The impact of diseases such as Phytophthora foot rot and the replacement of unproductive cultivars by high yielding ones has brought about the disappearance of varieties in Piper species, like any other crop. Black pepper (King of spices), is a major spice crop consumed throughout the world. It is widely cultivated across various parts of the world apart from India. The different cultivars may be genetically related and could be a source of valuable genes for disease resistance and an increase in quantity and quality. Even though Western Ghats in India is believed to be the site of origin of this crop, numerous accessions from the NBPGR have not yet been evaluated. Our study aims to investigate the genetic relatedness in major cultivars of black pepper using Amplified Fragment Length Polymorphism.
Amplified Fragment Length Polymorphic (AFLP) DNA analysis was performed in thirty popular cultivars of black pepper from National Bureau of Plant Genetic Resources (NBPGR), India. Fingerprint profiles were generated initially with, five different primer combinations, from which three primer pair combinations (EAGC/MCAA, EAGG/MCTA and EAGC/MCTG) gave consistent and scorable banding patterns. From 173 scorable markers, 158(> 90%) were polymorphic which shows there is considerable variation in the available germplasm. The dendrogram derived by unweighted pair group method analysis (UPGMA) grouped the accessions into three major clusters and four diverse cultivars with only 30% similarity. Karimunda, a widely grown and popular cultivar was unique in the fingerprint profiles obtained.
There are currently few fingerprinting studies using the valuable spice crop black pepper. We found considerable genetic variability among cultivars of black pepper. Fingerprinting analysis with AFLP proved to be an ideal tool for cultivar identification and phylogenetic studies. It shows the high level of polymorphism and the unique characterization of the major cultivars. An extensive range of similarity value between the cultivars was noted (6.01 to 98.13). Further screening of more cultivars will provide valuable information for current breeding programmes.
Next generation sequencing has an advantageon transformational development of species with limited available sequence data as it helps to decode the genome and transcriptome. We carried out the de novo sequencing using illuminaHiSeq™ 2000 to generate the first leaf transcriptome of black pepper (Piper nigrum L.), an important spice variety native to South India and also grown in other tropical regions. Despite the economic and biochemical importance of pepper, a scientifically rigorous study at the molecular level is far from complete due to lack of sufficient sequence information and cytological complexity of its genome. The 55 million raw reads obtained, when assembled using Trinity program generated 2,23,386 contigs and 1,28,157 unigenes. Reports suggest that the repeat-rich genomic regions give rise to small non-coding functional RNAs. MicroRNAs (miRNAs) are the most abundant type of non-coding regulatory RNAs. In spite of the widespread research on miRNAs, little is known about the hair-pin precursors of miRNAs bearing Simple Sequence Repeats (SSRs). We used the array of transcripts generated, for the in silico prediction and detection of ‘43 pre-miRNA candidates bearing different types of SSR motifs’. The analysis identified 3913 different types of SSR motifs with an average of one SSR per 3.04 MB of thetranscriptome. About 0.033% of the transcriptome constituted ‘pre-miRNA candidates bearing SSRs’. The abundance, type and distribution of SSR motifs studied across the hair-pin miRNA precursors, showed a significant bias in the position of SSRs towards the downstream of predicted ‘pre-miRNA candidates’. The catalogue of transcripts identified, together with the demonstration of reliable existence of SSRs in the miRNA precursors, permits future opportunities for understanding the genetic mechanism of black pepper and likely functions of ‘tandem repeats’ in miRNAs.
Molecular breeding of pepper (Capsicum spp.) can be accelerated by developing DNA markers associated with transcriptomes in breeding germplasm. Before the advent of next generation sequencing (NGS) technologies, the majority of sequencing data were generated by the Sanger sequencing method. By leveraging Sanger EST data, we have generated a wealth of genetic information for pepper including thousands of SNPs and Single Position Polymorphic (SPP) markers. To complement and enhance these resources, we applied NGS to three pepper genotypes: Maor, Early Jalapeño and Criollo de Morelos-334 (CM334) to identify SNPs and SSRs in the assembly of these three genotypes.
Two pepper transcriptome assemblies were developed with different purposes. The first reference sequence, assembled by CAP3 software, comprises 31,196 contigs from >125,000 Sanger-EST sequences that were mainly derived from a Korean F1-hybrid line, Bukang. Overlapping probes were designed for 30,815 unigenes to construct a pepper Affymetrix GeneChip® microarray for whole genome analyses. In addition, custom Python scripts were used to identify 4,236 SNPs in contigs of the assembly. A total of 2,489 simple sequence repeats (SSRs) were identified from the assembly, and primers were designed for the SSRs. Annotation of contigs using Blast2GO software resulted in information for 60% of the unigenes in the assembly. The second transcriptome assembly was constructed from more than 200 million Illumina Genome Analyzer II reads (80–120 nt) using a combination of Velvet, CLC workbench and CAP3 software packages. BWA, SAMtools and in-house Perl scripts were used to identify SNPs among three pepper genotypes. The SNPs were filtered to be at least 50 bp from any intron-exon junctions as well as flanking SNPs. More than 22,000 high-quality putative SNPs were identified. Using the MISA software, 10,398 SSR markers were also identified within the Illumina transcriptome assembly and primers were designed for the identified markers. The assembly was annotated by Blast2GO and 14,740 (12%) of annotated contigs were associated with functional proteins.
Before availability of pepper genome sequence, assembling transcriptomes of this economically important crop was required to generate thousands of high-quality molecular markers that could be used in breeding programs. In order to have a better understanding of the assembled sequences and to identify candidate genes underlying QTLs, we annotated the contigs of Sanger-EST and Illumina transcriptome assemblies. These and other information have been curated in a database that we have dedicated for pepper project.
Pepper; Capsicum spp; Molecular Markers; EST; Transcriptome; RNAseq; Annotation; SNP; SSR; SPP
The badnavirus, piper yellow mottle virus (PYMoV) is known to infect black pepper (Piper nigrum), betelvine (P. betle) and Indian long pepper (P. longum) in India and other parts of the world. Occurrence of PYMoV or other badnaviruses in other species of Piper and its variability is not reported so far. We have analysed sequence variability in the conserved putative reverse transcriptase (RT)/ribonuclease H (RNase H) coding region of the virus using specific badnavirus primers from 13 virus isolates of black pepper collected from different cultivars and regions and one isolate each from 23 other species of Piper. Of these, four species failed to produce expected amplicon while amplicon from four other species showed more similarities to plant sequences than to badnaviruses. Of the remaining, isolates from black pepper, P. argyrophyllum, P. attenuatum, P. barberi, P. betle, P. colubrinum, P. galeatum, P. longum, P. ornatum, P. sarmentosum and P. trichostachyon showed an identity of >85 % at the nucleotide and >90 % at the amino acid level with PYMoV indicating that they are isolates of PYMoV. On the other hand high sequence variability of 21–43 % at nucleotide and 17–46 % at amino acid level compared to PYMoV was found among isolates infecting P. bababudani, P. chaba, P. peepuloides, P. mullesua and P. thomsonii suggesting the presence of new badnaviruses. Phylogenetic analyses showed close clustering of all PYMoV isolates that were well separated from other known badnaviruses. This is the first report of occurrence of PYMoV in eight Piper spp and likely occurrence of four new species in five Piper spp.
Piper yellow mottle virus; Sequence variability; Piper spp. RT/RNase region
Plant chitinases have been of particular interest since they are known to be induced upon pathogen invasion. Inoculation of Piper colubrinum leaves with the foot rot fungus, Phytophthora capsici leads to increase in chitinase activity. A marked increase in chitinase activity in the inoculated leaves was observed, with the maximum activity after 60 h of inoculation and gradually decreased thereafter. Older leaves showed more chitinase activity than young leaves. The level of chitinase in black pepper (Piper nigrum L.) upon inoculation was found to be substantially high when compared to P. colubrinum. RT–PCR using chitinase specific primers revealed differential accumulation of mRNA in P. colubrinum leaves inoculated with P. capsici. However, hyphal extension assays revealed no obvious differences in the ability of the protein extracts to inhibit growth of P. capsici in vitro.
Chitinase assay; Differential accumulation; Piper; Phytophthora capsici; Reverse transcription
The activities of enzymes responsible for lignification in pepper, pre-inoculation with arbuscular mycorrhizal (AM) fungus of Glomus intraradices and/or infection with pathogenic strain of Phytophthora capsici, and the biological control effect of G. intraradices on Phytophthora blight in pepper were investigated. The experiment was carried out with four treatments: (1) plants pre-inoculated with G. intraradices (Gi), (2) plants pre-inoculated with G. intraradices and then infected with P. capsici (Gi+Pc), (3) plants infected with P. capsici (Pc), and (4) plants without any of the two microorganisms (C). Mycorrhizal colonization rate was reduced by about 10% in pathogen challenged plants. Root mortality caused by infection of P. capsici was completely eliminated by pre-inoculation with antagonistic G. intraradices. On the ninth day after pathogen infection, Peroxidase (POD) activity increased by 116.9% in Pc-treated roots but by only 21.2% in Gi+Pc-treated roots, compared with the control, respectively. Polyphenol oxidase (PPO) and Phenylalanine ammonia-lyase (PAL) activities gradually increased during the first 3 d and dramatically decreased in Pc-treated roots but slightly decreased in Gi+Pc-treated roots, respectively. On the ninth day after pathogen infection, PPO and PAL decreased by 62.8% and 73.9% in Pc-treated roots but by only 19.8% and 19.5% in Gi+Pc-treated roots, compared with the control, respectively. Three major POD isozymes (45 000, 53 000 and 114 000) were present in Pc-treated roots, while two major bands (53 000 and 114 000) and one minor band (45 000) were present in spectra of Gi+Pc-treated roots, the 45 000 POD isozyme was significantly suppressed by G. intraradices, suggesting that the 45 000 POD isozyme was induced by the pathogen infection but not induced by the antagonistic G. intraradices. A 60 000 PPO isozyme was induced in Pc-treated roots but not induced in Gi+Pc-treated roots. All these results showed the inoculation of antagonistic G. intraradices alleviates root mortality, activates changes of lignification-related enzymes and induces some of the isozymes in pepper plants infected by P. capsici. The results suggested that G. intraradices is a potentially effective protection agent against P. capsici.
Arbuscular mycorrhizal (AM) fungus; Glomus intraradices; Phytophthora capsici; Peroxidase (POD); Polyphenol oxidase (PPO); Phenylalanine ammonia-lyase (PAL)
Different herbs and spices have been used as feed additives for various purposes in poultry production. This study was conducted to assess the effect of feed supplemented with black pepper (Piper nigrum), turmeric powder (Curcuma longa), coriander seeds (Coriandrum sativum) and their combinations on the performance of broilers. A total of 210 (Cobb) one-d-old chicks were divided into seven groups of 30 birds each. The treatments were: a control group received no supplement, 0.5% black pepper (T1), 0.5% turmeric powder (T2), 2% coriander seeds (T3), a mixture of 0.5% black pepper and 0.5% turmeric powder (T4), a mixture of 0.5% black pepper and 2% coriander seed (T5), and a mixture of 0.5% black pepper, 0.5% turmeric powder and 2% coriander seeds (T6). Higher significant values of body weight gain during the whole period of 5 weeks (p<0.001) were observed in broilers on T1, T3, T5, and T6 compared to control. Dietary supplements with T1, T2, T3, and T6 improved the cumulative G:F of broilers during the whole period of 5 weeks (p<0.001) compared with control. The dressing percentage and edible giblets were not influenced by dietary supplements, while higher values of relative weight of the liver (p<0.05) were obtained in T5 and T6 compared to control. The addition of feed supplements in T5 and T6 significantly increased serum total protein and decreased serum glucose, triglycerides and alkaline phosphatase concentrations compared with the control group (p<0.05). Broilers on T6 showed significant decrease in the serum glutamate pyruvate transaminase concentration (p<0.05) compared to control. The broilers having T5 and T6 supplemented feed had relatively greater antibody titre (p<0.001) at 35 d of age than control. It is concluded that dietary supplements with black pepper or coriander seeds or their combinations enhanced the performance and health status of broiler chickens.
Antibody Titre; Blood Biochemistry; Carcass; Chicken; Herbs; Performance
Peroxidases are involved in many plant processes including plant defense responses to biotic and abiotic stresses. We isolated a novel peroxidase gene CanPOD from leaves of pepper cultivar A3. The full-length gene has a 1353-bp cDNA sequence and contains an open reading frame (ORF) of 975-bp, which encodes a putative polypeptide of 324 amino acids with a theoretical protein size of 34.93 kDa. CanPOD showed diverse expression levels in different tissues of pepper plants. To evaluate the role of CanPOD in plant stress responses, the expression patterns of CanPOD were examined using Real-Time RT-PCR. The results indicated that CanPOD was significantly induced by Phytophtora capsici. Moreover, CanPOD was also up-regulated in leaves after salt and drought stress treatments. In addition, CanPOD expression was strongly induced by signaling hormones salicylic acid (SA). In contrast, CanPOD was not highly expressed after treatment with cold. Meanwhile, in order to further assess the role of gene CanPOD in defense response to P. capsici attack, we performed a loss-of-function experiment using the virus-induced gene silencing (VIGS) technique in pepper plants. In comparison to the control plant, the expression levels of CanPOD were obviously decreased in CanPOD-silenced pepper plants. Furthermore, we analyzed the effect of P. capsici on detached-leaves and found that the CanPOD-silenced plant leaves were highly susceptible to P. capsici infection. Taken together, our results suggested that CanPOD is involved in defense responses to P. capsici infection as well as abiotic stresses in pepper plants.
pepper; peroxidase; Phytophtora capsici; abiotic stress; virus-induce gene silencing (VIGS)
MicroRNAs (miRNAs) are a class of non-coding RNAs approximately 21 nt in length which play important roles in regulating gene expression in plants. Although many miRNA studies have focused on a few model plants, miRNAs and their target genes remain largely unknown in hot pepper (Capsicum annuum), one of the most important crops cultivated worldwide. Here, we employed high-throughput sequencing technology to identify miRNAs in pepper extensively from 10 different libraries, including leaf, stem, root, flower, and six developmental stage fruits. Based on a bioinformatics pipeline, we successfully identified 29 and 35 families of conserved and novel miRNAs, respectively. Northern blot analysis was used to validate further the expression of representative miRNAs and to analyze their tissue-specific or developmental stage-specific expression patterns. Moreover, we computationally predicted miRNA targets, many of which were experimentally confirmed using 5′ rapid amplification of cDNA ends analysis. One of the validated novel targets of miR-396 was a domain rearranged methyltransferase, the major de novo methylation enzyme, involved in RNA-directed DNA methylation in plants. This work provides the first reliable draft of the pepper miRNA transcriptome. It offers an expanded picture of pepper miRNAs in relation to other plants, providing a basis for understanding the functional roles of miRNAs in pepper.
Black pepper (Piper nigrum) is one of the well-known spices extensively used worldwide especially in India, and Southeast Asia. The presence of alkaloids in the pepper, namely, piperine and its three stereoisomers, isopiperine, chavicine and isochavicine are well noticed.
The current study evaluated the effect of lyophilization and oven drying on the stability and decomposition of constituents of black pepper ethanolic extract.
Materials and Methods
In the current study ethanolic extract of black pepper obtained by maceration method was dried using two methods. The effect of freeze and oven drying on the chemical composition of the extract especially piperine and its three isomers were evaluated by HPLC analysis of the ethanolic extract before and after drying processes using diode array detector. The UV Vis spectra of the peaks at piperine retention time before and after each drying methods indicated maximum absorbance at 341.2 nm corresponding to standard piperine.
The results indicated a decrease in intensity of the chromatogram peaks at approximately all retention times after freeze drying, indicating a few percent loss of piperine and its isomers upon lyophilization. Two impurity peaks were completely removed from the extract.
In oven dried samples two of the piperine stereoisomers were completely removed from the extract and the intensity of piperine peak was increased.
Freeze Drying; Black Pepper; Piperine
There is no dedicated database available for Expressed Sequence Tags (EST) of the chili pepper (Capsicum annuum), although the interest in a chili pepper EST database is increasing internationally due to the nutritional, economic, and pharmaceutical value of the plant. Recent advances in high-throughput sequencing of the ESTs of chili pepper cv. Bukang have produced hundreds of thousands of complementary DNA (cDNA) sequences. Therefore, a chili pepper EST database was designed and constructed to enable comprehensive analysis of chili pepper gene expression in response to biotic and abiotic stresses.
We built the Pepper EST database to mine the complexity of chili pepper ESTs. The database was built on 122,582 sequenced ESTs and 116,412 refined ESTs from 21 pepper EST libraries. The ESTs were clustered and assembled into virtual consensus cDNAs and the cDNAs were assigned to metabolic pathway, Gene Ontology (GO), and MIPS Functional Catalogue (FunCat). The Pepper EST database is designed to provide a workbench for (i) identifying unigenes in pepper plants, (ii) analyzing expression patterns in different developmental tissues and under conditions of stress, and (iii) comparing the ESTs with those of other members of the Solanaceae family. The Pepper EST database is freely available at .
The Pepper EST database is expected to provide a high-quality resource, which will contribute to gaining a systemic understanding of plant diseases and facilitate genetics-based population studies. The database is also expected to contribute to analysis of gene synteny as part of the chili pepper sequencing project by mapping ESTs to the genome.
Intercropping systems could increase crop diversity and avoid vulnerability to biotic stresses. Most studies have shown that intercropping can provide relief to crops against wind-dispersed pathogens. However, there was limited data on how the practice of intercropping help crops against soil-borne Phytophthora disease.
Compared to pepper monoculture, a large scale intercropping study of maize grown between pepper rows reduced disease levels of the soil-borne pepper Phytophthora blight. These reduced disease levels of Phytophthora in the intercropping system were correlated with the ability of maize plants to form a “root wall” that restricted the movement of Phytophthora capsici across rows. Experimentally, it was found that maize roots attracted the zoospores of P. capsici and then inhibited their growth. When maize plants were grown in close proximity to each other, the roots produced and secreted larger quantities of 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) and 6-methoxy-2-benzoxazolinone (MBOA). Furthermore, MBOA, benzothiazole (BZO), and 2-(methylthio)-benzothiazole (MBZO) were identified in root exudates of maize and showed antimicrobial activity against P. capsici.
Maize could form a “root wall” to restrict the spread of P. capsici across rows in maize and pepper intercropping systems. Antimicrobe compounds secreted by maize root were one of the factors that resulted in the inhibition of P. capsici. These results provide new insights into plant-plant-microbe mechanisms involved in intercropping systems.
Phytophthora capsici, the causal agent of Phytophthora blight, is a major concern in vegetable production in Georgia and many other states in the United States. Contamination of irrigation water sources by P. capsici may be an important source of inoculum for the pathogen. A simple method was developed in this study to improve the efficiency of recovering P. capsici from fruits used as baits in irrigation ponds. In contrast to direct isolation on agar plates, infected fruit tissues were used to inoculate stems of pepper seedlings, and the infected pepper stems were used for isolation on agar plates. With isolation through inoculation of pepper stems, the frequency of recovering P. capsici from infected eggplant and pear fruits increased from 13.9% to 77.7% and 8.1% to 53.5%, respectively, compared with direct isolation on agar plates. P. capsici was isolated from seven out of nine irrigation ponds evaluated, with most of the ponds containing both A1 and A2 mating types and a 4:5 ratio of A1 to A2 when isolates from all ponds were calculated. All P. capsici isolates were pathogenic on squash plants, and only a small proportion (8.2%) of the isolates were resistant or intermediately sensitive to mefenoxam. Simple sequence repeats (SSRs) were identified through bioinformatics mining of 55,848 publicly available expressed sequence tags of P. capsici in dbEST GenBank. Thirty-one pairs of SSR primers were designed, and SSR analysis indicated that the 61 P. capsici isolates from irrigation ponds were genetically distinct. Cluster analysis separated the isolates into five genetic clusters with no more than two genetic groups in one pond, indicating relatively low P. capsici genetic diversity in each pond. The isolation method and SSR markers developed for P. capsici in this study could contribute to a more comprehensive understanding of the genetic diversity of this important pathogen.
Geminiviruses are a large and important family of plant viruses that infect a wide range of crops throughout the world. The Begomovirus genus contains species that are transmitted by whiteflies and are distributed worldwide causing disease on an array of horticultural crops. Symptom remission, in which newly developed leaves of systemically infected plants exhibit a reduction in symptom severity (recovery), has been observed on pepper (Capsicum annuum) plants infected with Pepper golden mosaic virus (PepGMV). Previous studies have shown that transcriptional and post-transcriptional gene silencing mechanisms are involved in the reduction of viral nucleic acid concentration in recovered tissue. In this study, we employed deep transcriptome sequencing methods to assess transcriptional variation in healthy (mock), symptomatic, and recovered pepper leaves following PepGMV infection.
Differential expression analyses of the pepper leaf transcriptome from symptomatic and recovered stages revealed a total of 309 differentially expressed genes between healthy (mock) and symptomatic or recovered tissues. Computational prediction of differential expression was validated using quantitative reverse-transcription PCR confirming the robustness of our bioinformatic methods. Within the set of differentially expressed genes associated with the recovery process were genes involved in defense responses including pathogenesis-related proteins, reactive oxygen species, systemic acquired resistance, jasmonic acid biosynthesis, and ethylene signaling. No major differences were found when compared the differentially expressed genes in symptomatic and recovered tissues. On the other hand, a set of genes with novel roles in defense responses was identified including genes involved in histone modification. This latter result suggested that post-transcriptional and transcriptional gene silencing may be one of the major mechanisms involved in the recovery process. Genes orthologous to the C. annuum proteins involved in the pepper-PepGMV recovery response were identified in both Solanum lycopersicum and Solanum tuberosum suggesting conservation of components of the viral recovery response in the Solanaceae.
These data provide a valuable source of information for improving our understanding of the underlying molecular mechanisms by which pepper leaves become symptomless following infection with geminiviruses. The identification of orthologs for the majority of genes differentially expressed in recovered tissues in two major solanaceous crop species provides the basis for future comparative analyses of the viral recovery process across related taxa.
Differential expression; Geminiviruses; Pepper golden mosaic virus; Plant defense; Recovery
Background and Aims
Anatomy had been one of the foundations in our understanding of plant evolutionary trends and, although recent evo-devo concepts are mostly based on molecular genetics, classical structural information remains useful as ever. Of the various plant organs, the roots have been the least studied, primarily because of the difficulty in obtaining materials, particularly from large woody species. Therefore, this review aims to provide an overview of the information that has accumulated on the anatomy of angiosperm roots and to present possible evolutionary trends between representatives of the major angiosperm clades.
This review covers an overview of the various aspects of the evolutionary origin of the root. The results and discussion focus on angiosperm root anatomy and evolution covering representatives from basal angiosperms, magnoliids, monocots and eudicots. We use information from the literature as well as new data from our own research.
The organization of the root apical meristem (RAM) of Nymphaeales allows for the ground meristem and protoderm to be derived from the same group of initials, similar to those of the monocots, whereas in Amborellales, magnoliids and eudicots, it is their protoderm and lateral rootcap which are derived from the same group of initials. Most members of Nymphaeales are similar to monocots in having ephemeral primary roots and so adventitious roots predominate, whereas Amborellales, Austrobaileyales, magnoliids and eudicots are generally characterized by having primary roots that give rise to a taproot system. Nymphaeales and monocots often have polyarch (heptarch or more) steles, whereas the rest of the basal angiosperms, magnoliids and eudicots usually have diarch to hexarch steles.
Angiosperms exhibit highly varied structural patterns in RAM organization; cortex, epidermis and rootcap origins; and stele patterns. Generally, however, Amborellales, magnoliids and, possibly, Austrobaileyales are more similar to eudicots, and the Nymphaeales are strongly structurally associated with the monocots, especially the Acorales.
Anatomy; angiosperms; cortex; epidermis; evolution; roots; vascular tissue
Saponins are mainly amphipathic glycosides that posses many biological activities and confer potential health benefits to humans. Inspite of its medicinal attributes most of the triterpenes and enzymes involved in the saponin biosynthesis remains uncharacterized at the molecular level. Since the major steroidal components are present in the roots of A. racemosus our study is focussed on the comparative denovo transcriptome analysis of root versus leaf tissue and identifying some root specific transcripts involved in saponin biosynthesis using high-throughput next generation transcriptome sequencing.
After sequencing, de novo assembly and quantitative assessment, 126861 unigenes were finally generated with an average length of 1200 bp. Then functional annotation and GO enrichment analysis was performed by aligning all-unigenes with public protein databases including NR, SwissProt, and KEGG. Differentially expressed genes in root were initially identified using the RPKM method using digital subtraction between root and leaf. Twenty seven putative secondary metabolite related transcripts were experimentally validated for their expression in root or leaf tissue using q-RT PCR analysis. Most of the above selected transcripts showed preferential expression in root as compared to leaf supporting the digitally subtracted result obtained. The methyl jasmonate application induces the secondary metabolite related gene transcripts leading to their increased accumulation in plants. Therefore, the identified transcripts related to saponin biosynthesis were further analyzed for their induced expression after 3, 5 and 12 hours of exogenous application of Methyl Jasmonate in tissue specific manner.
In this study, we have identified a large set of cDNA unigenes from A. racemosus leaf and root tissue. This is the first transcriptome sequencing of this non-model species using Illumina, a next generation sequencing technology. The present study has also identified number of root specific transcripts showing homology with saponin biosynthetic pathway. An integrated pathway of identified saponin biosynthesis transcripts their tissue specific expression and induced accumulation after methyl jasmonate treatment was discussed.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-746) contains supplementary material, which is available to authorized users.
Asparagus racemosus; saponin; Transcriptome; De novo assembly; Unigenes
There are several plant extractions which are being used for mosquito control. The aim of this study was to evaluate the efficacy of Olea vera, Linum usitatissimum and Piper nigera against Anopheles stephensi and Aedes aegypti under laboratory conditions.
These tests were carried out using WHO recommended bioassay method for adult mosquitoes.
The extracts from black pepper was more effective as adulticide with lowest LC50 values (2.26% and 8.4%) against Aedes aegypti and Anopheles stephensi after 24 h of exposure while after 48h (1.56% and 5.11%) respectively. In terms of LC90 value black pepper was best with (8.66% and 30.1%) against Ae. aegypti and An. stephensi after 24 h of exposure while after 48h (4.59% and 17.3%) respectively. In terms of LT50 black pepper took 15 h to kill 50% tested population of Ae. aegypti while against An. stephensi it took more than 2 days. In terms of percentage mortality black pepper kill 84% of the population of Ae. aegypti and 44.75% of the An. stephensi population.
Black pepper showed best results in term of LC50, LC90, LT50 and percentage mortality against Ae. aegypti and An. stephensi. Our study suggested that the plant extracts have potential to kill adult mosquitoes, are environment friendly and can be used for the control of mosquitoes.
Adulticide; Plant extracts; Anopheles stephensi
Some Saudi herbs and spices were analyzed. The results indicated that mustard, black cumin, and cress seeds contain high amount of fat 38.45%, 31.95% and 23.19%, respectively, as compared to clove (16.63%), black pepper (5.34%) and fenugreek (4.51%) seeds. Cress, mustard, black cumin and black pepper contain higher protein contents ranging from 26.61 to 25.45%, as compared to fenugreek (12.91%) and clove (6.9%). Crude fiber and ash content ranged from 6.36 to 23.6% and from 3.57 to 7.1%, respectively. All seeds contain high levels of potassium (ranging from 383 to 823 mg/100g), followed by calcium (ranging from 75 to 270 mg/100g), Magnesium (ranged from 42 to 102 mg/100g) and iron (ranged from 20.5 to 65 mg/100g). However, zinc, manganese and copper were found at low levels. The major fatty acids in cress and mustard were linolenic acid (48.43%) and erucic acid (29.81%), respectively. The lenoleic acid was the major fatty acid in black cumin, fenugreek, black pepper and clove oils being 68.07%, 34.85%, 33.03% and 44.73%, respectively. Total unsaturated fatty acids were 83.24, 95.62, 86.46, 92.99, 81.34 and 87.82% for cress, mustard, black cumin, fenugreek, black pepper and clove, respectively. The differences in the results obtained are due to environmental factors, production areas, cultivars used to produce seeds and also due to the different methods used to prepare these local spices.
The widely cultivated pepper, Capsicum spp., important as a vegetable and spice crop world-wide, is one of the most diverse crops. To enhance breeding programs, a detailed characterization of Capsicum diversity including morphological, geographical and molecular data is required. Currently, molecular data characterizing Capsicum genetic diversity is limited. The development and application of high-throughput genome-wide markers in Capsicum will facilitate more detailed molecular characterization of germplasm collections, genetic relationships, and the generation of ultra-high density maps. We have developed the Pepper GeneChip® array from Affymetrix for polymorphism detection and expression analysis in Capsicum. Probes on the array were designed from 30,815 unigenes assembled from expressed sequence tags (ESTs). Our array design provides a maximum redundancy of 13 probes per base pair position allowing integration of multiple hybridization values per position to detect single position polymorphism (SPP). Hybridization of genomic DNA from 40 diverse C. annuum lines, used in breeding and research programs, and a representative from three additional cultivated species (C. frutescens, C. chinense and C. pubescens) detected 33,401 SPP markers within 13,323 unigenes. Among the C. annuum lines, 6,426 SPPs covering 3,818 unigenes were identified. An estimated three-fold reduction in diversity was detected in non-pungent compared with pungent lines, however, we were able to detect 251 highly informative markers across these C. annuum lines. In addition, an 8.7 cM region without polymorphism was detected around Pun1 in non-pungent C. annuum. An analysis of genetic relatedness and diversity using the software Structure revealed clustering of the germplasm which was confirmed with statistical support by principle components analysis (PCA) and phylogenetic analysis. This research demonstrates the effectiveness of parallel high-throughput discovery and application of genome-wide transcript-based markers to assess genetic and genomic features among Capsicum annuum.
Forty-three bacterial isolates in five genera were recovered from naturally infected green pepper fruits (38 samples) showing dark brown, irregular-shaped splotches. The pathogenicity test was performed on healthy green pepper fruits and red colonies were from inoculated fruits showing the same symptoms and the infected area developed into soft rot. Their identification was based on phenotypic characters and sequence of the gene fragment coding 16S rRNA. Of 43 isolates, 10 showing splotches on green pepper fruits belonged to genus Serratia on the basis of phenotypic characters. One representative isolate of the genus Serratia has been identified by partial 16S rRNA gene sequencing and phylogenetic analysis as belonging to the Serratia rubidaea and has the potential to cause spot on green pepper. Eleven phytopathogenic bacterial isolates were also obtained at the same time but did not induce any splotch symptoms on artificially infected green pepper. Five out of 11 bacterial isolates were identified as Ralstonia on the basis of biochemical tests. Partial sequencing of 16S ribosomal gene of representative isolate revealed that the isolate is Ralstonia solanacearum. The six remaining isolates were related to Xanthomonas vesicatoria on the basis of biochemical tests. Twenty-two of opportunistic human pathogens were isolated at the same time and related to Proteus and Klebsiella. Opportunistic human pathogens did not produce any symptoms on artificially infected green pepper. One representative isolate for each genus was identified as Klebsiella oxytoca and Proteus mirabilis based on their partial 16S rRNA gene sequences. The virulence of the S. rubidaea, the causal agent of green pepper fruits splotches was attributed to the production and secretion of a large variety of enzymes capable of degrading the complex polysaccharides of the plant cell wall and membrane constituents.
Green pepper; Pathogenic bacteria; Klebsiella oxytoca; Proteus mirabilis
The capsaicinoids are a group of compounds produced by chili pepper fruits and are used widely in many fields, especially in medical purposes. The capsaicinoid biosynthetic pathway has not yet been established clearly. To understand more knowledge in biosynthesis of capsaicinoids, we applied RNA-seq for the mixture of placenta and pericarp of pungent pepper (Capsicum frutescens L.). We have assessed the effect of various assembly parameters using different assembly software, and obtained one of the best strategies for de novo assembly of transcriptome data. We obtained a total 54,045 high-quality unigenes (transcripts) using Trinity software. About 92.65% of unigenes showed similarity to the public protein sequences, genome of potato and tomato and pepper (C. annuum) ESTs databases. Our results predicted 3 new structural genes (DHAD, TD, PAT), which filled gaps of the capsaicinoid biosynthetic pathway predicted by Mazourek, and revealed new candidate genes involved in capsaicinoid biosynthesis based on KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. A significant number of SSR (Simple Sequence Repeat) and SNP (Single Nucleotide Polymorphism) markers were predicted in C. frutescens and C. annuum sequences, which will be helpful in the identification of polymorphisms within chili pepper populations. These data will provide new insights to the pathway of capsaicinoid biosynthesis and subsequent research of chili peppers. In addition, our strategy of de novo transcriptome assembly is applicable to a wide range of similar studies.
PURPOSE: Piperine, a major alkaloid of black pepper (Piper nigrum) and long pepper (Piper longum), was shown to have anti-inflammatory activity through the suppression of cyclooxygenase (COX)-2 gene expression and enzyme activity. It is also reported to exhibit anti-platelet activity, but the mechanism underlying this action remains unknown. In this study, we investigated a putative anti-platelet aggregation mechanism involving arachidonic acid (AA) metabolism and how this compares with the mechanism by which it inhibits macrophage inflammatory responses; METHODS: Rabbit platelets and murine macrophage RAW264.7 cells were treated with piperine, and the effect of piperine on the activity of AA-metabolizing enzymes, including cytosolic phospholipase A2 (cPLA2), COX-1, COX-2, and thromboxane A2 (TXA2) synthase, as well as its effect on AA liberation from the plasma membrane components, were assessed using isotopic labeling methods and enzyme immunoassay kit; RESULTS: Piperine significantly suppressed AA liberation by attenuating cPLA2 activity in collagen-stimulated platelets. It also significantly inhibited the activity of TXA2 synthase, but not of COX-1, in platelets. These results suggest that piperine inhibits platelet aggregation by attenuating cPLA2 and TXA2 synthase activities, rather than through the inhibition of COX-1 activity. On the other hand, piperine significantly inhibited lipopolysaccharide-induced generation of prostaglandin (PG)E2 and PGD2 in RAW264.7 cells by suppressing the activity of COX-2, without effect on cPLA2; CONCLUSION: Our findings indicate that piperine inhibits platelet aggregation and macrophage inflammatory response by different mechanisms.
pepper; piperine; platelet aggregation; arachidonic acid; cyclooxygenase; phospholipase A2; thromboxane A2 synthase; prostaglandins
Plants protect themselves against a variety of invading pathogenic organisms via sophisticated defence mechanisms. These responses include deployment of specialized antimicrobial compounds, such as phytoalexins, that rapidly accumulate at pathogen infection sites. However, the extent to which these compounds contribute to species-level resistance and their spectrum of action remain poorly understood. Capsidiol, a defense related phytoalexin, is produced by several solanaceous plants including pepper and tobacco during microbial attack. Interestingly, capsidiol differentially affects growth and germination of the oomycete pathogens Phytophthora infestans and Phytophthora capsici, although the underlying molecular mechanisms remain unknown. In this study we revisited the differential effect of capsidiol on P. infestans and P. capsici, using highly pure capsidiol preparations obtained from yeast engineered to express the capsidiol biosynthetic pathway. Taking advantage of transgenic Phytophthora strains expressing fluorescent markers, we developed a fluorescence-based method to determine the differential effect of capsidiol on Phytophtora growth. Using these assays, we confirm major differences in capsidiol sensitivity between P. infestans and P. capsici and demonstrate that capsidiol alters the growth behaviour of both Phytophthora species. Finally, we report intraspecific variation within P. infestans isolates towards capsidiol tolerance pointing to an arms race between the plant and the pathogens in deployment of defence related phytoalexins.
Expressed sequence tag (EST)-based markers are preferred because they reflect transcribed portions of the genome. We report the development of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers derived from transcriptome sequences in cabbage, and their utility for map construction.
Transcriptome sequences were obtained from two cabbage parental lines, C1184 and C1234, which are susceptible and resistant to black rot disease, respectively, using the 454 platform. A total of 92,255 and 127,522 reads were generated and clustered into 34,688 and 40,947 unigenes, respectively. We identified 2,405 SSR motifs from the unigenes of the black rot-resistant parent C1234. Trinucleotide motifs were the most abundant (66.15%) among the repeat motifs. In addition, 1,167 SNPs were detected between the two parental lines. A total of 937 EST-based SSR and 97 SNP-based dCAPS markers were designed and used for detection of polymorphism between parents. Using an F2 population, we built a genetic map comprising 265 loci, and consisting of 98 EST-based SSRs, 21 SNP-based dCAPS, 55 IBP markers derived from B. rapa genome sequence and 91 public SSRs, distributed on nine linkage groups spanning a total of 1,331.88 cM with an average distance of 5.03 cM between adjacent loci. The parental lines used in this study are elite breeding lines with little genetic diversity; therefore, the markers that mapped in our genetic map will have broad spectrum utility.
This genetic map provides additional genetic information to the existing B. oleracea map. Moreover, the new set of EST-based SSR and dCAPS markers developed herein is a valuable resource for genetic studies and will facilitate cabbage breeding. Additionally, this study demonstrates the usefulness of NGS transcriptomes for the development of genetic maps even with little genetic diversity in the mapping population.
Cabbage; EST; Genetic linkage map; SSR; SNP; Transcriptome sequencing
Low temperature is one of the major factors limiting pepper (Capsicum annuum L.) production during winter and early spring in non-tropical regions. Application of exogenous abscisic acid (ABA) effectively alleviates the symptoms of chilling injury, such as wilting and formation of necrotic lesions on pepper leaves; however, the underlying molecular mechanism is not understood. The aim of this study was to identify genes that are differentially up- or downregulated in ABA-pretreated hot pepper seedlings incubated at 6°C for 48 h, using a suppression subtractive hybridization (SSH) method. A total of 235 high-quality ESTs were isolated, clustered and assembled into a collection of 73 unigenes including 18 contigs and 55 singletons. A total of 37 unigenes (50.68%) showed similarities to genes with known functions in the non-redundant database; the other 36 unigenes (49.32%) showed low similarities or unknown functions. Gene ontology analysis revealed that the 37 unigenes could be classified into nine functional categories. The expression profiles of 18 selected genes were analyzed using quantitative RT-PCR; the expression levels of 10 of these genes were at least two-fold higher in the ABA-pretreated seedlings under chilling stress than water-pretreated (control) plants under chilling stress. In contrast, the other eight genes were downregulated in ABA-pretreated seedlings under chilling stress, with expression levels that were one-third or less of the levels observed in control seedlings under chilling stress. These results suggest that ABA can positively and negatively regulate genes in pepper plants under chilling stress.