RESUMO
The secondary metabolism of Piper species is known to produce a myriad of natural products from various biosynthetic pathways which, represent a rich source of previously uncharacterized chemical compounds. The determination of gene expression profiles in multiple tissue/organ samples could provide valuable clues towards understanding the potential biological functions of chemical changes in these plants. Studies on gene expression by RT-qPCR require particularly careful selection of suitable reference genes as a control for normalization. Here, we provide a study for the identification of reliable reference genes in P. arboreum, P. gaudichaudianum, P. malacophyllum, and P. tuberculatum, at two different life stages: 2-month-old seedlings and adult plants. To do this, annotated sequences were recovered from transcriptome datasets of the above listed Piper spp. These sequences were subjected to expression analysis using RT-qPCR, followed by analysis using the geNorm and NormFinder algorithms. A set of five genes were identified showing stable expression: ACT7 (Actin-7), Cyclophilin (Peptidyl-prolyl cis-trans isomerase), EF1α (Elongation factor 1-alpha), RNABP (RNA-binding protein), and UBCE (Ubiquitin conjugating enzyme). The universality of these genes was then validated using two target genes, ADC (arginine decarboxylase) and SAMDC (S-adenosylmethionine decarboxylase), which are involved in the biosynthesis of polyamines. We showed that normalization genes varied according to Piper spp., and we provide a list of recommended pairs of the best combination for each species. This study provides the first set of suitable candidate genes for gene expression studies in the four Piper spp. assayed, and the findings will facilitate subsequent transcriptomic and functional gene research.
Assuntos
Piper , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Piper/genética , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , TranscriptomaRESUMO
The species Piper hispidinervum, Piper aduncum, and Piper affinis hispidinervum have essential oils with high levels of safrole, dillapiole, and sarisan, respectively. Safrole is important for pharmaceutical and chemical industries, while dillapiole and sarisan are promising compounds to control insects and fungi. These species are very similar morphologically and their taxonomy is controversial. Divergent hypotheses consider P. aduncum and P. hispidinervum either as a single species or as distinct taxa, while P. affinis hispidinervum is inferred to be a natural hybrid or a chemotype of P. hispidinervum. Delimiting the taxonomic boundaries would be helpful for germplasm conservation and breeding programs. This study aimed to undertake a detailed analysis of P. aduncum, P. hispidinervum, and P. affinis hispidinervum karyotype and rDNA sites. Genomic in situ hybridization (GISH) was used to establish genomic homology among species and to test the natural hybridization hypothesis for origin of P. affinis hispidinervum. Karyotype traits were similar for all three species: 2n = 26 small chromosomes, predominantly metacentric. All three species exhibited CMA+ bands on the secondary constriction of chromosome pair 4. A size-heteromorphic 35S rDNA site was co-localized with the CMA+ band. A 5S rDNA site was located in the proximal region of chromosome pair 7. The patterns of genomic hybridization revealed that the repetitive DNA fraction of the species is highly similar in terms of proportion of genome, sequence type, and distribution. Our findings did not allow us to differentiate the three species and point to the importance of deeper genomic studies to elucidate the taxonomic controversy.
Assuntos
Piper , Brasil , DNA Ribossômico/genética , Cariotipagem , Piper/química , Piper/genética , Melhoramento Vegetal , SafrolRESUMO
Piper is one of two large genera in the Piperaceae, and with ca. 2600 species, is one of the largest plant genera in the world. Species delimitation and evaluation of genetic diversity among populations are important requisites for conservation and adequate exploitation of economically important species. DNA barcoding has been used as a powerful tool and a practical method for species characterization and delimitation. The present work aims to evaluate molecular markers for barcoding three Piper species native to Brazil: P. gaudichaudianum ("jaborandi" or "pariparoba"), P. malacophyllum ("pariparoba-murta") and P. regnellii ("caapeba" or "pariparoba"). A reference DNA barcode library was developed using sequences of three candidate regions: ITS2, trnH-psbA and rbcL. Transferability of the microsatellite (SSR) primers Psol 3, Psol 6 and Psol 10, designed originally for Piper solmsianum, to the three Piper species was also evaluated. The discriminatory power of the markers was based on the determination of inter- and intraspecific distances, phylogenetic reconstruction, and clustering analysis, as well as BLASTn comparison. Sequences of ITS2 enabled efficient species identification by means of the BLASTn procedure. Based on these sequences, intraspecific divergence was lower than interspecific variation. Maximum Parsimony analyses based on ITS2 sequences provided three resolved clades, each corresponding to one of the three analysed species. Sequences of trnH-psbA and rbcL had lower discriminatory value. Analyses combining sequences of these regions were less effective toward the attainment of resolved and strongly supported clades of all species. In summary, robustly supported clades of P. regnellii were obtained in most of the analyses, based either on isolated or combined sequences. The SSRs primers Psol 3, Psol 6 and Psol 10 were shown to be transferable to P. gaudichaudianum and P. regnellii, but not to P. malacophyllum. Preliminary cluster analyses based on the polymorphism of the amplified products suggested that Psol 3 has lower potential than Psol 6 and Psol 10 for discrimination of Piper species.
Assuntos
DNA de Plantas/genética , Piper/classificação , Piper/genética , Brasil , Análise por Conglomerados , Código de Barras de DNA Taxonômico , DNA Espaçador Ribossômico/genética , Marcadores Genéticos , Variação Genética , Repetições de Microssatélites , Filogenia , Floresta Úmida , Especificidade da EspécieRESUMO
2,4-Diacetylphloroglucinol (DAPG) (1) is a phenolic polyketide produced by some plant-associated Pseudomonas species, with many biological activities and ecological functions. Here, we aimed at reconstructing the natural history of DAPG using phylogenomics focused at its biosynthetic gene cluster or phl genes. In addition to around 1500 publically available genomes, we obtained and analyzed the sequences of nine novel Pseudomonas endophytes isolated from the antidiabetic medicinal plant Piper auritum. We found that 29 organisms belonging to six Pseudomonas species contain the phl genes at different frequencies depending on the species. The evolution of the phl genes was then reconstructed, leading to at least two clades postulated to correlate with the known chemical diversity surrounding DAPG biosynthesis. Moreover, two of the newly obtained Pseudomonas endophytes with high antiglycation activity were shown to exert their inhibitory activity against the formation of advanced glycation end-products via DAPG and related congeners. Its isomer, 5-hydroxyferulic acid (2), detected during bioactivity-guided fractionation, together with other DAPG congeners, were found to enhance the detected inhibitory activity. This report provides evidence of a link between the evolution and chemical diversity of DAPG and congeners.
Assuntos
Endófitos/química , Floroglucinol/análogos & derivados , Piper/microbiologia , Plantas Medicinais/microbiologia , Policetídeos/isolamento & purificação , Policetídeos/farmacologia , Pseudomonas/química , Ácidos Cumáricos/química , Ácidos Cumáricos/isolamento & purificação , México , Estrutura Molecular , Família Multigênica , Floroglucinol/química , Floroglucinol/isolamento & purificação , Floroglucinol/farmacologia , Piper/genética , Componentes Aéreos da Planta/química , Plantas Medicinais/genética , Policetídeos/química , EstereoisomerismoRESUMO
Black pepper is a perennial climbing vine. It is widely cultivated because its berries can be utilized not only as a spice in food but also for medicinal use. This study aimed to construct a standardized, high-quality cDNA library to facilitated identification of new Piper hainanense transcripts. For this, 262 unigenes were used to generate raw reads. The average length of these 262 unigenes was 774.8 bp. Of these, 94 genes (35.9%) were newly identified, according to the NCBI protein database. Thus, identification of new genes may broaden the molecular knowledge of P. hainanense on the basis of Clusters of Orthologous Groups and Gene Ontology categories. In addition, certain basic genes linked to physiological processes, which can contribute to disease resistance and thereby to the breeding of black pepper. A total of 26 unigenes were found to be SSR markers. Dinucleotide SSR was the main repeat motif, accounting for 61.54%, followed by trinucleotide SSR (23.07%). Eight primer pairs successfully amplified DNA fragments and detected significant amounts of polymorphism among twenty-one piper germplasm. These results present a novel sequence information of P. hainanense, which can serve as the foundation for further genetic research on this species.
Assuntos
Etiquetas de Sequências Expressas , Piper/genética , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Genoma de Planta/genética , Polimorfismo Genético/genéticaRESUMO
PREMISE OF THE STUDY: Documented fossil floras in the neotropics are sparse, yet their records provide evidence on the spatial and temporal occurrence of taxa, allowing for testing of biogeographical and diversification scenarios on individual lineages. A new fossil Piper from the Late Cretaceous of Colombia is described here, and its importance for assessing diversification patterns in the genus is addressed. METHODS: Leaf architecture of 32 fossil leaf compressions from the Guaduas Formation was compared with that of 294 extant angiosperm species. The phylogenetic position of the fossil named Piper margaritae sp. nov. was established based on leaf traits and a molecular scaffold of Piper. The age of the fossil was independently used as a calibration point for divergence time estimations. KEY RESULTS: Natural affinities of P. margaritae to the Schilleria clade of Piper indicate that the genus occurred in tropical America by the Late Cretaceous. Estimates of age divergence and lineage accumulation reveal that most of the extant diversity of the genus accrued during the last â¼30 Myr. CONCLUSIONS: The recent radiation of Piper is coeval with both the Andean uplift and the emergence of Central America, which have been proposed as important drivers of diversity. This pattern could exemplify a recurrent theme among many neotropical plant lineages.
Assuntos
Fósseis , Fenótipo , Filogenia , Piper/genética , Folhas de Planta/anatomia & histologia , Clima Tropical , Colômbia , Especificidade da EspécieRESUMO
Host-parasite systems have been models for understanding the connection between shifts in resource use and diversification. Despite theoretical expectations, ambiguity remains regarding the frequency and importance of host switches as drivers of speciation in herbivorous insects and their parasitoids. We examine phylogenetic patterns with multiple genetic markers across three trophic levels using a diverse lineage of geometrid moths (Eois), specialist braconid parasitoids (Parapanteles) and plants in the genus Piper. Host-parasite associations are mapped onto phylogenies, and levels of cospeciation are assessed. We find nonrandom patterns of host use within both the moth and wasp phylogenies. The moth-plant associations in particular are characterized by small radiations of moths associated with unique host plants in the same geographic area (i.e. closely related moths using the same host plant species). We suggest a model of diversification that emphasizes an interplay of factors including host shifts, vicariance and adaptation to intraspecific variation within hosts.
Assuntos
Cadeia Alimentar , Interações Hospedeiro-Patógeno/genética , Mariposas/genética , Mariposas/parasitologia , Piper/genética , Árvores , Vespas/genética , Animais , Sequência de Bases , Teorema de Bayes , Biologia Computacional , Costa Rica , DNA Mitocondrial/genética , Equador , Evolução Molecular , Especiação Genética , Interações Hospedeiro-Patógeno/fisiologia , Larva/parasitologia , Larva/fisiologia , Modelos Genéticos , Dados de Sequência Molecular , Mariposas/classificação , Mariposas/fisiologia , Filogenia , Análise de Sequência de DNA , Vespas/fisiologiaRESUMO
Random amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) analysis were applied to 74 individual plants of Piper spp in Hainan Island. The results showed that the SRAP technique may be more informative and more efficient and effective for studying genetic diversity of Piper spp than the RAPD technique. The overall level of genetic diversity among Piper spp in Hainan was relatively high, with the mean Shannon diversity index being 0.2822 and 0.2909, and the mean Nei's genetic diversity being 0.1880 and 0.1947, calculated with RAPD and SRAP data, respectively. The ranges of the genetic similarity coefficient were 0.486-0.991 and 0.520-1.000 for 74 individual plants of Piper spp (the mean genetic distance was 0.505 and 0.480) and the within-species genetic distance ranged from 0.063 to 0.291 and from 0.096 to 0.234, estimated with RAPD and SRAP data, respectively. These genetic indices indicated that these species are closely related genetically. The dendrogram generated with the RAPD markers was topologically different from the dendrogram based on SRAP markers, but the SRAP technique clearly distinguished all Piper spp from each other. Evaluation of genetic variation levels of six populations showed that the effective number of alleles, Nei's gene diversity and the Shannon information index within Jianfengling and Diaoluoshan populations are higher than those elsewhere; consequently conservation of wild resources of Piper in these two regions should have priority.
Assuntos
Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/métodos , DNA de Plantas/genética , Marcadores Genéticos , Variação Genética , Piper/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Alelos , Cruzamento , China , Filogenia , Piper/classificação , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
In vitro activity of the essential oil from Piper diospyrifolium leaves was tested using disk diffusion techniques. The antifungal assay showed significant potencial antifungal activity: the oil was effective against several clinical fungal strains. The majority compounds in the essential oil were identified as sesquiterpenoids by GC-MS and GC-FID techniques.
Assuntos
Antifúngicos , Técnicas In Vitro , Estruturas Vegetais , Piper/crescimento & desenvolvimento , Piper/genética , Piperaceae/genética , Sesquiterpenos/análise , Árvores , Oceano Atlântico , Métodos , Óleos Voláteis , Folhas de Planta , Preparações de Plantas , MétodosRESUMO
Piper tuberculatum is an exotic Piper from the Amazon region that shows resistance to infection by Fusarium solani f. sp. piperis, causal agent of Fusarium disease in black pepper (Piper nigrum L.). In this work we aimed to study the interaction between P. tuberculatum and F. solani f. sp. piperis at a molecular level, using suppression subtractive hybridization to identify genes potentially related to Fusarium disease resistance. Comparative sequence analysis confirmed that clones isolated here show a high identity with genes coding for proteins that have a known role in plant defense response mechanisms, such as peroxidase, hydroxyproline-rich glycoprotein and CBL-interacting protein kinase. The present study constitutes the first effort to understand the molecular basis of this plant-pathogen interaction, identifying genes which may be used in the future genetic improvement of black pepper.