Effectiveness of an innovative prototype subtracted diversity array (SDA) for fingerprinting plant species of medicinal importance.

The accurate identification of medicinal plants is becoming increasingly important due to reported concerns about purity, quality and safety. The previously developed prototype subtracted diversity array (SDA) had been validated for the ability to distinguish clade-level targets in a phylogenetically accurate manner. This study represents the rigorous investigation of the SDA for genotyping capabilities, including the genotyping of plant species not included during the construction of the SDA, as well as to lower classification levels including family and species. The results show that the SDA, in its current form, has the ability to accurately genotype species not included during SDA development to clade level. Additionally, for those species that were included during SDA development, genotyping is successful to the family level, and to the species level with minor exceptions. Twenty polymorphic SDA features were sequenced in a first attempt to characterize the polymorphic DNA between species, which showed that transposon-like sequences may be valuable as polymorphic features to differentiate angiosperm families and species. Future refinements of the SDA to allow more sensitive genotyping are discussed with the overall goal of accurate medicinal plant identification in mind.

Construction and validation of a prototype microarray for efficient and high-throughput genotyping of angiosperms.

Until recently, the identification of plants relied on conventional techniques, such as morphological, anatomical and chemical profiling, that are often inefficient or unfeasible in certain situations. Extensive literature exists describing the use of polymerase chain reaction (PCR) DNA-based identification techniques, which offer a reliable platform, but their broad application is often limited by a low throughput. However, hybridization-based microarray technology represents a rapid and high-throughput tool for genotype identification at a molecular level. Using an innovative technique, a 'Subtracted Diversity Array' (SDA) of 376 features was constructed from a pooled genomic DNA library of 49 angiosperm species, from which pooled non-angiosperm genomic DNA was subtracted. Although not the first use of a subtraction technique for genotyping, the SDA method was superior in accuracy, sensitivity and efficiency, and showed high-throughput capacity and broad application. The SDA technique was validated for potential genotyping use, and the results indicated a successful subtraction of non-angiosperm DNA. Statistical analysis of the polymorphic features from the pilot study enabled the establishment of accurate phylogenetic relationships, confirming the potential use of the SDA technique for genotyping. Further, the technique substantially enriched the presence of polymorphic sequences; 68% were polymorphic when using the array to differentiate six angiosperm clades (Asterids, Rosids, Caryophyllids, Ranunculids, Monocots and Eumagnoliids). The 'proof of concept' experiments demonstrate the potential of establishing a highly informative, reliable and high-throughput microarray-based technique for novel application to sequence independent genotyping of major angiosperm clades.