The mitogenome of the black yabby, Geocharax gracilis, was sequenced using the MiSeq Personal Sequencer. It has 15,924 base pairs consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 23 transfer RNAs, and a non-coding AT-rich region. The base composition of G. gracilis mitogenome is 32.18% for T, 22.32% for C, 34.83% for A, and 10.68% for G, with an AT bias of 67.01%. The mitogenome gene order is typical for that of parastacid crayfish with the exception of some minor rearrangements involving tRNA genes.
This study provides a first description of the phylogeographic patterns and evolutionary history of two species of the mudskipper genus Periophthalmus. These amphibious gobies are distributed throughout the whole Indo-Pacific region and Atlantic coast of Africa, in peritidal habitats of soft-bottom coastal ecosystems. Three sequence datasets of two widely distributed species, Periophthalmus argentilineatus and P. kalolo, were obtained by amplifying and sequencing two mtDNA markers (D-loop and 16S rDNA) and the nDNA rag1 region. The three datasets were then used to perform phylogeographic, demographic and population genetic analyses. Our results indicate that tectonic events and past climatic oscillations strongly contributed to shape present genetic differentiation, phylogeographic and demographic patterns. We found support for the monophyly of P. kalolo, and only shallow genetic differentiation between East-African and Indo-Malayan populations of this species. However, our collections of the morphospecies P. argentilineatus include three molecularly distinct lineages, one of them more closely related to P. kalolo. The presence of Miocenic timings for the most recent common ancestors of some of these morphologically similar clades, suggests the presence of strong stabilising selection in mudskippers' habitats. At population level, demographic analyses and palaeoecological records of mangrove ecosystems suggest that Pleistocene bottlenecks and expansion plus secondary contact events of the studied species were associated with recurrent sea transgressions during interglacials, and sea regressions or stable regimes during glacials, respectively.
This report documents an emerging trend of identification of Megalocytivirus-like inclusions in a range of ornamental fish species intercepted during quarantine detention at the Australian border. From September 2012 to February 2013, 5 species of fish that had suffered mortality levels in excess of 25% whilst in the post-entry quarantine and had Megalocytivirus-like inclusion bodies in histological sections were examined by PCR. The fish had been imported from Singapore, Malaysia and Sri Lanka. Ninety-seven of 111 individual fish from affected tanks of fish tested were positive for the presence of Megalocytivirus by PCR. Sequence analysis of representative PCR products revealed an identical sequence of 621 bp in all cases which was identical to a previously characterized Megalocytivirus (Sabah/RAA1/2012 strain BMGIV48). Phylogenetic analysis of available Megalocytivirus major capsid protein (MCP) sequences confirmed the existence of 3 major clades of Megalocytivirus. The virus detected in this study was identified as a member of Genotype II. The broad host range and pathogenicity of megalocytiviruses, coupled to the documented spread of ornamental fish into the environment, render this a significant and emerging biosecurity threat to Australia.
Matched MeSH terms: DNA Virus Infections/transmission; DNA Virus Infections/veterinary*; DNA Virus Infections/virology
Gracilaria tenuistipitata is an agarophyte with substantial economic potential because of its high growth rate and tolerance to a wide range of environment factors. This red seaweed is intensively cultured in China for the production of agar and fodder for abalone. Microsatellite markers were developed from the chloroplast genome of G. tenuistipitata var. liui to differentiate G. tenuistipitata obtained from six different localities: four from Peninsular Malaysia, one from Thailand and one from Vietnam. Eighty G. tenuistipitata specimens were analyzed using eight simple sequence repeat (SSR) primer-pairs that we developed for polymerase chain reaction (PCR) amplification.
Matched MeSH terms: DNA Primers; DNA, Chloroplast/genetics*; Random Amplified Polymorphic DNA Technique
The knowledge of genetic diversity of tree crop is very important for breeding and improvement program for the purpose of improving the yield and quality of its produce. Genetic diversity study and analysis of genetic relationship among 20 Moringa oleifera were carried out with the aid of twelve primers from, random amplified polymorphic DNA marker. The seeds of twenty M. oleifera genotypes from various origins were collected and germinated and raised in nursery before transplanting to the field at University Agricultural Park (TPU). Genetic diversity parameter, such as Shannon's information index and expected heterozygosity, revealed the presence of high genetic divergence with value of 1.80 and 0.13 for Malaysian population and 0.30 and 0.19 for the international population, respectively. Mean of Nei's gene diversity index for the two populations was estimated to be 0.20. In addition, a dendrogram constructed, using UPGMA cluster analysis based on Nei's genetic distance, grouped the twenty M. oleifera into five distinct clusters. The study revealed a great extent of variation which is essential for successful breeding and improvement program. From this study, M. oleifera genotypes of wide genetic origin, such as T-01, T-06, M-01, and M-02, are recommended to be used as parent in future breeding program.
Matched MeSH terms: DNA Primers/metabolism; Random Amplified Polymorphic DNA Technique/methods*
Burkholderia pseudomallei is the causative agent of melioidosis. We initiated this investigation with a virulent and an attenuated strain of B. pseudomallei. Pulsed-field gel electrophoresis was carried out initially for macrogenomic comparison of both strains of B. pseudomallei. However, the pulsotypes obtained were identical and therefore we applied a subtractive hybridization technique to distinguish and determine the possible differences between the two strains. Six virulence strain-specific DNA fragments were obtained and the encoding homolog proteins were identified as a xenobiotic-responsive element family of transcriptional regulator, a hypothetical protein, an unknown protein, a plasmid recombination enzyme, a regulatory protein and a putative hemolysin activator protein. A combination of at least three of these determinants was identified in 45 clinical isolates when screening was carried out with self-designed multiplex PCR targeting the six putative virulent determinants. Our data demonstrated that different combinations of the six putative virulence genes were present in the clinical isolates indicating their probable role in the pathogenesis of B. pseudomallei infections.
Matched MeSH terms: DNA, Bacterial/genetics; Sequence Analysis, DNA; DNA Fragmentation
Saliva has been suggested as an attractive resource for evaluating physiological and pathological conditions in humans. This study aims to evaluate saliva sampling as an alternative to blood sampling for molecular testing.
Microsatellite markers were developed for Johannesteijsmannia lanceolata to assess the genetic diversity and mating system of this alarmingly endangered species.
Matched MeSH terms: DNA Primers/genetics*; DNA, Plant/genetics*
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.
Matched MeSH terms: DNA, Mitochondrial/genetics; DNA Primers; Random Amplified Polymorphic DNA Technique
This study was done to evaluate various DNA and RNA extractions from archival FFPE tissues. A total of 30 FFPE blocks from the years of 2004 to 2006 were assessed with each modified and adapted method. Extraction protocols evaluated include the modified enzymatic extraction method (Method A), Chelex-100 extraction method (Method B), heat-induced retrieval in alkaline solution extraction method (Methods C and D) and one commercial FFPE DNA Extraction kit (Qiagen, Crawley, UK). For RNA extraction, 2 extraction protocols were evaluated including the enzymatic extraction method (Method 1), and Chelex-100 RNA extraction method (Method 2). Results show that the modified enzymatic extraction method (Method A) is an efficient DNA extraction protocol, while for RNA extraction, the enzymatic method (Method 1) and the Chelex-100 RNA extraction method (Method 2) are equally efficient RNA extraction protocols.
We used a combined microscopy-molecular approach to determine the occurrence and identities of waterborne Giardia sp. cysts isolated from 18 separate, 10l grab samples collected from a Malaysian zoo. Microscopy revealed that 17 of 18 samples were Giardia cyst positive with concentrations ranging from 1 to 120 cysts/l. Nine (52.9%) of the 17 cyst positive samples produced amplicons of which 7 (77.8%) could be sequenced. Giardia duodenalis assemblage A (6 of 7) and assemblage B (1 of 7), both infectious to humans, were identified at all sampling sites at the zoo. The presence of human infectious cysts raises public health issues, and their occurrence, abundance and sources should be investigated further. In this zoo setting, our data highlight the importance of incorporating environmental sampling (monitoring) in addition to routine faecal examinations to determine veterinary and public health risks, and water monitoring should be considered for inclusion as a separate element in hazard analysis, as it often has a historical (accumulative) connotation.
Matched MeSH terms: DNA, Protozoan/genetics*; DNA, Protozoan/chemistry; Sequence Analysis, DNA
The nuclear envelope presents a major barrier to transgene delivery and expression using a non-viral vector. Virus is capable of overcoming the barrier to deliver their genetic materials efficiently into the nucleus by virtue of the specialized protein components with the unique amino acid sequences recognizing cellular nuclear transport machinery. However, considering the safety issues in the clinical gene therapy for treating critical human diseases, non-viral systems are highly promising compared with their viral counterparts. This review summarizes the progress on exploring the nuclear traffic mechanisms for the prominent viral vectors and the technological innovations for the nuclear delivery of non-viral DNA by mimicking those natural processes evolved for the viruses as well as for many cellular proteins.
Two forms of Staurois that are differentiated by body size occur parapatrically in the Crocker Range, Sabah, Borneo. Analyses of a total of 1,499 bp of the mitochondrial cytochrome b, 12S rRNA, and 16S rRNA genes revealed that the two forms could be completely split genetically. The two forms could be also clearly differentiated morphologically, not only by snout-vent length but also by the relative sizes of snout, eye, and finger disk. Comparisons of the two forms with all known species of the genus revealed the large and small forms to be S. tuberilinguis and S. parvus, respectively. The latter species has long been synonymized with the former, but we here consider them to represent different species.
Matched MeSH terms: DNA, Mitochondrial/genetics; Sequence Analysis, DNA; DNA Primers
Mitochondrial DNA sequences of the hypervariable regions HV1 and HV2 were analyzed in 205 unrelated ethnic Malays residing in Singapore as an initial effort to generate a database for forensic identification purposes. Sequence polymorphism was detected using PCR and direct sequencing analysis. A total of 152 haplotypes was found containing 152 polymorphisms. Out of the 152 haplotypes, 115 were observed only once and 37 types were seen in multiple individuals. The most common haplotype (16223T, 16295T, 16362C, 73G, 146C, 199C, 263G, and 315.1C) was shared by 7 (3.41%) individuals, two haplotypes were shared by 4 individuals, seven haplotypes were shared by 3 individuals, and 27 haplotypes by 2 individuals. Haplotype diversity and random match probability were estimated to be 0.9961% and 0.87%, respectively.
Matched MeSH terms: DNA, Mitochondrial/genetics*; DNA Fingerprinting; Sequence Analysis, DNA
An 835 base pair (bp) fragment of mitochondrial DNA (mtDNA) was sequenced to characterize genetic variation within and among 1,053 samples comprising five regional populations each of longtail macaques (Macaca fascicularis) and rhesus macaques (Macaca mulatta), and one sample each of Japanese (M. fuscata) and Taiwanese (M. cyclopis) macaques. The mtDNA haplotypes of longtail macaques clustered in two large highly structured clades (Fas1 and Fas2) of a neighbor-joining tree that were reciprocally monophyletic with respect to those representing rhesus macaques, Japanese macaques, and Taiwanese macaques. Both clades exhibited haplotypes of Indonesian and Malaysian longtail macaques widely dispersed throughout them; however, longtail macaques from Indochina, Philippines, and Mauritius each clustered in a separate well-defined clade together with one or a few Malaysian and/or Indonesian longtail macaques, suggesting origins on the Sunda shelf. Longtail macaques from Malaysia and Indonesia were far more genetically diverse, and those from Mauritius were far less diverse than any other population studied. Nucleotide diversity between mtDNA sequences of longtail macaques from different geographic regions is, in some cases, greater than that between Indian and Chinese rhesus macaques. Approximately equal amounts of genetic diversity are due to differences among animals in the same regional population, different regional populations, and different species. A greater proportion of genetic variance was explained by interspecies differences when Japanese and Taiwanese macaques were regarded as regional populations of rhesus macaques than when they were treated as separate species. Rhesus macaques from China were more closely related to both Taiwanese and Japanese macaques than to their own conspecifics from India.
Matched MeSH terms: DNA, Mitochondrial/classification; DNA, Mitochondrial/chemistry*; Sequence Analysis, DNA
Giardia duodenalis causes diarrhoea and malabsorption. The objectives of the study were to detect local isolates of G. doudenalis by polymerase chain reaction (PCR) and to determine their restriction fragment length polymorphisms (RFLP). G. doudenalis isolated from stools of patients from Hospital Orang Asli Gombak were cultured axenically using TYI-S-33 medium with 10% foetal calf serum. The commercially designed primer-pair 432/433 was used to amplify a 0.52 kb segment known to encode the homologous cysteine-rich trophozoite surface antigen (tsp11 and tsa417). Results showed that the primer-pair 432/433 could amplify the target region of the local isolates. RFLP study on the identical isolates showed that all the restriction enzymes tested ( HindIII, ClaI, PstI and Kpn) gave a banding pattern similar to that of the WB strain a reference pathogenic strain from human. The reference pathogenic strain were commercially obtained from the American Type Culture Collection (ATCC).
The species diversity and genetic structure of mosquitoes belonging to the Anopheles maculatus group in Southeast Asia were investigated using the internal transcribed spacer 2 (ITS2) of ribosomal DNA (rDNA). A molecular phylogeny indicates the presence of at least one hitherto unrecognised species. Mosquitoes of chromosomal form K from eastern Thailand have a unique ITS2 sequence that is 3.7% divergent from the next most closely related taxon (An. sawadwongporni) in the group. In the context of negligible intraspecific variation at ITS2, this suggests that chromosomal form K is most probably a distinct species. Although An. maculatus sensu stricto from northern Thailand and southern Thailand/peninsular Malaysia differ from each other in chromosomal banding pattern and vectorial capacity, no intraspecific variation was observed in the ITS2 sequences of this species over this entire geographic area despite an extensive survey. A PCR-based identification method was developed to distinguish five species of the group (An. maculatus, An. dravidicus, An. pseudowillmori, An. sawadwongporni and chromosomal form K) to assist field-based studies in northwestern Thailand. Sequences from 187 mosquitoes (mostly An. maculatus and An. sawadwongporni) revealed no intraspecific variation in specimens from Thailand, Cambodia, mainland China, Malaysia, Taiwan and Vietnam, suggesting that this identification method will be widely applicable in Southeast Asia. The lack of detectable genetic structure also suggests that populations of these species are either connected by gene flow and/or share a recent common history.
Many species of Trichoderma have attracted interest as agents for the biological control of soil borne fungal pathogens of a range of crop plants. Research on the biochemical mechanisms associated with this application has focused on the ability of these fungi to produce enzymes which lyse fungal cell walls, and antifungal antibiotics. An important group of the latter are the non-ribosomal peptides called peptaibols. In this study Trichoderma asperellum, a strain used in biological control in Malaysia, was found to produce the peptaibol, trichotoxin. This type of peptide molecule is synthesised by a peptide synthetase (PES) enzyme template encoded by a peptide synthetase (pes) gene. Using nucleotide sequences amplified from adenylation (A-) domains as probes, to hybridise against a lambda FIXII genomic library from T. asperellum, 25 clones were recovered. These were subsequently identified as representative of four groups based on their encoding properties for specific amino acid incorporation modules in a PES. This was based on analysis of their amino acid sequences which showed up to 86% identity to other PESs including TEX 1.
Matched MeSH terms: DNA, Fungal/genetics; DNA, Fungal/chemistry; Sequence Analysis, DNA
Giardia duodenalis is a protozoan parasite that can cause significant diarrhoeal diseases. Knowledge of population genetics is a prerequisite for ascertaining the invasion patterns of this parasite. In order to infer evolutionary patterns that could not be uncovered based on the morphological features, a population genetic study with the incorporation of molecular marker was carried out to access the genetic structure of G. duodenalis isolated from the Malaysian population and the global populations.
Matched MeSH terms: DNA, Protozoan/genetics; DNA, Protozoan/chemistry; Sequence Analysis, DNA