Metabolite profiling of Piper longum L. fruit volatiles by two-dimensional gas chromatography and time-of-flight mass spectrometry: Insights into the chemical complexity.

Piper longum L. (long pepper) is an economically and industrially important medicinal plant. However, the characterization of its volatiles has only been analyzed by gas chromatography-mass spectrometry (GC-MS). In the present study, precise characterization of P. longum fruit volatiles has been performed for the first time through advanced two-dimensional gas chromatography-time-of-flight spectrometry (GC×GC-TOFMS). A total of 146 constituents accounting for 93.79% were identified, of which 30 were reported for the first time. All these constituents were classified into alcohols (4.5%), alkanes (8.9%), alkenes (6.71%), esters (6.15%), ketones (0.58%), monoterpene hydrocarbons (1.64%), oxygenated monoterpenes (2.24%), sesquiterpene hydrocarbons (49.61%), oxygenated sesquiterpenes (13.03%), phenylpropanoid (0.23%), and diterpenes (0.2%). Among all the classes, sesquiterpene hydrocarbons were abundant, with germacrene-D (2.87% ± 0.01%) as the major one, followed by 8-heptadecene (2.69% ± 0.03%), ß-caryophyllene (2.43% ± 0.03%), n-heptadecane (2.4% ± 0.04%), n-pentadecane (2.11% ± 0.05%), and so forth. Further, 20 constituents were observed to be coeluted and separated precisely in the two-dimensional column. The investigation provides an extensive metabolite profiling of P. longum fruit volatiles, which could be helpful to improve its therapeutic potential.

Derivatives of Cinnamic Acid Esters and Terpenic Diversity in Volatiles of Thirty-Six Sand Ginger (Kaempferia galanga L.) Accessions of Eastern India Revealing Quality Chemovars.

The essential oil of Kaempferia galanga L. commonly known as sand ginger has increased its demand in national and international market for decades. Cinnamic acid esters like ethyl-p-methoxy cinnamate (EPMC) and ethyl cinnamate (EC) are major constituents in its essential oil. In spite of the high demand for the plant as raw material, identification of quality chemovars having high essential oil (EO) yield and constituents is still at an infant stage. With this in mind, we have evaluated the EO yield of 36 accessions from three provinces of Eastern India, which varied within a range of 0.41 ± 0.01 to 2.63 ± 0.03 v/w. Further, a total of 65 compounds were detected by gas chromatography and mass spectrometry (GC-MS) with area percentages varying from 76.16 to 97.3%. EPMC was found to be the major component in 14 accessions with area percentages varying from 10.7% to 41.1%, whereas other 22 accessions showed EC as the major constituent, varying from 16% to 29.1%. Further, a diversity study among accessions was performed by agglomerative hierarchical clustering (AHC) and principal component analysis (PCA) analysis based on the abundance of identified constituents, which categorized all 36 accessions into three clusters. Thus, the present study helps to identify quality chemovar K.g16 and K.g14 with respect to oil yield and constituents, respectively, which could be used to guide commercial cultivation and further improvement of the taxa.

Application of a Multilayer Perceptron Artificial Neural Network for the Prediction and Optimization of the Andrographolide Content in Andrographis paniculata.

Andrographolide, the principal secondary metabolite of Andrographis paniculata, displays a wide spectrum of medicinal activities. The content of andrographolide varies significantly in the species collected from different geographical regions. Therefore, this study aims at investigating the role of different abiotic factors and selecting suitable sites for the cultivation of A. paniculata with high andrographolide content using a multilayer perceptron artificial neural network (MLP-ANN) approach. A total of 150 accessions of A. paniculata collected from different regions of Odisha and West Bengal in eastern India showed a variation in andrographolide content in the range of 0.28-5.45% on a dry weight basis. The MLP-ANN was trained using climatic factors and soil nutrients as the input layer and the andrographolide content as the output layer. The best topological ANN architecture, consisting of 14 input neurons, 12 hidden neurons, and 1 output neuron, could predict the andrographolide content with 90% accuracy. The developed ANN model showed good predictive performance with a correlation coefficient (R2) of 0.9716 and a root-mean-square error (RMSE) of 0.18. The global sensitivity analysis revealed nitrogen followed by phosphorus and potassium as the predominant input variables influencing the andrographolide content. The andrographolide content could be increased from 3.38% to 4.90% by optimizing these sensitive factors. The result showed that the ANN approach is reliable for the prediction of suitable sites for the optimum andrographolide yield in A. paniculata.

Assessment of genetic diversity in Zingiberaceae through nucleotide binding site-based motif-directed profiling.

Functional motif-directed profiling was performed with 15 nucleotide binding site (NBS) primer-enzyme combinations to identify and elucidate the phylogenetic relationships among 15 genotypes of the family Zingiberaceae. We retrieved 167 polymorphic bands (24.85 %), with an average of 11.13 bands per primer. Mean polymorphism rates were detected using MseI (26 %), RsaI (21 %), and AluI (28 %) as restriction enzymes. The polymorphism information content (PIC) for each NBS primer-enzyme combination ranged from 0.48 to 0.76 with a mean value of 0.65. The 38 NBS profiling markers had PIC values ranging from 0.3 to 0.6 and exhibited good power to discriminate between genotypes. Comparison of NBS profiling with microsatellite data for the same set of genotypes exhibited a correlation value of 0.78, P ≤ 0.001. Our study suggests that genetic variability assessment could be more efficient if it targeted genes that exhibit functionally relevant variation, rather than random markers.

Potential role of Indian long pepper (Piper longum L.) volatiles against free radicals and multidrug resistant isolates.

In the present study, the extracted volatiles from dried leaf and fruit of Piper longum were analysed by gas chromatography-mass spectrometry (GC-MS) and each detected 53 constituents having 92.41% and 96.31% of the total volatiles respectively. E-nerolidol (19.56%), ß-pinene (17.07%) and α-pinene (6.8%) were main constituents in leaf volatiles whereas the fruit volatiles dominated by germacrene-D (23.38%), 8-heptadecene (8.95%) and ß-caryophyllene (8.20%). Antioxidant potential of the volatiles were assessed by DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) assays. The fruit volatiles revealed higher radical scavenging activities as compared to leaf. The samples were also evaluated against multidrug resistant (MDR) isolates including one non MDR fungal strain. The fruit volatiles showed a very strong activity against Acinetobacter baumannii than others whereas leaf volatiles possessed strong activity against Klebsiella pneumoniae as compared to other strains. Thus, the dried fruits can be exploited for drug development towards therapeutic purpose.

Thermal desorption modulation based detection of volatile constituents of Alpinia galanga by two dimensional gas chromatography and time of flight mass spectrometry.

Alpinia galanga Wild.(L.) is well known for its aromatic constituents. Though the aromatic composition is already known, but lots of constituents which contributing overall aroma of the oil are still unknown due to the co-eluting factor of single column in GC-MS. Thus the current study aims to characterise maximum volatile constituents present in the essential oil of A. galanga using thermal desorption modulator of two-dimensional gas chromatography and time-of-flight mass spectrometry. The 102 compounds with good match and high probability value were identified out of which 42 were identified for the first time. The total identified compounds include 47 hydrocarbons 25 alcohols, 7 ketones, 7 esters, 3 aldehyde, 4 ethers and 9 other classified aromatic compounds. It was further categorised into Monoterpene Hydrocarbons, Oxygenated Monoterpenes, Sesquiterpene Hydrocarbons and Oxygenated Sesquiterpenes. The major constituent also varies with respect to area percentage. The in-depth characterisation will help in its qualitative analysis.

Rapid plant regeneration in industrially important Curcuma zedoaria revealing genetic and biochemical fidelity of the regenerants.

The present investigation was carried out to establish an efficient and reproducible micropropagation protocol for the production of morphologically, genetically and chemically uniform plants of Curcuma zedoaria. Axillary bud explants of C. zedoaria were inoculated into MS basal medium supplemented with various combinations and concentrations of 6-benzyladenine (2.2-22.2 µM, BA), kinetin (2.3-23.2 µM, Kin), indole-3-acetic acid (2.9-11.4 µM, IAA), α-naphthalene acetic acid (2.7-10.2 µM, NAA) and adenine sulphate (33.9-203.6 µM, Ads). Almost 95% of rhizome buds sprouted on MS medium supplemented with 13.3 µM BA, 5.7 µM IAA and 63.9 µM Ads giving rise to an average of 12.89 ± 0.02 shoots within 6 weeks. However, the maximum number of roots (25.8 ± 0.07 roots per explant) was obtained on half strength MS medium supplemented with 7.4 µM of IBA after 4 weeks of inoculation. Morphological characteristics were similar in both conventionally propagated and micropropagated plants. Additionally, genetic homogeneity of in vitro plants was further confirmed through ISSR and flow cytometry analysis. A total of 27 ISSR primers were screened, out of which 13 ISSR primers generated 58 monomorphic and reproducible bands thereby confirming the genetic uniformity of obtained plants. The mean 2C DNA content of the mother plant (2.96 pg) was similar to that of in vitro derived plants (3.07 pg). Gas chromatography-mass spectrometry (GC-MS) analysis showed similarity in the qualitative profile of chemical constituents of essential oil and high-performance liquid chromatography analysis revealed no significant differences in curcumin content in the tissue culture regenerants and mother plants of C. zedoaria. Therefore, the present micropropagation protocol could be effectively employed to generate true to type plantlets of C. zedoaria.

Chemical composition and antioxidant activities of essential oil of Hedychium greenii and Hedychium gracile from India.

The chemical constituents of the essential oils hydrodistilled from rhizome parts of Hedychium greenii W.W. Sm. and Hedychium gracile Roxb, of family Zingiberaceae, growing in India, were analysed for the first time by GC-FID and GC-MS, respectively. A total of 30 and 29 components representing 99.62 and 96.74% of the total oil were identified in the essential oils of H. greenii and H. gracile, respectively. The major components of H. greenii were bornyl acetate (31.32%), α-pinene (14.49%), camphene (12.81%) and limonene (10.55%), whereas H. gracile was dominated by ß-pinene (25.24%), γ-terpinene (24.62%), terpinen-4-ol (14.87%) and 1,8-cineole (7.51%). Essential oils were assessed for antioxidant potential using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging assay. H. greenii oil exhibited stronger antioxidant potential as compared to H. gracile oil and butylated hydroxytoluene (BHT). Thus, H. greenii rhizome oil has the potential to be used as an alternative source of antioxidant.

Hedychium coronarium extract arrests cell cycle progression, induces apoptosis, and impairs migration and invasion in HeLa cervical cancer cells.

BACKGROUND: Hedychium coronarium Koen. (Zingiberaceae) is traditionally used as medicine in countries such as India, China, and Vietnam for treatment of various ailments including cancer. However, in spite of its implied significance in cancer treatment regimes, there are no reports so far involving the anticancerous attributes of H, coronarium ethanol extract (HCEE) on cancer cells and a more comprehensive study on its mechanism is still lacking. MATERIALS AND METHODS: The cytotoxicity of HCEE was evaluated by MTT and clonogenic survival assay. Annexin V/propidium iodide (PI), Hoechst 33342 staining, and TUNEL assay were performed to detect apoptosis. Cell cycle analysis was performed using PI staining. JC-1 and 2',7'-dichlorodihydrofluorescein diacetate assay were used to check the levels of MMP and ROS, respectively. Western blot analysis was carried out to measure the expression levels of proteins. Migration and invasion activity were assessed by wound healing and Transwell membrane assay, respectively. RESULTS: Antiproliferative effect of HCEE was investigated in various cancerous and normal cell lines. Among these, HCEE significantly inhibited the survival of HeLa cells without affecting the viability of normal human umbilical vein endothelial cells. Annexin V/PI, Hoechst staining, and TUNEL assay showed HCEE induced apoptosis in HeLa cells in a dose-dependent manner. HCEE promoted cell cycle arrest at G1 phase in HeLa cells by upregulating the levels of p53 and p21 and downregulating the levels of cyclin D1, CDK-4, and CDK-6. Moreover, HCEE treatment upregulated the expression of Bax and downregulated the expression of Bcl-2. Additionally, HCEE activated the caspase cascade by increasing the activities of caspase-9, caspase-8, and caspase-3. The expression levels of Fas ligand and Fas were also upregulated. Further, HCEE inhibited the migratory potential of HeLa cells by downregulating MMP-2 and MMP-9 expression levels. CONCLUSION: Our results indicate H. coronarium exerts antiproliferative and apoptotic effects against HeLa cells, and therefore may be used for treatment against cervical cancer.

Phytochemical analysis of flower from Pandanus odorifer (Forssk.) Kuntze for industrial application.

Pandanus odorifer (Forssk.) Kuntze is an economically important aromatic plant. The essential oil from male flowers is widely used in aromatherapy, cosmetics and as food flavouring agent. Phenylethyl methyl ether (PEME), the major constituent of essential oil, gives the chief characteristic fragrance to the oil. In the present study, 180 samples from 12 different regions were collected and hydrodistilled for essential oil isolation. The oil was then subjected to GC and GC-MS analysis to find out the percentage of the constituents. The results revealed PEME as the major constituent ranging from 58.03 to 81.86% and terpinen-4-ol, the second major constituent ranging from 7.81 to 21.46%. Soil nitrogen was found to be the most influential factor for oil yield and PEME content. The flowers containing high essential oil yield and PEME content could be used as elite chemotypes with enough potential for large-scale commercial cultivation to meet the demand of kewda industries.

EST-SSR marker revealed effective over biochemical and morphological scepticism towards identification of specific turmeric (Curcuma longa L.) cultivars.

Turmeric (Curcuma longa L., family Zingiberaceae) is one of the most economically important plants for its use in food, medicine, and cosmetic industries. Cultivar identification is a major constraint in turmeric, owing to high degree of morphological similarity that in turn, affects its commercialization. The present study addresses this constraint, using EST-SSR marker based, molecular identification of 8 elite cultivars and 88 accessions in turmeric. Fifty EST-SSR primers were screened against eight cultivars of turmeric (Suroma, Roma, Lakadong, Megha, Alleppey Supreme, Kedaram, Pratibha, and Suvarna); out of which 11 primers showed polymorphic banding pattern. The polymorphic information content (PIC) of these primers ranged from 0.13 to 0.48. However, only three SSR loci (CSSR 14, CSSR 15, and CSSR 18) gave reproducible unique banding pattern clearly distinguishing the cultivars 'Lakadong' and 'Suvarna' from other cultivars tested. These three unique SSR markers also proved to be effective in identification of 'Lakadong' cultivars when analysed with 88 accessions of turmeric collected from different agro-climatic regions. Furthermore, two identified cultivars (Lakadong and Suvarna) could also be precisely differentiated when analysed and based on phylogenetic tree, with other 94 genotypes of turmeric. The novel SSR markers can be used for identification and authentication of two commercially important turmeric cultivars 'Lakadong' and 'Suvarna'.

Characterization of Kewda volatile components by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry.

Kewda (Pandanus fascicularis Lam.) is a well known medicinal and aromatic plant. The paper aims to precisely characterize volatile constituents present in Kewda flower oil using comprehensive two-dimensional gas chromatography coupled to time of flight mass spectrometry (GCxGC-TOFMS). A total of 159 components were identified due to enhanced chromatographic separation and mass spectral deconvolution of GCxGC-TOFMS. On the basis of its chemical structure, the identified compounds were grouped into hydrocarbons, alcohols, ethers, ketones, esters, nitrogen compounds, aldehydes, acids, lactones, halides and sulfur containing compounds. Ethers were the major components. The predominant compounds identified by GCxGC-TOFMS were kewda ether, ortho-cymene and terpinen-4-ol. A database containing retention indices of compounds was created for the bi-dimensional column, thus proving to be a remarkable step for analysis of constituents using a GCxGC system. GCxGC-TOFMS separated a number of co-eluting components which were unresolved on a single GC column.

Chemical composition and antioxidant activity of essential oil from leaves and rhizomes of Curcuma angustifolia Roxb.

The essential oil extracted from rhizome and leaf of Curcuma angustifolia Roxb. (Zingiberaceae) was characterised by gas chromatography-mass spectrometry (GC-MS). The GC-MS analysis revealed the presence of 32 and 35 identified constituents, comprising 92.6% and 92% of total leaf and rhizome oil, respectively. Curzerenone (33.2%), 14-hydroxy-δ-cadinene (18.6%) and γ-eudesmol acetate (7.3%) were the main components in leaf oil. In rhizome oil, curzerenone (72.6%), camphor (3.3%) and germacrone (3.3%) were found to be the major constituents. Antioxidant capacities of oil were assessed by various methods, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and reducing power ability (RPA). Based on the results, the leaf oil showed more antioxidant potential as compared to rhizome oil and reference standards (ascorbic acid and butylated hydroxytoluene (BHT)). Thus, the leaf essential oil of C. angustifolia can be used as an alternative source of natural antioxidant.

Transcriptome profiling of Curcuma longa L. cv. Suvarna.

Turmeric is an economically valued crop, because of its utility in the food, pharmaceutical industries and Ayurvedic medicine, attracts the attention in many areas of research work. In the present study, we executed resequencing through transcriptome assembly of the turmeric cultivar Suvarna (CL_Suv_10). Resequencing of Suvarna variety has generated 5 Gbases raw data with 75 bp paired-end sequence. The raw data has been submitted to SRA database of NCBI with accession number SRR4042181. Reads were assembled using Cufflinks-2.2.1 tool which ended up with 42994 numbers of transcripts. The length of transcripts ranged from 83 to15565, with a N50 value 1216 and median transcript length 773. The transcripts were annotated through number of databases. For the first time transcriptome profiling of cultivar Suvarna has been done, which could help towards identification of single nucleotide polymorphisms (SNPs) between Suvarna and other turmeric cultivars for its authentic identification.

Resequencing of Curcuma longa L. cv. Kedaram through transcriptome profiling reveals various novel transcripts.

Curcuma longa L. (Turmeric), of the family Zingiberaceae, is one of the economically as well as medicinally important plant species. It is a sterile, polyploid and vegetatively propagated spice crop cultivated usually in Southeast Asia. In the current study, we carried out re-sequencing through transcriptome profiling of Curcuma longa cv. Kedaram (Cl_Ked_6). We acquired a total of 1 GB raw data by resequencing through paired-end sequencing using Nextseq 500 platform. The raw data obtained in this study can be accessible in NCBI database with accession number of SRR3928562 with bioproject accession number PRJNA324755. Cufflinks-2.2.1 tool was used for transcriptome assembly which resulted in 39,554 numbers of transcripts. The transcript length ranged from 76 to 15,568, having N50 value of 1221 and median transcript length of 860. We annotated the transcripts using multiple databases. This data will be beneficial for studying sequence variations particularly SNPs between cultivars of turmeric towards authentic identification and discovery of novel functional transcripts in Kedaram.

De Novo transcriptome assembly of Zingiber officinale cv. Suruchi of Odisha.

Zingiber officinale Rosc., known as ginger, is an Asian crop, popularly used in every household kitchen and commercially used in bakery, beverage, food and pharmaceutical industries. The present study deals with de novo transcriptome assembly of an elite ginger cultivar Suruchi by next generation sequencing methodology. From the analysis 10.9 GB raw data was obtained which can be available in NCBI accession number SAMN03761185. We identified 41,969 transcripts using Trinity RNA-Seq from ginger rhizome of Suruchi variety from Odisha. The transcript length varied from 300 bp to 8404 bp with a total length of 3,96,40,526 bp and N50 of 1251 bp. To the best of our knowledge, this is the first transcriptome data of an elite ginger cultivar Suruchi released for Odisha state of India which will help molecular biologists to develop genetic markers for identification of cultivars.

Molecular modeling and docking characterization of CzR1, a CC-NBS-LRR R-gene from Curcuma zedoaria Loeb. that confers resistance to Pythium aphanidermatum.

Plant NBS-LRR R-genes recognizes several pathogen associated molecular patterns (PAMPs) and limit pathogen infection through a multifaceted defense response. CzR1, a coiled-coil-nucleotide-binding-site-leucine-rich repeat R-gene isolated from Curcuma zedoaria L exhibit constitutive resistance to different strains of P. aphanidermatum. Majority of the necrotrophic oomycetes are characterized by the presence of carbohydrate PAMPs ß-glucans in their cell walls which intercat with R-genes. In the present study, we predicted the 3D (three dimensional) structure of CzR1 based on homology modeling using the homology module of Prime through the Maestro interface of Schrodinger package ver 2.5. The docking investigation of CzR1 with ß-glucan using the Glide software suggests that six amino acid residues, Ser186, Glu187, Ser263, Asp264, Asp355 and Tyr425 act as catalytic residues and are involved in hydrogen bonding with ligand ß-(1,3)-D-Glucan. The calculated distance between the carboxylic oxygen atoms of Glu187-Asp355 pair is well within the distance of 5Å suggesting a positive glucanase activity of CzR1. Elucidation of these molecular characteristics will help in in silico screening and understanding the structural basis of ligand binding to CzR1 protein and pave new ways towards a broad spectrum rhizome rot resistance development in the cultivated turmeric.

Classification and comparative analysis of Curcuma longa L. expressed sequences tags (ESTs) encoding glycine-rich proteins (GRPs).

Glycine-rich proteins (GRPs) are a group of proteins characterized by their high content of glycine residues often occurring in repetitive blocs. The diverse expression pattern and sub cellular localization of various GRPs suggest their implication in different physiological processes. Several GRPs has been isolated and characterized from different monocots and dicots. However, little or no information is available about the structure and function of GRPs in asexually reproducing plants. In this study, in-silico analysis of expressed sequence tag database resulted in the isolation of fifty-one GRPs from Curcuma longa L., an asexually reproducible plant of great medicinal and economic significance. Phylogenetic analysis grouped the GRPs into four distinct classes based on conserved motifs and nature of glycine-rich repeats. Majority of the isolated GRPs exhibited high homology with known GRPs from other plants that are expressed in response to various stresses. The presence of high structural diversity and signal peptide in some GRPs suggest their diverse physiological role and tissue specific localization. The isolated sequences can be used as a framework for cloning, characterization and expressional analysis of GRPs in response to various biotic and abiotic stresses in Curcuma longa as well as other asexually reproducing plants.

Molecular cloning, characterization, and expression analysis of resistance gene candidates in Kaempferia galanga L.

Majority of the plant disease resistance genes expresses cytoplasmic receptor-like proteins characterized by an N-terminal nucleotide-binding site (NBS) and a leucine-rich repeat (LRR) domain. Degenerative primers based on these conserved motifs were used to isolate NBS type sequences in Kaempferia galanga. Cloning and sequencing identified 12 Kaempferia NBS-type sequences called resistance gene candidates (RGCs) classified into four classes. The amino acid sequences of the RGCs detected the presence of conserved domains, viz., kinase-1a, kinase-2, and hydrophobic GLPL, categorizing them with the NBS-LRR class gene family. Structural and phylogenetic characterization grouped the RGCs with the non-toll interleukin receptor (non-TIR) subclasses of the NBS sequences. Reverse transcription PCR with 10 Kaempferia RGC specific primers revealed 7 out of 10 Kaempferia RGCs to be expressive. The isolation and characterization of Kaempferia RGCs has been reported for the first time in this study. This will provide a starting point towards characterization of candidate resistance genes in Kaempferia and can act as a source pool for disease resistance development in other asexually reproducing plants.

Survey and characterization of NBS-LRR (R) genes in Curcuma longa transcriptome.

Resistance genes are among the most important gene classes for plant breeding purposes being responsible for activation of plant defense mechanisms. Among them, the nucleotide binding site-leucine rich repeat (NBS-LRR) class R-genes are the most abundant and actively found in all types of plants. Insilico characterization of EST database resulted in the detection of 28 NBS types R-gene sequences in Curcuma longa. All the 28 sequences represented the NB-ARC domain, 21 of which were found to have highly conserved motif characteristics and categorized as regular NBS genes. The Open Reading Frames varied from 361 (CL.CON.3566) to 112 (CL.CON.1267) with an average of 279 amino acids. Most alignment occurred with monocots (67.8%) with emphasis on Oryza sativa and Zingiber sequences. All best alignments with dicots occurred with Arabidopsis thaliana, Populus trichocarpa and Medicago sativa. These detected NBS type Rgenes from Curcuma longa can be used as a valuable resource for molecular marker development, molecular mapping of R-genes, and identification of resistance gene analogs and functional and evolutionary characterization of NBS-LRR-encoding resistance genes in asexually reproducing plants.