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1.  A preliminary assessment of genetic relationships among agronomically important cultivars of black pepper 
BMC Genetics  2007;8:42.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2156-8-42
PMCID: PMC1948014  PMID: 17603884
2.  De novo Transcriptome Sequencing Reveals a Considerable Bias in the Incidence of Simple Sequence Repeats towards the Downstream of ‘Pre-miRNAs’ of Black Pepper 
PLoS ONE  2013;8(3):e56694.
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.
doi:10.1371/journal.pone.0056694
PMCID: PMC3587635  PMID: 23469176
3.  De novo assembly of the pepper transcriptome (Capsicum annuum): a benchmark for in silico discovery of SNPs, SSRs and candidate genes 
BMC Genomics  2012;13:571.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2164-13-571
PMCID: PMC3545863  PMID: 23110314
Pepper; Capsicum spp; Molecular Markers; EST; Transcriptome; RNAseq; Annotation; SNP; SSR; SPP
4.  Sequence diversity among badnavirus isolates infecting black pepper and related species in India 
Virusdisease  2014;25(3):402-407.
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.
doi:10.1007/s13337-014-0221-0
PMCID: PMC4188196
Piper yellow mottle virus; Sequence variability; Piper spp. RT/RNase region
5.  Differential induction of chitinase in Piper colubrinum in response to inoculation with Phytophthora capsici, the cause of foot rot in black pepper 
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.
doi:10.1016/j.sjbs.2009.07.002
PMCID: PMC3730965  PMID: 23961037
Chitinase assay; Differential accumulation; Piper; Phytophthora capsici; Reverse transcription
6.  De Novo Assembly and Characterization of Fruit Transcriptome in Black Pepper (Piper nigrum) 
PLoS ONE  2015;10(6):e0129822.
Black pepper is one of the most popular and oldest spices in the world and valued for its pungent constituent alkaloids. Pinerine is the main bioactive compound in pepper alkaloids, which perform unique physiological functions. However, the mechanisms of piperine synthesis are poorly understood. This study is the first to describe the fruit transcriptome of black pepper by sequencing on Illumina HiSeq 2000 platform. A total of 56,281,710 raw reads were obtained and assembled. From these raw reads, 44,061 unigenes with an average length of 1,345 nt were generated. During functional annotation, 40,537 unigenes were annotated in Gene Ontology categories, Kyoto Encyclopedia of Genes and Genomes pathways, Swiss-Prot database, and Nucleotide Collection (NR/NT) database. In addition, 8,196 simple sequence repeats (SSRs) were detected. In a detailed analysis of the transcriptome, housekeeping genes for quantitative polymerase chain reaction internal control, polymorphic SSRs, and lysine/ornithine metabolism-related genes were identified. These results validated the availability of our database. Our study could provide useful data for further research on piperine synthesis in black pepper.
doi:10.1371/journal.pone.0129822
PMCID: PMC4488137  PMID: 26121657
7.  Active changes of lignification-related enzymes in pepper response to Glomus intraradices and/or Phytophthora capsici *  
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.
doi:10.1631/jzus.2005.B0778
PMCID: PMC1389859  PMID: 16052711
Arbuscular mycorrhizal (AM) fungus; Glomus intraradices; Phytophthora capsici; Peroxidase (POD); Polyphenol oxidase (PPO); Phenylalanine ammonia-lyase (PAL)
8.  Effects of Black Pepper (Piper Nigrum), Turmeric Powder (Curcuma Longa) and Coriander Seeds (Coriandrum Sativum) and Their Combinations as Feed Additives on Growth Performance, Carcass Traits, Some Blood Parameters and Humoral Immune Response of Broiler Chickens 
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.
doi:10.5713/ajas.2013.13644
PMCID: PMC4093167  PMID: 25050023
Antibody Titre; Blood Biochemistry; Carcass; Chicken; Herbs; Performance
9.  A Novel Peroxidase CanPOD Gene of Pepper Is Involved in Defense Responses to Phytophtora capsici Infection as well as Abiotic Stress Tolerance 
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.
doi:10.3390/ijms14023158
PMCID: PMC3588037  PMID: 23380961
pepper; peroxidase; Phytophtora capsici; abiotic stress; virus-induce gene silencing (VIGS)
10.  The Hot Pepper (Capsicum annuum) MicroRNA Transcriptome Reveals Novel and Conserved Targets: A Foundation for Understanding MicroRNA Functional Roles in Hot Pepper 
PLoS ONE  2013;8(5):e64238.
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.
doi:10.1371/journal.pone.0064238
PMCID: PMC3667847  PMID: 23737975
11.  Evaluation of Drying Process on the Composition of Black Pepper Ethanolic Extract by High Performance Liquid Chromatography With Diode Array Detector 
Background
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.
Objectives
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.
Results
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.
Conclusions
In oven dried samples two of the piperine stereoisomers were completely removed from the extract and the intensity of piperine peak was increased.
PMCID: PMC3941875  PMID: 24624176
Freeze Drying; Black Pepper; Piperine
12.  Pepper EST database: comprehensive in silico tool for analyzing the chili pepper (Capsicum annuum) transcriptome 
BMC Plant Biology  2008;8:101.
Background
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.
Results
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 .
Conclusion
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.
doi:10.1186/1471-2229-8-101
PMCID: PMC2575210  PMID: 18844979
13.  Plant-Plant-Microbe Mechanisms Involved in Soil-Borne Disease Suppression on a Maize and Pepper Intercropping System 
PLoS ONE  2014;9(12):e115052.
Background
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.
Principal Findings
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.
Conclusions
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.
doi:10.1371/journal.pone.0115052
PMCID: PMC4281244  PMID: 25551554
14.  Influence of Six Vegetable Cultivars on Reproduction of Meloidogyne javanica 
Journal of Nematology  1983;15(4):559-564.
Replicated field and greenhouse experiments were used to evaluate the effect of tomato, cabbage, cucumber, carrot, Amaranthus hybridus, and pepper on growth and fecundity of Meloidogyne spp., particularly M. javanica. In the field tests, tomato, cucumber, and carrot favored population increases of Meloidogyne spp., while Amaranthus, pepper, and cabbage limited them. Some cropping sequences that included crops from the latter group had a suppressive effect on population growth. Thus, of the 36 cropping sequences that were investigated, the following kept the pests in check: tomato-pepper; tomato-Amaranthus; cabbage-pepper; Amaranthus-pepper; carrot-cabbage; pepper-pepper; pepper-Amaranthus; and Amaranthus-pepper. In the greenhouse tests, tomato, cucumber, and carrot had a high number of galls per 50 cm of root, large, conspicuous galls and egg masses, and a high number of larvae per egg mass. Thus, they were highly susceptible. Cabbage and Amaranthus were unsuitable hosts as reflected in the absence of galls or a low number per 50 cm of root. small size of galls and egg masses, and few progeny on the subsequent crop of pepper. The length of time required for eggs to hatch on different hosts varied considerably and is thought to be a significant factor in infection of hosts.
PMCID: PMC2618329  PMID: 19295847
rotations; pest management
15.  Development of an Improved Isolation Approach and Simple Sequence Repeat Markers To Characterize Phytophthora capsici Populations in Irrigation Ponds in Southern Georgia▿  
Applied and Environmental Microbiology  2009;75(17):5467-5473.
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.
doi:10.1128/AEM.00620-09
PMCID: PMC2737936  PMID: 19581483
16.  Transcriptome analysis of symptomatic and recovered leaves of geminivirus-infected pepper (Capsicum annuum) 
Virology Journal  2012;9:295.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1743-422X-9-295
PMCID: PMC3546870  PMID: 23185982
Differential expression; Geminiviruses; Pepper golden mosaic virus; Plant defense; Recovery
17.  Dynamic transcriptional profiling provides insights into tuberous root development in Rehmannia glutinosa 
Rehmannia glutinosa, an herb of the Scrophulariaceae family, is widely cultivated in the Northern part of China. The tuberous root has well-known medicinal properties; however, yield and quality are threatened by abiotic and biotic stresses. Understanding the molecular process of tuberous root development may help identify novel targets for its control. In the present study, we used Illumina sequencing and de novo assembly strategies to obtain a reference transcriptome that is relevant to tuberous root development. We then conducted RNA-seq quantification analysis to determine gene expression profiles of the adventitious root (AR), thickening adventitious root (TAR), and the developing tuberous root (DTR). Expression profiling identified a total of 6794 differentially expressed unigenes during root development. Bioinformatics analysis and gene expression profiling revealed changes in phenylpropanoid biosynthesis, starch and sucrose metabolism, and plant hormone biosynthesis during root development. Moreover, we identified and allocated putative functions to the genes involved in tuberous root development, including genes related to major carbohydrate metabolism, hormone metabolism, and transcription regulation. The present study provides the initial description of gene expression profiles of AR, TAR, and DTR, which facilitates identification of genes of interest. Moreover, our work provides insights into the molecular mechanisms underlying tuberous root development and may assist in the design and development of improved breeding schemes for different R. glutinosa varieties through genetic manipulation.
doi:10.3389/fpls.2015.00396
PMCID: PMC4461823  PMID: 26113849
Rehmannia glutinosa; tuberous root; development; transcriptome; RNA-seq
18.  De Novo Characterization of the Mung Bean Transcriptome and Transcriptomic Analysis of Adventitious Rooting in Seedlings Using RNA-Seq 
PLoS ONE  2015;10(7):e0132969.
Adventitious rooting is the most important mechanism underlying vegetative propagation and an important strategy for plant propagation under environmental stress. The present study was conducted to obtain transcriptomic data and examine gene expression using RNA-Seq and bioinformatics analysis, thereby providing a foundation for understanding the molecular mechanisms controlling adventitious rooting. Three cDNA libraries constructed from mRNA samples from mung bean hypocotyls during adventitious rooting were sequenced. These three samples generated a total of 73 million, 60 million, and 59 million 100-bp reads, respectively. These reads were assembled into 78,697 unigenes with an average length of 832 bp, totaling 65 Mb. The unigenes were aligned against six public protein databases, and 29,029 unigenes (36.77%) were annotated using BLASTx. Among them, 28,225 (35.75%) and 28,119 (35.62%) unigenes had homologs in the TrEMBL and NCBI non-redundant (Nr) databases, respectively. Of these unigenes, 21,140 were assigned to gene ontology classes, and a total of 11,990 unigenes were classified into 25 KOG functional categories. A total of 7,357 unigenes were annotated to 4,524 KOs, and 4,651 unigenes were mapped onto 342 KEGG pathways using BLAST comparison against the KEGG database. A total of 11,717 unigenes were differentially expressed (fold change>2) during the root induction stage, with 8,772 unigenes down-regulated and 2,945 unigenes up-regulated. A total of 12,737 unigenes were differentially expressed during the root initiation stage, with 9,303 unigenes down-regulated and 3,434 unigenes up-regulated. A total of 5,334 unigenes were differentially expressed between the root induction and initiation stage, with 2,167 unigenes down-regulated and 3,167 unigenes up-regulated. qRT-PCR validation of the 39 genes with known functions indicated a strong correlation (92.3%) with the RNA-Seq data. The GO enrichment, pathway mapping, and gene expression profiles reveal molecular traits for root induction and initiation. This study provides a platform for functional genomic research with this species.
doi:10.1371/journal.pone.0132969
PMCID: PMC4503682  PMID: 26177103
19.  De novo leaf and root transcriptome analysis identified novel genes involved in Steroidal sapogenin biosynthesis in Asparagus racemosus 
BMC Genomics  2014;15(1):746.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2164-15-746
PMCID: PMC4162912  PMID: 25174837
Asparagus racemosus; saponin; Transcriptome; De novo assembly; Unigenes
20.  Host Plant Resistance as an Alternative to Methyl Bromide for Managing Meloidogyne incognita in Pepper 
Journal of Nematology  2002;34(4):374-377.
Pre-plant soil fumigation with methyl bromide and host resistance were compared for managing the southern root-knot nematode (Meloidogyne incognita) in pepper. Three pepper cultivars (Carolina Cayenne, Keystone Resistant Giant, and California Wonder) that differed in resistance to M. incognita were grown in field plots that had been fumigated with methyl bromide (98% CH₃Br : 2% CCl₃NO₂ [w/w]) before planting or left untreated. Carolina Cayenne is a well-adapted cayenne-type pepper that is highly resistant to M. incognita. The bell-type peppers Keystone Resistant Giant and California Wonder are intermediate to susceptible and susceptible, respectively. None of the cultivars exhibited root galling in the methyl bromide fumigated plots and nematode reproduction was minimal (<250 eggs/g fresh root), indicating that the fumigation treatment was highly effective in controlling M. incognita. Root galling of Carolina Cayenne and nematode reproduction were minimal, and fruit yields were not reduced in the untreated plots. The root-galling reaction for Keystone Resistant Giant was intermediate (gall index = 2.9, on a scale of 1 to 5), and nematode reproduction was moderately high. However, yields of Keystone Resistant Giant were not reduced in untreated plots. Root galling was severe (gall index = 4.3) on susceptible California Wonder, nematode reproduction was high, and fruit yields were reduced (P ≤ 0.05) in untreated plots. The resistance exhibited by Carolina Cayenne and Keystone Resistant Giant provides an alternative to methyl bromide for reducing yield losses by southern root-knot nematodes in pepper. The high level of resistance of Carolina Cayenne also suppresses population densities of M. incognita.
PMCID: PMC2620592  PMID: 19265959
Capsicum annuum; Meloidogyne incognita; methyl bromide alternatives; nematode management; nematode resistance; pepper; root-knot nematodes
21.  Adulticidal Activity of Olea vera, Linum usitatissimum and Piper nigera against Anopheles stephensi and Aedes aegypti under Laboratory Conditions 
Background:
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.
Methods:
These tests were carried out using WHO recommended bioassay method for adult mosquitoes.
Results:
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.
Conclusion:
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.
PMCID: PMC3385576  PMID: 22808413
Adulticide; Plant extracts; Anopheles stephensi
22.  Specific requirement for translation initiation factor 4E or its isoform drives plant host susceptibility to Tobacco etch virus 
BMC Plant Biology  2014;14:67.
Background
In plants, eIF4E translation initiation factors and their eIFiso4E isoforms are essential susceptibility factors for many RNA viruses, including potyviruses. Mutations altering these factors are a major source of resistance to the viruses. The eIF4E allelic series is associated with specific resistance spectra in crops such as Capsicum annum. Genetic evidence shows that potyviruses have a specific requirement for a given 4E isoform that depends on the host plant. For example, Tobacco etch virus (TEV) uses eIF4E1 to infect Capsicum annuum but uses eIFiso4E to infect Arabidopsis thaliana. Here, we investigated how TEV exploits different translation initiation factor isoforms to infect these two plant species.
Results
A complementation system was set up in Arabidopsis to test the restoration of systemic infection by TEV. Using this system, Arabidopsis susceptibility to TEV was complemented with a susceptible pepper eIF4E1 allele but not with a resistant allele. Therefore, in Arabidopsis, TEV can use the pepper eIF4E1 instead of the endogenous eIFiso4E isoform so is able to switch between translation initiation factor 4E isoform to infect the same host. Moreover, we show that overexpressing the pepper eIF4E1 alleles is sufficient to make Arabidopsis susceptible to an otherwise incompatible TEV strain. Lastly, we show that the resistant eIF4E1 allele is similarly overcome by a resistance-breaking TEV strain as in pepper, confirming that this Arabidopsis TEV-susceptibility complementation system is allele-specific.
Conclusion
We report here a complementation system in Arabidopsis that makes it possible to assess the role of pepper pvr2-eIF4E alleles in susceptibility to TEV. Heterologous complementation experiments showed that the idiosyncratic properties of the 4E and iso4E proteins create a major checkpoint for viral infection of different hosts. This system could be used to screen natural or induced eIF4E alleles to find and study alleles of interest for plant breeding.
doi:10.1186/1471-2229-14-67
PMCID: PMC3999954  PMID: 24645730
Potyvirus; Translation initiation factor; eIF4E; Arabidopsis thaliana; Capsicum annuum
23.  Chemical Composition and Fatty Acid Content of Some Spices and Herbs under Saudi Arabia Conditions 
The Scientific World Journal  2012;2012:859892.
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.
doi:10.1100/2012/859892
PMCID: PMC3540753  PMID: 23319888
24.  Genetic differentiation and recombination among geographic populations of the fungal pathogen Colletotrichum truncatum from chili peppers in China 
Evolutionary Applications  2014;8(1):108-118.
Colletotrichum truncatum is an extremely important fungal pathogen. It can cause diseases both in humans and in over 460 plant species. However, little is known about its genetic diversity within and among populations. One of the major plant hosts of C. truncatum is pepper, and China is one of the main pepper-producing countries in the world. Here, we propose the hypotheses that geography has a major influence on the relationships among populations of C. truncatum in China and that infections in different populations need to be managed differently. To test these hypotheses, we obtained and analyzed 266 C. truncatum isolates from 13 regions representing the main pepper-growing areas throughout China. The analysis based on nine microsatellite markers identified high intrapopulation genetic diversity, evidence of sexual recombination, and geographic differentiation. The genetic differentiation was positively correlated with geographic distance, with the southern and northern China populations grouped in two distinct clusters. Interestingly, isolates collected from the pepper-breeding center harbored the most private alleles. The results suggest that the geographic populations of C. truncatum on peppers in China are genetically differentiated and should be managed accordingly. Our study also provides a solid foundation from which to further explore the global genetic epidemiology of C. truncatum in both plants and humans.
doi:10.1111/eva.12233
PMCID: PMC4310585  PMID: 25667606
clustering analyses; Colletotrichum truncatum; genetic differentiation; phylogeny; population structure; private alleles
25.  Identification of Novel Pepper Genes Involved in Bax- or INF1-Mediated Cell Death Responses by High-Throughput Virus-Induced Gene Silencing 
Hot pepper is one of the economically important crops in Asia. A large number of gene sequences, including expressed sequence tag (EST) and genomic sequences are publicly available. However, it is still a daunting task to determine gene function due to difficulties in genetic modification of a pepper plants. Here, we show the application of the virus-induced gene silencing (VIGS) repression for the study of 459 pepper ESTs selected as non-host pathogen-induced cell death responsive genes from pepper microarray experiments in Nicotiana benthamiana. Developmental abnormalities in N. benthamiana plants are observed in the 32 (7%) pepper ESTs-silenced plants. Aberrant morphological phenotypes largely comprised of three groups: stunted, abnormal leaf, and dead. In addition, by employing the combination of VIGS and Agrobacterium-mediated transient assays, we identified novel pepper ESTs that involved in Bax or INF1-mediated cell death responses. Silencing of seven pepper ESTs homologs suppressed Bax or INF1-induced cell death, five of which suppressed both cell death responses in N. benthamiana. The genes represented by these five ESTs encode putative proteins with functions in endoplasmic reticulum (ER) stress and lipid signaling. The genes represented by the other two pepper ESTs showing only Bax-mediated cell death inhibition encode a CCCH-type zinc finger protein containing an ankyrin-repeat domain and a probable calcium-binding protein, CML30-like. Taken together, we effectively isolated novel pepper clones that are involved in hypersensitive response (HR)-like cell death using VIGS, and identified silenced clones that have different responses to Bax and INF1 exposure, indicating separate signaling pathways for Bax- and INF1-mediated cell death.
doi:10.3390/ijms141122782
PMCID: PMC3856090  PMID: 24256816
Bax-induced cell death; hot chili pepper (Capsicum annum); hypersensitive response; INF1; Nicotiana benthamiana; virus-induced gene silencing

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