A new patterning method using Deoxyribose Nucleic Acid (DNA) strands capable of producing nanogaps of less than 100 nm is proposed and investigated in this work. DNA strands from Bosenbergia rotunda were used as the fundamental element in patterning DNA on thin films of aluminium (Al) metal without the need for any lithographic techniques. The DNA strands were applied in buffer solutions onto thin films of Al on silicon (Si) and the chemical interactions between the DNA strands and Al creates nanometer scale arbitrary patterning by direct transfer of the DNA strands onto the substrate. This simple and cost-effective method can be utilized in the fabrication of various components in electronic chips for microelectronics and Nano Electronic Mechanical System (NEMS) applications in general.
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.
The mango (Mangifer indica L.) is an important species of the family Anacardiaceae and is one of the most important crops cultivated commercially in many parts of the world. Hence, a better understanding of the phylogeny in this species is crucial as it is the basis knowledge of improving its genetic resources which is beneficial for breeding programs. Phylogenetic relationships among 13 mango cultivars from Indonesia, Malaysia and Taiwan were carried out by comparing DNA sequence data sets derived from the Internal Transcribed Spacer (ITS) region pfnuclear ribosomal DNA (nrDNA). Analysis using parsimony method showed that the cultivars were classified into three major groups. The first group composed almost Malaysian cultivars although with low bootstrap value, the second group consisted of mainly Taiwan cultivars and the last group included mostly Indonesia one. The results indicated that some cultivars have a close relationships with each other even it is originated from different countries. With regards to the relationship among these cultivars, this gives better insight for generating new cultivar.
Direct assembly of multiple linear DNA fragments via homologous recombination, a phenomenon known as in vivo assembly or transformation associated recombination, is used in biotechnology to assemble DNA constructs ranging in size from a few kilobases to full synthetic microbial genomes. It has also enabled the complete replacement of eukaryotic chromosomes with heterologous DNA. The moss Physcomitrella patens, a non-vascular and spore producing land plant (Bryophyte), has a well-established capacity for homologous recombination. Here, we demonstrate the in vivo assembly of multiple DNA fragments in P. patens with three examples of effective genome editing: we (i) efficiently deleted a genomic locus for diterpenoid metabolism yielding a biosynthetic knockout, (ii) introduced a salt inducible promoter, and (iii) re-routed endogenous metabolism into the formation of amorphadiene, a precursor of high-value therapeutics. These proof-of-principle experiments pave the way for more complex and increasingly flexible approaches for large-scale metabolic engineering in plant biotechnology.
Curculin, a sweet protein found in Curculigo latifolia fruit has great potential for the pharmaceutical industry. This protein interestingly has been found to have both sweet taste and taste-modifying capacities comparable with other natural sweeteners. According to our knowledge this is the first reported case on the isolation of microsatellite loci in this genus. Hence, the current development of microsatellite markers for C. latifolia will facilitate future population genetic studies and breeding programs for this valuable plant. In this study 11 microsatellite markers were developed using 3' and 5' ISSR markers. The primers were tested on 27 accessions from all states of Peninsular Malaysia. The number of alleles per locus ranged from three to seven, with allele size ranging from 141 to 306 bp. The observed and expected heterozygosity ranged between 0.00-0.65 and 0.38-0.79, respectively. The polymorphic information content ranged from 0.35 to 0.74 and the Shannon's information index ranged from 0.82 to 1.57. These developed polymorphic microsatellites were used for constructing a dendrogram by unweighted pair group method with arithmetic mean cluster analysis using the Dice's similarity coefficient. Accessions association according to their geographical origin was observed. Based on characteristics of isolated microsatellites for C. latifolia accessions all genotype can be distinguished using these 11 microsatellite markers. These polymorphic markers could also be applied to studies on uniformity determination and somaclonal variation of tissue culture plantlets, varieties identification, genetic diversity, analysis of phylogenetic relationship, genetic linkage maps and quantitative trait loci in C. latifolia.
The mating system and seed variation of Acacia hybrid (A. mangium x A. auriculiformis) were studied using allozymes and random amplified polymorphic DNA (RAPD) markers, respectively. Multi-locus outcrossing rate estimations indicated that the hybrid was predominantly outcrossed (mean+/- s.e. t(m) = 0.86+/-0.01). Seed variation was investigated using 35 polymorphic RAPD fragments. An analysis of molecular variance (AMOVA) revealed the highest genetic variation among seeds within a pod (66%-70%), followed by among pods within inflorescence (29%-37%), and the least variation among inflorescences within tree (1%). In addition, two to four RAPD profiles could be detected among seeds within pod. Therefore, the results suggest that a maximum of four seeds per pod could be sampled for the establishment of a mapping population for further studies.
We examined differences in pollen dispersal efficiency between 2 years in terms of both spatial dispersal range and genetic relatedness of pollen in a tropical emergent tree, Dipterocarpus tempehes. The species was pollinated by the giant honeybee (Apis dorsata) in a year of intensive community-level mass-flowering or general flowering (1996), but by several species of moths in a year of less-intensive general flowering (1998). We carried out paternity analysis based on six DNA microsatellite markers on a total of 277 mature trees forming four spatially distinct subpopulations in a 70 ha area, and 147 and 188 2-year-old seedlings originating from seeds produced in 1996 and 1998 (cohorts 96 and 98, respectively). Outcrossing rates (0.93 and 0.96 for cohorts 96 and 98, respectively) did not differ between years. Mean dispersal distances (222 and 192 m) were not significantly different between the 2 years but marginally more biased to long distance in 1996. The mean relatedness among cross-pollinated seedlings sharing the same mothers in cohort 96 was lower than that in cohort 98. This can be attributed to the two facts that the proportion of intersubpopulations pollen flow among cross-pollination events was marginally higher in cohort 96 (44%) than in cohort 98 (33%), and that mature trees within the same subpopulations are genetically more related to each other than those between different subpopulations. We conclude that D. tempehes maintained effective pollen dispersal in terms of outcrossing rate and pollen dispersal distance in spite of the large difference in foraging characteristics between two types of pollinators. In terms of pollen relatedness, however, a slight difference was suggested between years in the level of biparental inbreeding.
Fruit type in the genus Lithocarpus (Fagaceae) includes both classic oak acorns and novel modifications. Bornean taxa with modified fruits can be separated into two sections (Synaedrys and Lithocarpus) based on subtle shape differences. By following strict criteria for homology and representation, this variation in shape can be captured and the sections distinguished by using elliptic Fourier or eigenshape analysis. Phenograms of fruit shape, constructed by using restricted maximum likelihood techniques and these morphometric descriptors, were incorporated into combined and comparative analyses with molecular sequence data from the internal transcribed spacer (ITS) region of the nuclear rDNA, using branch-weighted matrix representation. The combined analysis strongly suggested independent derivation of the novel fruit type in the two sections from different acornlike ancestors, while the comparative analysis indicated frequent decoupling between the molecular and morphological changes as inferred at well-supported nodes. The acorn fruit type has undergone little modification between ingroup and outgroup, despite large molecular distance. Greater morphological than molecular change was inferred at critical transitions between acorn and novel fruit types, particularly for section Lithocarpus. The combination of these two different types of data improved our understanding of the macroevolution of fruit type in this difficult group, and the comparative analysis highlighted the significant incongruities in evolutionary pattern between the two datasets.
Three species of Shorea (S. leprosula, S. acuminata and S. cursitii) were collected from a natural forest reserve of Malaysia and analyzed for genetic variation using the technique of random amplification of polymorphic DNA (RAPD) by the polymerase chain reaction (PCR). The average number of nucleotide substitutions was estimated. The nucleotide diversities within species were very similar and larger than those found in Drosophila melanogaster. The nucleotide divergences between these species are about 1.5 times the nucleotide diversities within the species, indicating that these species diverged from a common ancestor relatively recently.
We investigated the fine-scale genetic structure of three tropical-rainforest trees, Hopea dryobalanoides, Shorea parvifolia and S. acuminata (Dipterocarpaceae), in Peninsular Malaysia, all of which cooccurred within a 6-ha plot in Pasoh Forest Reserve. A significant genetic structure was found in H. dryobalanoides, weaker (but still significant) genetic structure in S. parvifolia and nonsignificant structure in S. acuminata. Seeds of all three species are wind dispersed, and their flowers are thought to be insect pollinated. The most obvious difference among these species is their height: S. parvifolia and S. acuminata are canopy species, whereas H. dryobalanoides is a subcanopy species. Clear differences were also found among these species in their range of seed dispersal, which depends on the height of the release point; so taller trees disperse their seed more extensively. The estimates of seed dispersal area were consistent with the degree of genetic structure found in the three species. Therefore, tree height probably had a strong influence on the fine-scale genetic structure of the three species.
Ginger is an economically important and valuable plant around the world. Ginger is used as a food, spice, condiment, medicine and ornament. There is available information on biochemical aspects of ginger, but few studies have been reported on its molecular aspects. The main objective of this review is to accumulate the available molecular marker information and its application in diverse ginger studies. This review article was prepared by combing material from published articles and our own research. Molecular markers allow the identification and characterization of plant genotypes through direct access to hereditary material. In crop species, molecular markers are applied in different aspects and are useful in breeding programs. In ginger, molecular markers are commonly used to identify genetic variation and classify the relatedness among varieties, accessions, and species. Consequently, it provides important input in determining resourceful management strategies for ginger improvement programs. Alternatively, a molecular marker could function as a harmonizing tool for documenting species. This review highlights the application of molecular markers (isozyme, RAPD, AFLP, SSR, ISSR and others such as RFLP, SCAR, NBS and SNP) in genetic diversity studies of ginger species. Some insights on the advantages of the markers are discussed. The detection of genetic variation among promising cultivars of ginger has significance for ginger improvement programs. This update of recent literature will help researchers and students select the appropriate molecular markers for ginger-related research.
Nepenthaceae is one of the largest carnivorous plant families and features ecological and morphological adaptations indicating an impressive adaptive radiation. However, investigation of evolutionary and taxonomic questions is hindered by poor phylogenetic understanding, with previous molecular studies based on limited loci and taxa. We use high-throughput sequencing with a target-capture methodology based on a 353-loci, probe set to recover sequences for 197 samples, representing 151 described or putative Nepenthes species. Phylogenetic analyses were performed using supermatrix and maximum quartet species tree approaches. Our analyses confirm five Western outlier taxa, followed by N. danseri, as successively sister to the remainder of the group. We also find mostly consistent recovery of two major Southeast Asian clades. The first contains common or widespread lowland species plus a Wallacean-New Guinean clade. Within the second clade, sects. Insignes and Tentaculatae are well supported, while geographically defined clades representing Sumatra, Indochina, Peninsular Malaysia, Palawan, Mindanao and Borneo are also consistently recovered. However, we find considerable conflicting signal at the site and locus level, and often unstable backbone relationships. A handful of Bornean taxa are inconsistently placed and require further investigation. We make further suggestions for a modified infra-generic classification of genus Nepenthes.
DNA barcoding is a fast and reliable tool to assess and monitor biodiversity and, via community phylogenetics, to investigate ecological and evolutionary processes that may be responsible for the community structure of forests. In this study, DNA barcodes for the two widely used plastid coding regions rbcL and matK are used to contribute to identification of morphologically undetermined individuals, as well as to investigate phylogenetic structure of tree communities in 70 subplots (10 × 10m) of a 25-ha forest-dynamics plot in Brunei (Borneo, Southeast Asia). The combined matrix (rbcL + matK) comprised 555 haplotypes (from ≥154 genera, 68 families and 25 orders sensu APG, Angiosperm Phylogeny Group, 2016), making a substantial contribution to tree barcode sequences from Southeast Asia. Barcode sequences were used to reconstruct phylogenetic relationships using maximum likelihood, both with and without constraining the topology of taxonomic orders to match that proposed by the Angiosperm Phylogeny Group. A third phylogenetic tree was reconstructed using the program Phylomatic to investigate the influence of phylogenetic resolution on results. Detection of non-random patterns of community assembly was determined by net relatedness index (NRI) and nearest taxon index (NTI). In most cases, community assembly was either random or phylogenetically clustered, which likely indicates the importance to community structure of habitat filtering based on phylogenetically correlated traits in determining community structure. Different phylogenetic trees gave similar overall results, but the Phylomatic tree produced greater variation across plots for NRI and NTI values, presumably due to noise introduced by using an unresolved phylogenetic tree. Our results suggest that using a DNA barcode tree has benefits over the traditionally used Phylomatic approach by increasing precision and accuracy and allowing the incorporation of taxonomically unidentified individuals into analyses.
Cambay amber originates from the warmest period of the Eocene, which is also well known for the appearance of early angiosperm-dominated megathermal forests. The humid climate of these forests may have triggered the evolution of epiphytic lineages of bryophytes; however, early Eocene fossils of bryophytes are rare. Here, we present evidence for lejeuneoid liverworts and pleurocarpous mosses in Cambay amber. The preserved morphology of the moss fossil is inconclusive for a detailed taxonomic treatment. The liverwort fossil is, however, distinctive; its zig-zagged stems, suberect complicate-bilobed leaves, large leaf lobules, and small, deeply bifid underleaves suggest a member of Lejeuneaceae subtribe Lejeuneinae (Harpalejeunea, Lejeunea, Microlejeunea). We tested alternative classification possibilities by conducting divergence time estimates based on DNA sequence variation of Lejeuneinae using the age of the fossil for corresponding age constraints. Consideration of the fossil as a stem group member of Microlejeunea or Lejeunea resulted in an Eocene to Late Cretaceous age of the Lejeuneinae crown group. This reconstruction is in good accordance with published divergence time estimates generated without the newly presented fossil evidence. Balancing available evidence, we describe the liverwort fossil as the extinct species Microlejeunea nyiahae, representing the oldest crown group fossil of Lejeuneaceae.
Genetic structure can be altered by chemical mutagenesis, which is a common method applied in molecular biology and genetics. Second-generation sequencing provides a platform to reveal base alterations occurring in the whole genome due to mutagenesis. A model legume, Lotus japonicus ecotype Miyakojima, was chemically mutated with alkylating ethyl methanesulfonate (EMS) for the scanning of DNA lesions throughout the genome. Using second-generation sequencing, two individually mutated third-generation progeny (M3, named AM and AS) were sequenced and analyzed to identify single nucleotide polymorphisms and reveal the effects of EMS on nucleotide sequences in these mutant genomes. Single-nucleotide polymorphisms were found in every 208 kb (AS) and 202 kb (AM) with a bias mutation of G/C-to-A/T changes at low percentage. Most mutations were intergenic. The mutation spectrum of the genomes was comparable in their individual chromosomes; however, each mutated genome has unique alterations, which are useful to identify causal mutations for their phenotypic changes. The data obtained demonstrate that whole genomic sequencing is applicable as a high-throughput tool to investigate genomic changes due to mutagenesis. The identification of these single-point mutations will facilitate the identification of phenotypically causative mutations in EMS-mutated germplasm.
Aggressive collections and trade activities in recent decades have resulted in heavy pressure on the natural stands of Aquilaria malaccensis and concerns over its long-term survival potential. To aid DNA profiling and assessment of its genetic diversity, microsatellite markers were developed for the species.
Frullania subgenus Microfrullania is a clade of ca. 15 liverwort species occurring in Australasia, Malesia, and southern South America. We used combined nuclear and chloroplast sequence data from 265 ingroup accessions to test species circumscriptions and estimate the biogeographic history of the subgenus. With dense infra-specific sampling, we document an important role of long-distance dispersal in establishing phylogeographic patterns of extant species. At deeper time scales, a combination of phylogenetic analyses, divergence time estimation and ancestral range estimation were used to reject vicariance and to document the role of long-distance dispersal in explaining the evolution and biogeography of the clade across the southern Hemisphere. A backbone phylogeny for the subgenus is proposed, providing insight into evolution of morphological patterns and establishing the basis for an improved sectional classification of species within Microfrullania. Several species complexes are identified, the presence of two undescribed but genetically and morphologically distinct species is noted, and previously neglected names are discussed.
DNA barcoding has been a major advancement in the field of taxonomy, seeing much effort put into the barcoding of wide taxa of organisms, macro and microalgae included. The mitochondrial-encoded cox1 and plastid-encoded rbcL has been proposed as potential DNA barcodes for rhodophytes, but are yet to be tested on the commercially important carrageenophytes Kappaphycus and Eucheuma. This study gauges the effectiveness of four markers, namely the mitochondrial cox1, cox2, cox2-3 spacer and the plastid rbcL in DNA barcoding on selected Kappaphycus and Eucheuma from Southeast Asia. Marker assessments were performed using established distance and tree-based identification criteria from earlier studies. Barcoding patterns on a larger scale were simulated by empirically testing on the commonly used cox2-3 spacer. The phylogeny of these rhodophytes was also briefly described. In this study, the cox2 marker which satisfies the prerequisites of DNA barcodes was found to exhibit moderately high interspecific divergences with no intraspecific variations, thus a promising marker for the DNA barcoding of Kappaphycus and Eucheuma. However, the already extensively used cox2-3 spacer was deemed to be in overall more appropriate as a DNA barcode for these two genera. On a wider scale, cox1 and rbcL were still better DNA barcodes across the rhodophyte taxa when practicality and cost-efficiency were taken into account. The phylogeny of Kappaphycus and Eucheuma were generally similar to those earlier reported. Still, the application of DNA barcoding has demonstrated our relatively poor taxonomic comprehension of these seaweeds, thus suggesting more in-depth efforts in taxonomic restructuring as well as establishment.
Di-nucleotide microsatellites were isolated from a genomic library of a tropical tree species, Dryobalanops lanceolata, in Sarawak, for the purpose of using them as hypervariable genetic markers to study the pollen-mediated gene flow. Among 1600 recombinant clones, in total 20 clones gave positive signals when hybridized with oligonucleotides with the three different repeat motifs, GT, CA and CT. Estimations of abundance of (GT)n/(CA)n and (GA)n/(CT)n dinucleotide repeats in D. lanceolata genome revealed to be one in every 84 kb and 80 kb, respectively. Among six sequenced microsatellite loci, one was selected to synthesize PCR primers to amplify the microsatellite. PCR product size of the locus was variable among different individuals, which is attributed to the different number of di-nucleotide repeats. The same microsatellite genotype was detected in the trunk and canopy of a single large tree, indicating the utility of trunk tissue as the source of DNA for the population genetic study of tropical tree species, the canopy of which is usually difficult to approach.
DNA is widely used in plant genetic and molecular biology studies. In this chapter, we describe how to extract DNA from wheat tissues. The tissue samples are ground to disrupt the cell wall. Then cetyltrimethylammonium bromide (CTAB) or sodium dodecyl sulfate (SDS) is used to disrupt the cell and nuclear membranes to release the DNA into solution. A reducing agent, β-mercaptoethanol, is added to break the disulfide bonds between the cysteine residues and to help remove the tanins and polyphenols. A high concentration of salt is employed to remove polysaccharides. Ethylenediaminetetraacetic acid (EDTA) stops DNase activity by chelating the magnesium ions. The nucleic acid solution is extracted with chloroform-isoamyl alcohol (24:1) or 6 M ammonium acetate. The DNA in aqueous phase is precipated with ethanol or isopropanol, which makes DNA less hydrophilic in the presence of sodium ions (Na+).