Nepentheceae, the most prominent carnivorous family in the Caryophyllales order, comprises the Nepenthes genus, which has modified leaf trap characteristics. Although most Nepenthes species have unique morphologies, their vegetative stages are identical, making identification based on morphology difficult. DNA barcoding is seen as a potential tool for plant identification, with small DNA segments amplified for species identification. In this study, three barcode loci; ribulose-bisphosphate carboxylase (rbcL), intergenic spacer 1 (ITS1) and intergenic spacer 2 (ITS2) and the usefulness of the ITS1 and ITS2 secondary structure for the molecular identification of Nepenthes species were investigated. An analysis of barcodes was conducted using BLASTn, pairwise genetic distance and diversity, followed by secondary structure prediction. The findings reveal that PCR and sequencing were both 100% successful. The present study showed the successful amplification of all targeted DNA barcodes at different sizes. Among the three barcodes, rbcL was the least efficient as a DNA barcode compared to ITS1 and ITS2. The ITS1 nucleotide analysis revealed that the ITS1 barcode had more variations compared to ITS2. The mean genetic distance (K2P) between them was higher for interspecies compared to intraspecies. The results showed that the DNA barcoding gap existed among Nepenthes species, and differences in the secondary structure distinguish the Nepenthes. The secondary structure generated in this study was found to successfully discriminate between the Nepenthes species, leading to enhanced resolutions.
Artemisia vulgaris L. belongs to Asteraceae, is a herbal plant that has various benefits in the medical field, so that its use in the medical field can be explored optimally, the plant must be thoroughly identified. This study aims to identify A. vulgaris both in terms of descriptive morpho-anatomy and DNA barcoding using BLAST and phylogenetic tree reconstruction. The morpho-anatomical character was observed on root, stem, and leaf. DNA barcoding analysis was carried out through amplification and alignment of the rbcL and matK genes. All studies were conducted on three samples from Taman Husada (Medicinal Plant Garden) Graha Famili Surabaya, Indonesia. The anatomical slide was prepared by the paraffin method. Morphological studies revealed that the leaves of A. vulgaris both on the lower-middle part and on the upper part of the stem have differences, especially in the character of the stipules, petioles, and incisions they have. Meanwhile, from the study of anatomy, A. vulgaris has an anomocytic type of stomata and its distribution is mostly on the ventral part of the leaves. Through the BLAST process and phylogenetic tree reconstruction, the plant sequences being studied are closely related to several species of the genus Artemisia as indicated by a percentage identity above 98% and branch proximity between taxa in the reconstructed phylogenetic tree.
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.
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.
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.
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.
Identification of plant variety and cultivar is pivotal in the agricultural sector due to the abundance of plant varieties and cultivars developed in many crop species. However, plant variety and cultivar identification via basic morphological features is problematic and challenging when differentiating closely related species not only due to their limited differences but also due to technical limitations of the process being time-consuming, labour-intensive and costly, and statistically imprecise information being available due to phenotypic plasticity. Therefore, it is imperative to have rapid and highly efficient techniques to mitigate these limitations. This review provides an overview and summarization of the development and application of molecular markers such as Random Amplified Polymorphic DNA (RAPD), Restriction Fragment Length Polymorphism (RFLP), Simple Sequence Repeats (SSR), Inter-simple sequence repeats (ISSR), Amplified Fragment Length Polymorphism (AFLP), Single nucleotide polymorphism (SNP) and DNA barcoding, High-resolution melting (HRM) and biosensor technology as potential tools in the identification of plant variety and cultivar.
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.
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.
Weedy rice is a close relative of domesticated rice (Oryza sativa) that competes aggressively with the crop and limits rice productivity worldwide. Most genetic studies of weedy rice have focused on populations in regions where no reproductively compatible wild Oryza species occur (North America, Europe and northern Asia). Here, we examined the population genetics of weedy rice in Malaysia, where wild rice (O. rufipogon) can be found growing in close proximity to cultivated and weedy rice. Using 375 accessions and a combined analysis of 24 neutral SSR loci and two rice domestication genes (sh4, controlling seed shattering, and Bh4, controlling hull colour), we addressed the following questions: (i) What is the relationship of Malaysian weedy rice to domesticated and wild rice, and to weedy rice strains in the USA? (ii) To what extent does the presence of O. rufipogon influence the genetic and phenotypic diversity of Malaysian weeds? (iii) What do the distributions of sh4 and Bh4 alleles and associated phenotypes reveal about the origin and contemporary evolution of Malaysian weedy rice? Our results reveal the following: independent evolutionary origins for Malaysian weeds and US strains, despite their very close phenotypic resemblance; wild-to-weed gene flow in Malaysian weed populations, including apparent adaptive introgression of seed-shattering alleles; and a prominent role for modern Malaysian cultivars in the origin and recent proliferation of Malaysian weeds. These findings suggest that the genetic complexity and adaptability of weedy crop relatives can be profoundly influenced by proximity to reproductively compatible wild and domesticated populations.
The effects of eight different doses (0, 10, 20, 25, 35, 40, 60, and 100 Gy) of acute gamma irradiation on 44 (three varieties of Curcuma alismatifolia: Chiang Mai Red, Sweet Pink, Kimono Pink, and one Curcuma hybrid (Doi Tung 554) individual plants were investigated. Radiation sensitivity tests revealed that the LD50 values of the varieties were achieved at 21 Gy for Chiang Mai Red, 23 Gy for Sweet Pink, 25 Gy for Kimono Pink, and 28 Gy for Doi Tung 554. From the analysis of variance (ANOVA), significant variations were observed for vegetative traits, flowering development, and rhizome characteristics among the four varieties of Curcuma alismatifolia and dose levels as well as the dose × variety interaction. In irradiated plants, the leaf length, leaf width, inflorescence length, the number of true flowers, the number of pink bracts, number of shoots, plant height, rhizome size, number of storage roots, and number of new rhizomes decreased significantly (P < 0.05) as the radiation dose increased. The cophenetic correlation coefficient (CCC) between genetic dissimilarity matrix estimated from the morphological characters and the UPGMA clustering method was r = 0.93, showing a proof fit. In terms of genetic variation among the acutely irradiated samples, the number of presumed alleles revealed by simple sequence repeats ranged from two to seven alleles with a mean value of 3.1, 4.5, and 5.3 alleles per locus for radiation doses of 0, 10, and 20 Gy, respectively. The average values of the effective number of alleles, Nei's gene diversity, and Shannon's information index were 2.5-3.2, 0.51-0.66, and 0.9-1.3, respectively. The constructed dendrogram grouped the entities into seven clusters. Principal component analysis (PCA) supported the clustering results. Consequently, it was concluded that irradiation with optimum doses of gamma rays efficiently induces mutations in Curcuma alismatifolia varieties.
Experiments were conducted to identify blast-resistant fragrant genotypes for the development of a durable blast-resistant rice variety during years 2012-2013. The results indicate that out of 140 test materials including 114 fragrant germplasms, 25 differential varieties (DVs) harbouring 23 blast-resistant genes, only 16 fragrant rice germplasms showed comparatively better performance against a virulent isolate of blast disease. The reaction pattern of single-spore isolate of Magnaporthe oryzae to differential varieties showed that Pish, Pi9, Pita-2 and Pita are the effective blast-resistant genes against the tested blast isolates in Bangladesh. The DNA markers profiles of selected 16 rice germplasms indicated that genotype Chinigura contained Pish, Pi9 and Pita genes; on the other hand, both BRRI dhan50 and Bawaibhog contained Pish and Pita genes in their genetic background. Genotypes Jirakatari, BR5, and Gopalbhog possessed Pish gene, while Uknimodhu, Deshikatari, Radhunipagol, Kalijira (3), Chinikanai each contained the Pita gene only. There are some materials that did not contain any target gene(s) in their genetic background, but proved resistant in pathogenicity tests. This information provided valuable genetic information for breeders to develop durable blast-resistant fragrant or aromatic rice varieties in Bangladesh.
Multivariate analyses were performed using 13 morphological traits and 13 molecular markers (10 SSRs and three ISSRs) to assess the phylogenetic relationship among tungro resistant genotypes. For morphological traits, the genotypes were grouped into six clusters, according to D(2) statistic and Canonical vector analysis. Plant height, days to flowering, days to maturity, panicle length, number of spikelet per panicle, number of unfilled grain per panicle and yield were important contributors to genetic divergence in 14 rice genotypes. Based on Nei's genetic distance for molecular studies, seven clusters were formed among the tungro resistant and susceptible genotypes. Mantel's test revealed a significant correlation (r = 0.834*) between the morphological and molecular data. To develop high yielding tungro resistant varieties based on both morphological and molecular analyses, crosses could be made with susceptible (BR10 and BR11) genotypes with low yielding but highly resistant genotypes, Sonahidemota, Kumragoir, Nakuchimota, Khaiyamota, Khairymota and Kachamota. The chi-square analysis for seven alleles (RM11, RM17, RM20, RM23, RM80, RM108 and RM531) of SSR and five loci (RY1, MR1, MR2, MR4 and GF5) of three ISSR markers in F2 population of cross, BR11×Sonahidemota, showed a good fit to the expected segregation ratio (1:2:1) for a single gene model.
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.
Species-specific simple sequence repeat (SSR) markers are favored for genetic studies and marker-assisted selection (MAS) breeding for oil palm genetic improvement. This report characterizes 20 SSR markers from an Elaeis oleifera genomic library (gSSR). Characterization of the repeat type in 2000 sequences revealed a high percentage of di-nucleotides (63.6%), followed by tri-nucleotides (24.2%). Primer pairs were successfully designed for 394 of the E. oleifera gSSRs. Subsequent analysis showed the ability of the 20 selected E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The average Polymorphism Information Content (PIC) value for the SSRs was 0.402, with the tri-repeats showing the highest average PIC (0.626). Low values of observed heterozygosity (H(o)) (0.164) and highly positive fixation indices (F(is)) in the E. oleifera germplasm collection, compared to the E. guineensis, indicated an excess of homozygosity in E. oleifera. The transferability of the markers to closely related palms, Elaeis guineensis, Cocos nucifera and ornamental palms is also reported. Sequencing the amplicons of three selected E. oleifera gSSRs across both species and palm taxa revealed variations in the repeat-units. The study showed the potential of E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The markers are also a valuable genetic resource for studying E. oleifera and other genus in the Arecaceae family.
Contamination of insect DNA for RAPD-PCR analysis can be a problem because many primers are non-specific and DNA from parasites or gut contents may be simultaneously extracted along with that of the insect. We measured the quantity of food ingested and assimilated by two sympatric populations of brown planthopper (BPH), Nilaparvata lugens, one from rice and the other from Leersia hexandra (Poaceae), a wetland forage grass, and we also investigated whether host plant DNA contaminates that of herbivore insects in extractions of whole insects. Ingestion and assimilation of food were reduced significantly when individuals derived from one host plant were caged on the other species. The bands, OPA3 (1.25), OPD3 (1.10), OPD3 (0.80), OPD3 (0.60), pUC/M13F (0.35), pUC/M13F (0.20), BOXAIR (0.50), peh#3 (0.50), and peh#3 (0.17) were found in both rice-infesting populations of brown planthopper and its host plant (rice). Similarly, the bands, OPA4 (1.00), OPB10 (0.70), OPD3 (0.90), OPD3 (0.80), OPD3 (0.60), pUC/ M13F (0.35), pUC/M13F (0.20), and BOXAIR (0.50) were found in both Leersia-infesting populations of brown planthopper and the host plant. So, it is clear that the DNA bands amplified in the host plants were also found in the extracts from the insects feeding on them.
Microsatellite markers were developed for Johannesteijsmannia lanceolata to assess the genetic diversity and mating system of this alarmingly endangered species.
Microsatellite markers of an important medicinal plant, Eurycoma longifolia (Simaroubaceae), were developed for DNA profiling and genetic diversity studies.
Plastid trnL-trnF and nuclear ribosomal ITS sequences were obtained from selected wild-type individuals of Polygonum minus Huds. in Peninsular Malaysia. The 380 bp trnL-trnF sequences of the Polygonum minus accessions were identical. Therefore, the trnL-trnF failed to distinguish between the Polygonum minus accessions. However, the divergence of ITS sequences (650 bp) among the Polygonum minus accessions was 1%, indicating that these accessions could be distinguished by the ITS sequences. A phylogenetic relationship based on the ITS sequences was inferred using neighbor-joining, maximum parsimony and Bayesian inference. All of the tree topologies indicated that Polygonum minus from Peninsular Malaysia is unique and different from the synonymous Persicaria minor (Huds.) Opiz and Polygonum kawagoeanum Makino.