Using ITS2 PCR-RFLP to generate molecular markers for authentication of Sophora flavescens Ait

• Published in: Journal of the science of food and agriculture 2012-03, Vol.92 (4), p.892-898
• Main Authors: Lin, Tzu Che, Yeh, Mau Shing, Cheng, Ya Ming, Lin, Li Chang, Sung, Jih Min
• Format: Article
• Language: English
• Subjects: Amplified Fragment Length Polymorphism Analysis; Base Sequence; Biological and medical sciences; China; Databases, Nucleic Acid; Deoxyribonucleic acid; DNA; DNA marker; DNA Restriction Enzymes - metabolism; DNA, Intergenic - chemistry; DNA, Intergenic - metabolism; DNA, Plant - chemistry; DNA, Plant - metabolism; Drugs, Chinese Herbal - analysis; Enzymes; Food industries; Food science; Fundamental and applied biological sciences. Psychology; General aspects; Genetic Markers; Glycyrrhiza uralensis - genetics; Glycyrrhiza uralensis - metabolism; ITS2; Methods of analysis, processing and quality control, regulation, standards; Molecular Sequence Data; PCR-RFLP; Phylogeny; Plant Roots - metabolism; Polymorphism, Restriction Fragment Length; Restriction Mapping; Sequence Alignment; Sophora - genetics; Sophora - metabolism; Sophora flavescens Ait; Species Specificity; Taiwan
• ISSN: 0022-5142; 1097-0010
• DOI: 10.1002/jsfa.4667

BACKGROUND: Dried root of Sophora flavescens Ait. is a medicinal material occasionally misused or adulterated by other species similar in appearance. In this study the internal transcribed spacer (ITS) regions of DNA samples of S. flavescens Ait. collected from different areas of Taiwan were amplified by polymerase chain reaction (PCR) and compared. The effectiveness of using ITS2 PCR restriction fragment length polymorphism (RFLP)‐generated markers to differentiate S. flavescens Ait. from possible adulterants was also evaluated. RESULTS: The S. flavescens Ait. samples collected from different areas were extremely low in ITS sequence variability at species level. ITS2 PCR‐RFLP coupled with restriction enzymes Sac I, Sac II, Xho I or Pvu I produced specific fragments for all tested variants. ITS2 PCR‐RFLP coupled with Sac II was further performed to identify mixtures of DNA extracts of S. flavescens Ait. and Sophora tomentosa L. in various ratios. The developed ITS2 PCR‐RFLP markers could detect mixed DNA samples of S. flavescens Ait./S. tomentosa L. up to a ratio of 10:1. CONCLUSION: The present study demonstrates the usefulness of ITS2 PCR‐RFLP coupled with pre‐selected restriction enzymes for practical and accurate authentication of S. flavescens Ait. The technique is also suitable for analysing S. flavescens Ait. mixed with other adulterants. Copyright © 2011 Society of Chemical Industry