PCR has been extensively used for amplification of DNA sequences. We conducted a study to obtain the best amplification conditions for cytochrome b (Cyt b), cytochrome c oxidase I (COI) and 12S rRNA (12S) gene fragments of Malayan gaur mtDNA. DNA from seven Malayan gaur samples were extracted for PCR amplification. Various trials and combinations were tested to determine the best conditions of PCR mixture and profile to obtain the best PCR products for sequencing purposes. Four selected target factors for enhancing PCR, annealing temperature, concentration of primer pairs, amount of Taq polymerase, and PCR cycle duration, were optimized by keeping the amount of DNA template (50 ng/μL) and concentration of PCR buffer (1X), MgCl(2) (2.5 mM) and dNTP mixture (200 μM each) constant. All genes were successfully amplified, giving the correct fragment lengths, as assigned for both forward and reverse primers. The optimal conditions were determined to be: 0.1 μM primers for Cyt b and COI, 0.3 μM primers for 12S, 1 U Taq polymerase for all genes, 30 s of both denaturation and annealing cycles for Cyt b, 1 min of both stages for 12S and COI and annealing temperature of 58.4 ° C for Cyt b, 56.1 ° C for 12S and 51.3 ° C for COI. PCR products obtained under these conditions produced excellent DNA sequences.
Mitochondrial DNA (mtDNA) is a useful genetic marker that can be used for species identification. The cytochrome b (Cyt b) gene is a suitable mtDNA candidate gene for use in phylogenetic analyses due to its sequence variability, which makes it appropriate for comparisons at the subspecies, species, and genus levels. This study was conducted to develop a rapid molecular method for species identification of Malayan gaur (Bos gaurus hubbacki), Kedah-Kelantan (KK) (Bos indicus), and Bali (Bos javanicus) cattle in Malaysia. DNA was extracted from blood samples of 8 Malayan gaurs, 30 KK, and 28 Bali cattle. A set of both specific and universal primers for the Cyt b gene were used in PCR amplification. DNA sequences obtained were then analyzed using BioEdit and Restriction Mapper softwares. The PCR products obtained from Cyt b gene amplification were then subjected to restriction enzyme digestion. The amplification, using both specific and universal primers, produced a 154- and a 603-bp fragment, respectively, in all three species. Two restriction enzymes, NlaIV and SspI, were used to obtain specific restriction profiles that allowed direct identification of Malayan gaur, KK, and Bali cattle. Our findings indicate that all three species can be identified separately using a combination of universal primers and the restriction enzyme SspI.
Advanced backcross families derived from Oryza sativa cv MR219/O. rufipogon IRGC105491 were utilized for identification of quantitative trait loci (QTL) for blast resistance using simple sequence repeat markers. Two hundred and sixty-one BC(2)F(3) families were used to construct a linkage map, using 87 markers, which covered 2375.2 cM of 12 rice chromosomes, with a mean density of 27.3 cM. The families were evaluated in a greenhouse for resistance to blast disease caused by pathotypes P7.2 and P5.0 of Magnaporthe oryzae. Five QTLs (qBL5.1, qBL5.2, qBL6.1, qBL8.1, and qBL10.1) for pathotype P5.0 and four QTLs (qBL5.3, qBL5.4, qBL7.1, and qBL8.2) for pathotype P7.2 were identified using the BC(2)F(3) families. Another linkage map was also constructed based on 31 BC(2)F(5) families, using 63 SSR markers, which covered 474.9 cM of 9 rice chromosomes, with a mean density of 8.01 cM. Five suggestive QTLs (qBL11.2, qBL11.3, qBL12.1, qBL12.2, qBL12.3) and one putative QTL (qBL2.1) were identified for pathotype P7.2. Also, seven suggestive QTLs (qBL1.1, qBL2.2, qBL4.1, qBL4.2, qBL5.3, qBL8.3, and qBL11.1) were detected for pathotype P5.0. We conclude that there is a non-race-specific resistance spectrum of O. rufipogon against M. oryzae pathotypes.
We evaluated 38 dura x pisifera (DP) oil palm progenies in four locations in Malaysia for genotype by environment interaction and genotypic stability studies. The DP progenies derived from crosses between pisifera palms of AVROS, Serdang S27B, Serdang 29/36, and Lever Cameroon were chosen to be the males' parent and Deli dura palms designated as females' parent. All the locations differed in terms of soil physical and chemical properties, and the soil types ranged from coastal clay to inland soils. The genotype by environment interaction and stability of the individual genotypes were analyzed for oil yield trait using several stability techniques. A genotype by environment interaction was detected for oil yield and it had a larger variance component than genotypic variance (σ(2)(gl)/σ(2)(g) = 139.7%). Genotype by environment interaction of oil yield was largely explained by a non-linear relationship between genotypic and environmental values. Overall assessment of individual genotypic stability showed that seven genotypes were highly stable and had consistent performance over the environments for the oil yield trait [total individual genotype stability scored more than 10 and mean oil yielded above the average of the environment (genotype means are more than 34.37 kg·palm(-1)·year(-1))]. These genotypes will be useful for oil palm breeding and tissue culture programs for developing high oil yielding planting materials with stable performance.
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.
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.
Twelve introgressed oil palm (Elaeis guineensis) progenies of Nigerian dura x Deli dura were evaluated for bunch yield, yield attributes, bunch quality components and vegetative characters at the Malaysian Palm Oil Board Research Station, in Keratong, Pahang, Malaysia. Analysis of variance revealed significant to highly significant genotypic differences, indicating sufficient genetic variability among the progenies for bunch yield and its attributes, vegetative characters and bunch quality components, except fruit to bunch ratio. Fresh fruit bunch yield ranged from 167 kg·palm(-1)·year(-1) in PK1330 to 212 kg·palm(-1)·year(-1) in PK1351, with a mean yield of 192 kg·palm(-1)·year(-1). Among the progeny, PK1313 had the highest oil to bunch ratio (19.36%), due to its high mesocarp to fruit ratio, fruit to bunch ratio and low shell to fruit ratio. Among the progenies, PK1313 produced the highest oil yield of 31.4 kg·palm(-1)·year(-1), due to a high mesocarp to fruit ratio (61.2%) and a low shell to fruit ratio (30.7%), coupled with high fruit to bunch ratio (65.6%). PK1330 was found promising for selection, as it had desirable vegetative characters, including smaller petiole cross section (27.15 cm2), short rachis length (4.83 m), short palm height (1.85 m), and the lowest leaf number (164.6), as these vegetative characters are prerequisites for selecting palms for high density planting and high yield per hectare. The genetic variability among the progenies was found to be high, indicating ample scope for further breeding, followed by selection.
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.
Diabetic retinopathy is the most common diabetic eye disease, occurring in about 60% of type 2 diabetic patients. Other than known clinical risk factors, the influence of genes has been suggested as part of the development of diabetic retinopathy. We investigated the association of Gly82Ser, 1704G/T and 2184A/G polymorphisms in the RAGE gene with retinopathy in type 2 diabetic patients in Malaysia. Ninety-eight unrelated retinopathy patients and 185 unrelated healthy controls from all over Malaysia were recruited in this study. The allele and genotype frequencies of the three gene polymorphisms were investigated using PCR-RFLP. The allele frequency of the three polymorphisms did not differ significantly between the control and the retinopathy group (P > 0.05). Analysis of the frequency of GA+AA, GT+TT and AG+GG in the retinopathy group did not reveal significant differences (P > 0.05) compared to the control group. We conclude that RAGE gene Gly82Ser, 1704G/T and 2184A/G polymorphisms are not associated with retinopathy development in the Malaysian population.
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 aim of this study was to determine whether C34T, a common polymorphism of the adenosine monophosphate deaminase 1 gene (AMPD1), is associated with essential hypertension (EH). We hypothesize that C34T is associated with the development of EH. A case-control design was used for this study. The DNA was extracted using a commercial kit from the whole blood of 200 patients with hypertension and 200 subjects without hypertension from selected Malaysian ethnicities (Malays, Chinese, and Indians). Polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) and agarose gel electrophoresis were used for genotyping. The C34T gene polymorphism of AMPD1 was significantly associated with EH in the Malaysian subjects (P < 0.0001). The genotype frequencies of CC, CT, and TT were 6%, 79%, and 15%, respectively, among hypertensive subjects, while no TT genotypes were observed in the normotensive subjects. Further, the frequency of hypertension was higher among T allele carriers than C carriers (OD = 9.94; 95%CI = 6.851-14.434). There were significant differences in the systolic blood pressure, diastolic blood pressure, and pulse pressure (P ˂ 0.05) between the normotensive and hypertensive Malaysian subjects; we believe those difference were caused by the C34T polymorphism. For the first time in Malaysia, the current study provides evidence that a common polymorphism of the AMPD1 gene (C34T) is strongly associated with EH.
This study was carried out to evaluate the genetic effect of quantitative trait loci (QTLs) conferring drought tolerance in wheat. A population of 120 F(2) individuals from the cross between the drought-tolerant S-78-11 and drought-sensitive Tajan cultivars were analyzed for their segregation under drought stress conditions. The relative water content under drought stress conditions exhibited continuous variation, indicating the minor gene effects on the trait. Single-marker analysis (SMA) was carried out to detect the main QTL association with drought tolerance. The SMA results revealed that the simple sequence repeat markers GWM182 and GWM292 on chromosome 5D and GWM410 on chromosome 5A exhibited significant association with drought tolerance, accounting for 30, 22, and 21% of the total variation, respectively. The 3 genetic loci, especially GWM182, can be used in marker-assisted selection methods in drought tolerance breeding in wheat.
The white-bellied sea eagle, Haliaeetus leucogaster, displays reversed sexual size dimorphism and is monomorphic for adult plumage coloration. Early attempts to identify sex in sexually monomorphic birds were based on morphological or chromosomal characters, but since avian W-specific DNA sequences were identified, PCR amplification has become commonly used for molecular sexing. We used a PCR test employing primers that amplify two homologous fragments of both the CHD-W gene, unique to females, and the CHD-Z gene, occurring in both sexes. This test was applied to five individuals of H. leucogaster from the Malacca Zoo and to male and female domestic chickens, Gallus domesticus, for comparison. All individuals were sexed successfully with high reproducibility. We conclude that this PCR-based test with feathers as the DNA source is a reliable sexing method for H. leucogaster. This sexing technique is objective and non-invasive and could be used to test sex ratio theories, as well as to help improve conservation and management actions for captive breeding program of this species in Malaysia.
Studies of genetic mutations that have been used in predicting glioma prognosis have revealed a complex relationship between clinical and genetic factors. Epidermal growth factor (EGF) and the NAT2 gene play a central role in carcinogenesis. An adenine (A) to guanine (G) single nucleotide polymorphism at position 61 in the 5'-untranslated region (5'-UTR) of the EGF gene has been found to be associated with levels of EGF production, and the mutations in the NAT2 gene have been postulated as a risk factor for cancer. We investigated EGF and the NAT2 gene in 13 glioma tissue samples and 12 normal controls. In the EGF 5'-UTR 61G polymorphism, the heterozygote GA was the most common genotype in the glioma patients. In the NAT2 polymorphism at nucleotide position 857G/A, the G allele and the GG genotype were the most prevalent forms in both the glioma and normal samples. We did not find any homozygous AA genotypes in the glioma patients. Based on this preliminary evidence, the EGF 5'-UTR at position 61 and the NAT2 SNP at position 857 polymorphisms are associated with increased risk for glioma.
Epinephelus fuscoguttatus is a commercially important marine fish species in southeast Asia. Due to overfishing and water pollution, this species has been declared as near-threatened. Thus, to provide information to help maintain and preserve the species, microsatellites were developed, using an enriched genomic library method. Thirty individuals were collected from the hatchery of the Fishery Research Institute, Terengganu, Malaysia. These individuals, from four to six years old, originated from Sabah and are maintained in captive culture as broodstock. Genomic DNA was extracted from the fins of selected individuals that weighed 3-8 kg. Ten microsatellite loci were found to be polymorphic in this population, with 5 to 21 alleles per locus. Observed and expected heterozygosities ranged from 0.53 to 0.97 and 0.59 to 0.95, respectively. Only one locus deviated significantly from Hardy-Weinberg equilibrium and no significant linkage disequilibrium was found among the pairs of loci. These polymorphic microsatellite loci will be used by the Malaysian Fishery Research Institute for investigating genetic diversity and for developing breeding strategies.
DNA extraction was carried out on 32 medicinal plant samples available in Malaysia using the TriOmic(TM) extraction kit. Amounts of 0.1 g flowers or young leaves were ground with liquid nitrogen, lysed at 65°C in RY1(plus) buffer and followed by RNAse treatment. Then, RY2 buffer was added to the samples and mixed completely by vortexing before removal of cell debris by centrifugation. Supernatants were transferred to fresh microcentrifuge tubes and 0.1 volume RY3 buffer was added to each of the transferred supernatant. The mixtures were applied to spin columns followed by a centrifugation step to remove buffers and other residues. Washing step was carried out twice by applying 70% ethanol to the spin columns. Genomic DNA of the samples was recovered by applying 50 μL TE buffer to the membrane of each spin column, followed by a centrifugation step at room temperature. A modification of the TriOmic(TM) extraction procedure was carried out by adding chloroform:isoamyl alcohol (24:1) steps in the extraction procedure. The genomic DNA extracted from most of the 32 samples showed an increase of total yield when chloroform:isoamyl alcohol (24:1) steps were applied in the TriOmicTM extraction procedure. This preliminary study is very important for molecular studies of medicinal plants available in Malaysia since the DNA extraction can be completed in a shorter period of time (within 1 h) compared to manual extraction, which entails applying phenol, chloroform and ethanol precipitation, and requires 1-2 days to complete.
Mitochondrial DNA cytochrome c oxidase II (COII) gene sequences of Malaysian Cercopithecidae were examined to ascertain their phylogenetic relationships. Colobinae were represented by the genera Presbytis, Trachypithecus and Nasalis, while the genus Macaca represented Cercopithecinae. DNA amplification and sequencing of the COII gene was performed on 16 samples. Symphalangus syndactylus (Hylobatidae) was used as the outgroup. Data were analyzed using both character (maximum parsimony) and distance (neighbor-joining) methods. Tree topologies indicated that Colobinae and Cercopithecinae have their own distinct monophyletic clade. This result was well supported by bootstrap values and genetic distances derived from the Kimura-2-parameter algorithm. Separation of Macaca nemestrina from M. fascicularis was also well supported by bootstrap values. In addition, tree topologies indicate a good resolution of the Colobinae phylogenetic relationships at the intergeneric level, but with low bootstrap support. The position of Nasalis remained problematic in both trees. Overall, COII is a good gene candidate for portraying the phylogenetic relationships of Malaysian primates at the inter- and intra-subfamily levels.
Malaysian arowana (dragonfish; Scleropages formosus) is an ancient osteoglossid fish from southeast Asia. Due to the high demand of the ornamental fish trade and because of habitat loss, the species is close to extinction. We isolated and characterized 10 polymorphic microsatellites of this species, using 5'-anchored PCR. The number of alleles at the 10 microsatellite loci ranged from 2 to 28, with a mean of 7.8/locus. The observed heterozygosity ranged from 0.03 to 0.93 (mean: 0.39), whereas the expected heterozygosity ranged from 0.03 to 0.94 (mean: 0.46). Seven microsatellites deviated from Hardy-Weinberg equilibrium, and three conformed to Hardy-Weinberg equilibrium and were in linkage equilibrium. These 10 novel microsatellites should facilitate studies of genetic diversity and population structure of arowana to help plan actions for the conservation of the indigenous Malaysian arowana.
A number of studies have pointed to the association of BDNF (brain-derived neurotrophic factor) and DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, 32 kDa) with schizophrenia. The purpose of this study was to determine whether these two genes are involved in the pathogenesis of schizophrenia in the Malay population. Two single nucleotide polymorphisms Val66Met of BDNF, -2036C>G and g.1238delG of DARPP-32 were genotyped in the Malay population in 200 patients with schizophrenia and 256 healthy controls. Analysis of allele and genotype frequencies in these two groups revealed no significant association of BDNF or DARPP-32 polymorphisms with schizophrenia in Malays. This is the first such association study in the Malay population.
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.