Validated Identity Test Method for Ginkgo biloba NHPs Using DNA-Based Species-Specific Hydrolysis PCR Probe.

Background: There is considerable risk of adulteration of Ginkgo biloba herbal products in the natural health product (NHP) industry. Authentication of G. biloba products is challenging because of the standard, complex, analytical chemistry methods that may be too costly and not appropriate for both raw and finished products. Objective: We sought to develop and validate an alternative method to authenticate G. biloba herbal dietary supplements, based on the use of a species-specific hydrolysis PCR probe assay. Methods: A species-specific hydrolysis probe assay was developed, validated, and evaluated for the performance of the assay in accurately identifying the species of interest using the following analytical validation criteria: (1) specificity (accuracy) in identifying the target species ingredient, while not identifying other nontarget species, (2) sensitivity in detecting the smallest amount of the target material, and (3) reliability (repeatability and reproducibility) in detecting the target species in raw materials on a real-time PCR platform. Results: The species-specific hydrolysis probe assay was successfully developed for raw materials of G. biloba. The specificity of the assay was 100% to the target species. Efficiency of the assay was observed to be 99%, and the reliability of the assay was 100% for the raw/starting materials. Conclusions and Highlights: The method developed in this study is simple, rapid, and easy for supplement manufacturers to perform in their laboratories to ensure that their G. biloba supplements are authentic.

DNA record of some traditional small millet landraces in India and Nepal.

Despite the extensive use of small millet landraces as an important source of nutrition for people living in semi-arid regions, they are presently marginalized and their diversity and distribution are threatened at a global scale. Local farmers have developed ancient breeding programs entrenched in traditional knowledge (TK) that has sustained rural cultures for thousands of years. The convention on biological diversity seeks fair and equitable sharing of genetic resources arising from local knowledge and requires signatory nations to provide appropriate policy and legal framework to farmers' rights over plant genetic resources and associated TK. DNA barcoding employed in this study is proposed as a model for conservation of genetic diversity and an essential step towards documenting and protecting farmers' rights and TK. Our study focuses on 32 landraces of small millets that are still used by indigenous farmers located in the rain fed areas of rural India and Nepal. Traditional knowledge of traits and utility was gathered using participatory methods and semi-structured interviews with key informants. DNA was extracted and sequenced (rbcL, trnH-psbA and ITS2) from 160 samples. Both multivariate analysis of traits and phylogenetic analyses were used to assess diversity among small millet landraces. Our research revealed considerable variation in traits and DNA sequences among the 32 small millet landraces. We utilized a tiered approach using ITS2 DNA barcode to make 100 % accurate landrace (32 landraces) and species (six species) assignments for all 160 blind samples in our study. We have also recorded precious TK of nutritional value, ecological and agricultural traits used by local farmers for each of these traditional landraces. This research demonstrates the potential of DNA barcoding as a reliable identification tool and for use in evaluating and conserving genetic diversity of small millets. We suggest ways in which DNA barcodes could be used in the Protection of Plant Varieties and Farmers' Rights in India and Nepal.

Genomic valorization of the fine scale classification of small millet landraces in southern India.

Our research seeks to investigate genomic diversity of landraces of millet, addressing a key uncertainty that will provide a framework for (i) a DNA barcode method that could be used for fast, sensitive, and accurate identification of millet landraces, and (ii) millet landrace conservation including biocultural diversity. We found considerable intraspecific variation among 15 landraces representing six species of small millets using nuclear regions (ITS, ITS1, and ITS2); there was no variation in plastid regions (rbcL, matK, and trnH-psbA). An efficacious ITS2 DNA barcode was used to make 100% accurate landrace assignments for 150 blind samples representing 15 landraces. Our research revealed that genomic variation is aligned with a fine-scale classification of landraces using traditional knowledge (TK) of local farmers. The landrace classification was highly correlated with traits (morphological, agricultural, and cultural utility) associated with considerable factors such as yield, drought tolerance, growing season, medicinal properties, and nutrition. This could provide a DNA-based model for conservation of genetic diversity and the associated bicultural diversity (TK) of millet landraces, which has sustained marginal farming communities in harsh environments for many generations.