DNA-Barcoding of Some Medicinal Plant Species in Saudi Arabia Using <i>rbcL</i> and <i>matK</i> Genes

In the Kingdom of Saudi Arabia (KSA), thousands of plants are considered to have therapeutic value. The ambiguous use of identification mainly morphological characteristics of many plants has resulted in the adulteration and displacement of plant products which undermine their therapeutic value and weak documentation of plant resources. The aims of this study were therefore to evaluate genetic variability and explore the phylogeographic architecture for Saudi medicinal plant samples using rbcL and matK genes as barcodes for genomic identification. The matK and rbcL sequences collected for these samples were used as key markers for examining the relationship between Saudi medicinal plant species based on genetic diversity. During our study we were successful in identifying and documenting 4 different species (Foeniculum vulgare, Nitraria retusa, Dodonaea viscosa, and Rumex nervosus) located in Saudi Arabia using DNA barcoding technique. A total number of 8 sequences were obtained with a total sequence length of 6176 bp, where it ranged from 617 bp to 878 bp with an average length of 772 bp. The total number of rbcL sequences length is 2801 bp, where it ranges from 617 bp to 807 bp with an average length of 700.2 bp. Out of the 4 plant samples used, only three samples were identified correctly on the species level with an identity percentage higher than 95% using rbcL gene. Additionally, 4 matK sequences have been retrieved belong to 4 species. The total number of matK sequences length is 3375 bp, where it ranges from 819 bp to 878 bp with an average length of 843.8 bp. Out of the 4 plant samples used, only two samples were identified correctly on the species level with an identity percentage higher than 98% using matK gene. Both rbcL and matK have been able to identify most of our collected plant samples by genus, and some by species. Using only one DNA-barcoding technique was not reliable for plant identification, where matK and rbcL must be used as a dual DNA-barcoding procedure.     

The Kautsky curve is a built-in barcode.

We identify objects from their visually observable morphological features. Automatic methods for identifying living objects are often needed in new technology, and these methods try to utilize shapes. When it comes to identifying plant species automatically, machine vision is difficult to implement because the shapes of different plants overlap and vary greatly because of different viewing angles in field conditions. In the present study we show that chlorophyll a fluorescence, emitted by plant leaves, carries information that can be used for the identification of plant species. Transient changes in fluorescence intensity when a light is turned on were parameterized and then subjected to a variety of pattern recognition procedures. A Self-Organizing Map constructed from the fluorescence signals was found to group the signals according to the phylogenetic origins of the plants. We then used three different methods of pattern recognition, of which the Bayesian Minimum Distance classifier is a parametric technique, whereas the Multilayer Perceptron neural network and k-Nearest Neighbor techniques are nonparametric. Of these techniques, the neural network turned out to be the most powerful one for identifying individual species or groups of species from their fluorescence transients. The excellent recognition accuracy, generally over 95%, allows us to speculate that the method can be further developed into an application in precision agriculture as a means of automatically identifying plant species in the field.     

Salicylic acid-inducible binding of a tobacco nuclear protein to a 10 bp sequence which is highly conserved amongst stress-inducible genes

A 10 bp sequence motif (TCATCTTCTT) which is repeated several times in the 5' non-transcribed region of a barley β-1,3-glucanase gene is also present in the non-translated regions of over 30 different plant genes which are known to be induced by one or more forms of stress. Gel retardation assays and South-western blotting experiments provide evidence that the motif is the binding site for a tobacco nuclear protein with an apparent molecular weight of 40 kDa. Binding activity is increased when nuclear extracts from salicylic acid-treated plants are analysed compared with extracts from control plants, indicating that the protein itself is either induced or modified under conditions of stress. These observations suggest roles for the 10 bp motif and its binding protein as cis - and trans -acting regulators of gene expression during response to stress.     

Plant genomic DNA isolation: an art or a science

Isolating quality DNA from tissues/cells presents a variety of problems in particular when plants are used as the source material. The specific characteristics of plants like the presence of rigid polysaccharide cell wall, pigments, chemical heterogeneity of secondary metabolites found in diverse species of plants, etc., necessitate special consideration and skill during isolation procedure. Until now, numerous protocols have been published for the purpose, but none is found to be universally applicable. Various factors starting from the selection of source material to the concentration of metabolites present in the plant decide the course of the isolation procedure. The present review is an update of various methods used for plant genomic DNA isolation, and it epitomizes the various problems faced and the solutions made to contend with them during DNA isolation from plant cells.     

Microcoding: the second step in DNA barcoding

After the process of DNA barcoding has become well advanced in a group of organisms, as it has in the economically important fungi, the question then arises as to whether shorter and literally more barcode-like DNA segments should be utilized to facilitate rapid identification and, where applicable, detection. Through appropriate software analysis of typical full-length barcodes (generally over 500 base pairs long), uniquely distinctive oligonucleotide 'microcodes' of less than 25 bp can be found that allow rapid identification of circa 100-200 species on various array-like platforms. Microarrays can in principle fulfill the function of microcode-based species identification but, because of their high cost and low level of reusability, they tend to be less cost-effective. Two alternative platforms in current use in fungal identification are reusable nylon-based macroarrays and the Luminex system of specific, colour-coded DNA detection beads analysed by means of a flow cytometer. When the most efficient means of rapid barcode-based species identification is sought, a choice can be made either for one of these methodologies or for basic high-throughput sequencing, depending on the strategic outlook of the investigator and on current costs. Arrays and functionally similar platforms may have a particular advantage when a biologically complex material such as soil or a human respiratory secretion sample is analysed to give a census of relevant species present.     

Utility of indels for species-level identification of a biologically complex plant group: a study with intergenic spacer in Citrus

The Consortium of Barcode of Life plant working group proposed to use the defined portion of plastid genes rbcL and matK either singly or in combination as the standard DNA barcode for plants. But DNA barcode based identification of biologically complex plant groups are always a challenging task due to the occurrence of natural hybridization. Here, we examined the use of indels polymorphism in trnH-psbA and trnL-trnF sequences for rapid species identification of citrus. DNA from young leaves of selected citrus species were isolated and matK gene (~800 bp) and trnH-psbA spacer (~450 bp) of Chloroplast DNA was amplified for species level identification. The sequences within the group taxa of Citrus were aligned using the ClustalX program. With few obvious misalignments were corrected manually using the similarity criterion. We identified a 54 bp inverted repeat or palindrome sequence (27–80 regions) and 6 multi residues indel coding regions. Large inverted repeats in cpDNA provided authentication at the higher taxonomic levels. These diagnostics indel marker from trnH-psbA were successful in identifying different species (5 out of 7) within the studied Citrus except Citrus limon and Citrus medica. These two closely related species are distinguished through the 6 bp deletion in trnL-trnF. This study demonstrated that the indel polymorphism based approach easily characterizes the Citrus species and the same may be applied in other complex groups. Likewise other indels occurring intergenic spacer of chloroplast regions may be tested for rapid identification of other secondary citrus species.     

Near Infrared Spectroscopy Facilitates Rapid Identification of Both Young and Mature Amazonian Tree Species

Precise identification of plant species requires a high level of knowledge by taxonomists and presence of reproductive material. This represents a major limitation for those working with seedlings and juveniles, which differ morphologically from adults and do not bear reproductive structures. Near-infrared spectroscopy (FT-NIR) has previously been shown to be effective in species discrimination of adult plants, so if young and adults have a similar spectral signature, discriminant functions based on FT-NIR spectra of adults can be used to identify leaves from young plants. We tested this with a sample of 419 plants in 13 Amazonian species from the genera Protium and Crepidospermum (Burseraceae). We obtained 12 spectral readings per plant, from adaxial and abaxial surfaces of dried leaves, and compared the rate of correct predictions of species with discriminant functions for different combinations of readings. We showed that the best models for predicting species in early developmental stages are those containing spectral data from both young and adult plants (98% correct predictions of external samples), but even using only adult spectra it is still possible to attain good levels of identification of young. We obtained an average of 75% correct identifications of young plants by discriminant equations based only on adults, when the most informative wavelengths were selected. Most species were accurately predicted (75–100% correct identifications), and only three had poor predictions (27–60%). These results were obtained despite the fact that spectra of young individuals were distinct from those of adults when species were analyzed individually. We concluded that FT-NIR has a high potential in the identification of species even at different ontogenetic stages, and that young plants can be identified based on spectra of adults with reasonable confidence.     

A PCR method for detection of plant meals from the guts of insects

The feeding behaviour of insects is a difficult ecological interaction to study. To date, entomologists have used biochemical and molecular techniques to identify the meals of predatory insects. We present here a molecular approach to identifying the DNA of plant species in the insect gut using the ribulose bisphosphate carboxylase gene large subunit (rbcL). A reference collection of 23 plant species from the southern Jordan Valley, Israel, was genetically characterized and employed. Insects belonging to eight different families were collected in the field along with the plants upon which they were found. After collection and prior to analysis, these insects were isolated on the plants they were found upon in the laboratory. This was to ensure that the insects had only one plant meal in their gut, as multiple plant meals would require additional techniques like cloning. A blind study was performed, genetically confirming plant DNA to species level from the processed gut contents of the insects. All reference plant species could be differentiated using a 157 bp long fragment of the rbcL gene. Plant DNA was identifiable, and the meal of the respective insect was accurately determined in each case. Analyses using experimentally fed crickets, Gryllodes hebraeus, determined that plant DNA was still detectable by PCR up to 12 h post-ingestion. This research proposes the application of molecular techniques for the identification of herbivorous insect feeding behaviour to increase understanding of plant–insect interactions.     

Specific detection of the toxigenic species Fusarium proliferatum and F. oxysporum from asparagus plants using primers based on calmodulin gene sequences

Fusarium proliferatum and Fusarium oxysporum are the causal agents of a destructive disease of asparagus called Fusarium crown and root rot. F. proliferatum from asparagus produces fumonisin B1 and B2, which have been detected as natural contaminants in infected asparagus plants. Polymerase chain reaction (PCR) assays were developed for the rapid identification of F. proliferatum and F. oxysporum in asparagus plants. The primer pairs are based on calmodulin gene sequences. The PCR products from F. proliferatum and F. oxysporum were 526 and 534 bp long, respectively. The assays were successfully applied to identify both species from the vegetative part of the plants.     

Identification and characterization of candidate Rlm4 blackleg resistance genes in Brassica napus using next-generation sequencing

A thorough understanding of the relationships between plants and pathogens is essential if we are to continue to meet the agricultural needs of the world's growing population. The identification of genes underlying important quantitative trait loci is extremely challenging in complex genomes such as Brassica napus (canola, oilseed rape or rapeseed). However, recent advances in next-generation sequencing (NGS) enable much quicker identification of candidate genes for traits of interest. Here, we demonstrate this with the identification of candidate disease resistance genes from B.?napus for its most devastating fungal pathogen, Leptosphaeria maculans (blackleg fungus). These two species are locked in an evolutionary arms race whereby a gene-for-gene interaction confers either resistance or susceptibility in the plant depending on the genotype of the plant and pathogen. Preliminary analysis of the complete genome sequence of Brassica rapa, the diploid progenitor of B.?napus, identified numerous candidate genes with disease resistance characteristics, several of which were clustered around a region syntenic with a major locus (Rlm4) for blackleg resistance on A7 of B.?napus. Molecular analyses of the candidate genes using B.?napus NGS data are presented, and the difficulties associated with identifying functional gene copies within the highly duplicated Brassica genome are discussed.     

Major intrinsic proteins (MIPs) in plants: a complex gene family with major impacts on plant phenotype

The ubiquitous cell membrane proteins called aquaporins are now firmly established as channel proteins that control the specific transport of water molecules across cell membranes in all living organisms. The aquaporins are thus likely to be of fundamental significance to all facets of plant growth and development affected by plant–water relations. A majority of plant aquaporins have been found to share essential structural features with the human aquaporin and exhibit water-transporting ability in various functional assays, and some have been shown experimentally to be of critical importance to plant survival. Furthermore, substantial evidence is now available from a number of plant species that shows differential gene expression of aquaporins in response to abiotic stresses such as salinity, drought, or cold and clearly establishes the aquaporins as major players in the response of plants to conditions that affect water availability. This review summarizes the function and regulation of these genes to develop a greater understanding of the response of plants to water insufficiency, and particularly, to identify tolerant genotypes of major crop species including wheat and rice and plants that are important in agroforestry. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Predicting Invasive Plants: Prospects for a General Screening System Based on Current Regional Models

Screening systems for predicting invasive plants have been independently developed for the non-indigenous floras of North America, the South African fynbos, and Australia. To evaluate the performance of these screening systems outside the regions for which they were developed, we tested them for the non-indigenous flora of the Hawaiian Islands. When known invasive plant species in the Hawaiian Islands were evaluated using the North American and Australian systems, 82% and 93% of the species were predicted to be invasive, respectively, and the remainder were classified as requiring further study. The South African fynbos system correctly predicted only 60% of the invasive species in the Hawaiian Islands. All three screening systems correctly classified a majority of the non-invaders as non-invasive. The Australian system has several advantages over the other systems, including the highest level of correct identification of invaders (>90%), ability to evaluate non-woody plants, and ability to evaluate a species even when the answers to some questions are unknown. Nevertheless, with the Australian system, a large fraction of species known not to be invasive were recommended for further study before importing, so there remains room for improvement in identifying non-invasive species. Based on our results for the Hawaiian Islands and a previous evaluation in New Zealand, the Australian system appears to be a promising template for building a globally applicable system for screening out invasive plant introductions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of a comprehensive detection method for medicinal and toxic plant species

Pharmacologically active ingredients in plants can cause significant morbidity through their increasingly common use in herbal alternative medicines and dietary supplements. Monitoring consumer products for the presence of toxic plants is encumbered by the lack of rapid and specific assays. To create a sensitive, reliable, fast, and broad-spectrum assay for medicinal or toxic plant species, we tested multiplexed ligation-dependent probe amplification (MLPA), which requires partial genomic DNA sequences from species of plants that are not well represented in currently available genetic databases. Genomic DNA was obtained from 21 species of medicinal and/or toxic plants. The PCR products were amplified from these plants and cloned for sequencing. The MLPA method was successful with DNA samples from many different species. The use of a microarray to facilitate screening of potentially thousands of plants in a single assay also was successful. The combination of the specificity of the MLPA assay with the broad-scale capabilities of microarray technology should make this an especially useful tool in screening in foods and commercial herbal preparations to identify the plant compounds actually present. Other applications could potentially extend to the identification of any plant species in samples for academic botanical studies and for biodefense and forensics applications.     

Can aphid-induced plant signals be transmitted aerially and through the rhizosphere?

Aphids, through their close association with plants, cause systemic release of semiochemicals. These may have negative effects on subsequent aphid colonisation and can also have positive roles with insects that are antagonistic to aphid development, for example parasitoids. One of the semiochemicals involved in host selection by aphids is methyl salicylate, and since this compound was shown to have a role as a plant stress signal, the hypothesis that aphids might facilitate identification of new plant signals was examined. Confirmation was obtained during an investigation of avoidance of unsuitable hosts by the lettuce aphid, Nasonovia ribis-nigri. (Z)-Jasmone was identified as a plant-derived semiochemical acting negatively for a number of aphid species, and positively for insect antagonists such as parasitoids and predators. However, when the compound was employed at 0.1 ppm in air above intact plants, these plants then attracted aphid parasitoids long after the (Z)-jasmone itself was no longer detectable. A specific interaction was proposed, since the (Z)-jasmone appeared to be selectively taken up by the plants. Aerial interactions between intact barley plants from different cultivars, which may be differentially releasing stress associated signals, can also influence acceptability to aphids. Furthermore, it has been shown that exudates from the roots of aphid-infested plants, grown hydroponically or in soil, cause intact plants to become more attractive to parasitoids.     

Evaluation of the use of the tobacco PR-1a promoter to monitor defense gene expression by the luciferase bioluminescence reporter system

Because of their marked responsiveness to induction signals, genes encoding pathogenesis-related proteins are used as markers to monitor defense gene expression in plants. To develop a non-invasive bioluminescence reporter assay system, we tested acidic PR-1 gene promoters from tobacco and Arabidopsis. These two promoters share common regulatory elements and are believed to show similar responsiveness to various stimuli but the results of transient expression assays by microprojectile bombardment of various plant cells and npr1 mutant Arabidopsis suggest that the tobacco PR-1a promoter is superior to its Arabidopsis counterpart in terms of responsiveness to salicylic acid treatment. Transgenic Arabidopsis seedlings harboring the tobacco PR-1a promoter fused to firefly luciferase showed marked induction in response to treatment with chemicals that induce defense gene expression in plants. These results suggest that the tobacco PR-1a promoter is applicable in monitoring defense-gene expression in various plant species.     

Acetaldehyde and ethanol biosynthesis in leaves of plants

Leaves of terrestrial plants are aerobic organs, and are not usually considered to possess the enzymes necessary for biosynthesis of ethanol, a product of anaerobic fermentation. We examined the ability of leaves of a number of plant species to produce acetaldehyde and ethanol anaerobically, by incubating detached leaves in N₂ and measuring headspace acetaldehyde and ethanol vapors. Greenhouse-grown maize and soybean leaves produced little or no acetaldehyde or ethanol, while leaves of several species of greenhouse-grown woody plants produced up to 241 nanograms per milliliter headspace ethanol in 24 hours, corresponding to a liquid-phase concentration of up to 3 milligrams per gram dry weight. When leaves of 50 plant species were collected in the field and incubated in N₂, all higher plants produced acetaldehyde and ethanol, with woody plants generally producing greater amounts (up to 1 microgram per milliliter headspace ethanol concentration). Maize and soybean leaves from the field produced both acetaldehyde and ethanol. Production of fermentation products was not due to phylloplane microbial activity: surface sterilized leaves produced as much acetaldehyde and ethanol as did unsterilized controls. There was no relationship between site flooding and foliar ethanol biosynthesis: silver maple and cottonwood from upland sites produced as much acetaldehyde and ethanol anaerobically as did plants from flooded bottomland sites. There was no relationship between flood tolerance of a species and ethanol biosynthesis rates: for example, the flood intolerant species Quercus rubra and the flood tolerant species Quercus palustris produced similar amounts of ethanol. Cottonwood leaves produced more ethanol than did roots, in both headspace and enzymatic assays. These results suggest a paradox: that the plant organ least likely to be exposed to anoxia or hypoxia is rich in the enzymes necessary for fermentation.     

基于连续统去除法的水生植物提取及其时空变化分析——以官厅水库库区为例

光谱特征变量的筛选作为水生植物识别的重要手段之一, 在水生植物种类识别研究中应用广泛。该研究将实测光谱特征提取与多时相Landsat 8 OLI影像数据分析相结合, 找到一种有效识别不同种类水生植物的特征变量。在水生植物反射光谱特征分析中引入矿质分析中普遍使用的连续统去除法, 对光谱重采样结果作连续统去除处理后提取光谱吸收深度特征。采用单因素方差分析法对比7个光谱重采样波段和3个连续统去除吸收深度敏感波段, 发现经连续统去除处理的短波红外1波段(SWIR1CR)对于不同类型的水生植物区分效果最佳。将连续统去除法应用到遥感影像处理上, 发现SWIR1CR波段能较好区分沉水植物和挺水植物; 结合影像归一化植被指数和SWIR1CR波段可较好区分三类水生植物。结合特征波段筛选结果采用支持向量机分类方法, 得到水生植物的分类结果精度为86.33%, 对比全生长期12期影像提取的水生植物分布图, 发现水生植物主要分布于官厅水库库区南北岸浅水区, 水生植物面积最大时约占库区总面积的35.13%; 其中沉水植物年内生长分布变化幅度较大, 6月上旬开始迅速生长; 10月份水生植物开始衰减; 11月份水生植物占库区面积的20%, 沉水、浮水植物大幅衰减消失。     

Extraction of aquatic plants based on continuous removal method and analysis of its temporal and spatial changes—A case study of Guanting Reservoir

光谱特征变量的筛选作为水生植物识别的重要手段之一, 在水生植物种类识别研究中应用广泛。该研究将实测光谱特征提取与多时相Landsat 8 OLI影像数据分析相结合, 找到一种有效识别不同种类水生植物的特征变量。在水生植物反射光谱特征分析中引入矿质分析中普遍使用的连续统去除法, 对光谱重采样结果作连续统去除处理后提取光谱吸收深度特征。采用单因素方差分析法对比7个光谱重采样波段和3个连续统去除吸收深度敏感波段, 发现经连续统去除处理的短波红外1波段(SWIR1CR)对于不同类型的水生植物区分效果最佳。将连续统去除法应用到遥感影像处理上, 发现SWIR1CR波段能较好区分沉水植物和挺水植物; 结合影像归一化植被指数和SWIR1CR波段可较好区分三类水生植物。结合特征波段筛选结果采用支持向量机分类方法, 得到水生植物的分类结果精度为86.33%, 对比全生长期12期影像提取的水生植物分布图, 发现水生植物主要分布于官厅水库库区南北岸浅水区, 水生植物面积最大时约占库区总面积的35.13%; 其中沉水植物年内生长分布变化幅度较大, 6月上旬开始迅速生长; 10月份水生植物开始衰减; 11月份水生植物占库区面积的20%, 沉水、浮水植物大幅衰减消失。     

Specific interactions between arbuscular mycorrhizal fungi and plant growth-promoting bacteria: as revealed by different combinations

The interactions between two plant growth-promoting rhizobacteria (PGPR, Pseudomonas fluorescens SBW25 and Paenibacillus brasilensis PB177), two arbuscular mycorrhizal (AM) fungi (Glomus mosseae and Glomus intraradices) and one pathogenic fungus (Microdochium nivale) were investigated on winter wheat (Triticum aestivum cultivar Tarso) in a greenhouse trial. PB177, but not SBW25, had strong inhibitory effects on M. nivale in dual culture plate assays. The results from the greenhouse experiment show very specific interactions; for example, the two AM fungi react differently when interacting with the same bacteria on plants. Glomus intraradices (single inoculation or together with SBW25) increased plant dry weight on M. nivale-infested plants, suggesting that the pathogenic fungus is counteracted by G. intraradices, but PB177 inhibited this positive effect. This is an example of two completely different reactions between the same AM fungus and two species of bacteria, previously known to enhance plant growth and inhibit pathogens. When searching for plant growth-promoting microorganisms, it is therefore important to test for the most suitable combination of plant, bacteria and fungi in order to achieve satisfactory plant growth benefits.     

Accurate identification of taxon-specific molecular markers in plants based on DNA signature sequence

Accurate identification of plants remains a significant challenge for taxonomists and is the basis for plant diversity conservation. Although DNA barcoding methods are commonly used for plant identification, these are limited by the low amplification success and low discriminative power of selected genomic regions. In this study, we developed a k-mer–based approach, the DNA signature sequence (DSS), to accurately identify plant taxon-specific markers, especially at the species level. DSS is a constant-length nucleotide sequence capable of identifying a taxon and distinguishing it from other taxa. In this study, we performed the first large-scale study of DSS markers in plants. DSS candidates of 3899 angiosperm plant species were calculated based on a chloroplast data set with 4356 assemblies. Using Sanger sequencing of PCR amplicons and high-throughput sequencing, DSSs were validated in four and 165 species, respectively. Based on this, the universality of the DSSs was over 79.38%. Several indicators influencing DSS marker identification and detection have also been evaluated, and common criteria for DSS application in plant identification have been proposed.