, morphology-based fish identification has been applied without molecular evaluation. Many studies showed that specimens with a similar morphology are frequently found to be quite genetically distinct. One of the fish species that still remains taxonomically problematic is a commercial snapper species, Lutjanus johnii. Because of morphological ambiguities among local fish taxonomists in Malaysia, we examined the ability of the cytochrome oxidase I (COI) gene to genetically examine the taxonomic status of L. johnii. A 626-base pair COI region was successfully amplified and aligned with conspecific sequences that were retrieved from GenBank. The phylogenetic tree obtained showed two major clusters; the first cluster consists of L. johnii from Straits of Malacca, Thailand, Australia, and China while the second cluster comprises L. johnii from China and India. The latter group showed sequence divergence greater than 3.5%. After observing this, we suspected that there might be a cryptic species between the South China Sea and Indian Ocean. This is the first molecular report concerning the commercial species of snapper, L. johnii, in Malaysia, which had only gained provisional recognition from morphological examination.
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.
The genus Curcuma is a member of the ginger family (Zingiberaceae) that has recently become popular for use as flowering pot plants, both indoors and as patio and landscape plants. We used PCR-based molecular markers (SSRs) to elucidate genetic variation and relationships between five varieties of Curcuma (Curcuma alismatifolia) cultivated in Malaysia. Of the primers tested, 8 (of 17) SSR primers were selected for their reproducibility and high rates of polymorphism. The number of presumed alleles revealed by the SSR analysis ranged from two to six alleles, with a mean value of 3.25 alleles per locus. The values of HO and HE ranged from 0 to 0.8 (mean value of 0.2) and 0.1837 to 0.7755 (mean value of 0.5102), respectively. Eight SSR primers yielded 26 total amplified fragments and revealed high rates of polymorphism among the varieties studied. The polymorphic information content varied from 0.26 to 0.73. Dice's similarity coefficient was calculated for all pairwise comparisons and used to construct an unweighted pair group method with arithmetic average (UPGMA) dendrogram. Similarity coefficient values from 0.2105 to 0.6667 (with an average of 0.4386) were found among the five varieties examined. A cluster analysis of data using a UPGMA algorithm divided the five varieties/hybrids into 2 groups.
This study was directed at the understanding of the function of CCoAOMT isolated from Acacia auriculiformis x Acacia mangium. Full length cDNA of the Acacia hybrid CCoAOMT (AhCCoAOMT) was 1024-bp long, containing 750-bp coding regions, with one major open reading frame of 249 amino acids. On the other hand, full length genomic sequence of the CCoAOMT (AhgflCCoAOMT) was 2548 bp long, containing three introns and four exons with a 5' untranslated region (5'UTR) of 391 bp in length. The 5'UTR of the characterized CCoAOMT gene contains various regulatory elements. Southern analysis revealed that the Acacia hybrid has more than three copies of the CCoAOMT gene. Real-time PCR showed that this gene was expressed in root, inner bark, leaf, flower and seed pod of the Acacia hybrid. Downregulation of the homologous CCoAOMT gene in tobacco by antisense (AS) and intron-containing hairpin (IHP) constructs containing partial AhCCoAOMT led to reduction in lignin content. Expression of the CCoAOMT in AS line (pART-HAS78-03) and IHP line (pART-HIHP78-06) was reduced respectively by 37 and 75% compared to the control, resulting in a decrease in the estimated lignin content by 24 and 56%, respectively. AhCCoAOMT was found to have altered not only S and G units but also total lignin content, which is of economic value to the pulp industry. Subsequent polymorphism analysis of this gene across eight different genetic backgrounds each of A. mangium and A. auriculiformis revealed 47 single nucleotide polymorphisms (SNPs) in A. auriculiformis CCoAOMT and 30 SNPs in A. mangium CCoAOMT.