ABSTRACT
Plant DNA barcoding has proven to be a handy tool for identifying botanical species. However, extracting DNA from woody materials is often a challenging task. Forensic applications, therefore, must be able to overcome the technical difficulties of this nature. A simple and successful adaptation, through a widely used method in forensic laboratories, using chemistry based on magnetic DNA isolation technology and a robotic platform, is presented here. The model case was the application of this adapted DNA extraction method for the identification of Aspidosperma spp., a genus comprising several species in all biomes of Brazil, including Cerrado and Rain Forest. Such technology adaptation can aid in the identification of seized material and help in investigations involving illegal logging and deforestation, ultimately contributing to environmental protection.
Subject(s)
Aspidosperma/genetics , DNA, Plant/isolation & purification , Wood/genetics , Brazil , Conservation of Natural Resources , Databases, Nucleic Acid , Forensic Genetics/methods , Humans , Pilot Projects , Polymerase Chain Reaction , Sequence Analysis, DNA , Species Specificity , Specimen Handling/methodsABSTRACT
Monoterpenoid indole alkaloids (MIAs) possess a diversity of alkaloid skeletons whose biosynthesis is poorly understood. A bioinformatic search of candidate genes, combined with their virus-induced gene silencing, targeted MIA profiling and in vitro/in vivo pathway reconstitution identified and functionally characterized six genes as well as a seventh enzyme reaction required for the conversion of 19E-geissoschizine to tabersonine and catharanthine. The involvement of pathway intermediates in the formation of four MIA skeletons is described, and the role of stemmadenine-O-acetylation in providing necessary reactive substrates for the formation of iboga and aspidosperma MIAs is described. The results enable the assembly of complex dimeric MIAs used in cancer chemotherapy and open the way to production of many other biologically active MIAs that are not easily available from nature.
Subject(s)
Carbolines/metabolism , Catharanthus/metabolism , Indole Alkaloids/metabolism , Plant Proteins/genetics , Aspidosperma/genetics , Aspidosperma/metabolism , Catharanthus/genetics , Enzymes/genetics , Enzymes/metabolism , Gene Expression Regulation, Plant , Gene Silencing , NADP/metabolism , Plant Proteins/metabolism , Quinolines/metabolism , Strychnos/metabolism , Tabernaemontana/metabolism , Vinca Alkaloids/metabolismABSTRACT
PREMISE OF THE STUDY: This study was designed to develop and characterize a preliminary set of nuclear microsatellite markers for Aspidosperma polyneuron, an endangered tropical tree species. METHODS AND RESULTS: Leaf samples were collected from a group of mature A. polyneuron trees in a large area along the Pardo and Mogi-Guaçu river basins in São Paulo State in the southeastern region of Brazil. Twenty-five simple sequence repeat (SSR) markers were developed and 16 of 30 samples had polymorphic loci. The number of alleles per locus ranged from two to 17 (average = 7.06). A high diversity (H(e) = 0.65) and parentage exclusion probability for the first (0.9995) and second (0.9999) parents were measured. CONCLUSIONS: These 16 polymorphic markers suggest a high potential for genetic diversity, gene flow, and mating system analyses in A. polyneuron.
Subject(s)
Aspidosperma/genetics , DNA, Plant/analysis , Trees/genetics , Aspidosperma/classification , Brazil , Endangered Species , Gene Flow , Genetic Variation , Microsatellite Repeats , Species Specificity , Trees/classificationABSTRACT
Studies on intraspecific variation can contribute to the development of conservation strategies by identifying units of conservation for threatened species. Aspidosperma polyneuron is a tropical tree of seasonal semideciduous forests that is currently endangered and protected because it has been heavily logged for lumber, although it was once common in Brazil and neighboring countries. We investigated genetic structure in two samples of A. polyneuron collected from steep hillsides and from flat areas of a natural forest fragment in northern Paraná State, Brazil. Seven AFLP primer combinations yielded 200 markers, with a polymorphic rate of 88.5% for samples from the flat area and 99% for samples from the high declivity area. Total genetic diversity (H(T)) was 0.387, while the genetic diversity within the populations (H(S)) was 0.307 and 0.372, for samples from the flat and the high declivity areas, respectively. Genetic differentiation between samples was high, with a mean F(ST) of 0.265 and a genetic distance of 0.148, indicative of a high degree of genetic structure over a short distance. Principal coordinate analysis separated the samples into three groups of individuals; the first group included individuals from the high declivity area, the second group consisted of individuals only from the flat area, and the third group had individuals from both areas. Bayesian analysis also showed K = 3 clusters. The unexpected high level of intraspecific variation of A. polyneuron in this small forest fragment should be taken into account when evaluating the genetic impact of forest degradation on this species in other semideciduous forest fragments.
