Selection And Identification Of DNA Barcodes In The Genus Epimedium From Sichuan Province, China

The genus Epimedium (Beberaceae) as a temperate genus of perennial herbs, comprises54species in the world and43in central-southeastern China. It is effective in strengthening kidneys, enhancing sexual performance, treating cardiovascular diseases and improving immunity. Certain species of Epimedium have been used as a tonic in China for over2000years. The official species listed in the Chinese Pharmacopoeia (2010) are E. brevicornu, E. sagittatum, E. koreanum and E. pubescens.However, the tranditional identification is inadequate due to the morphological variation, complicated procedure and diverse distribution. A new efficient identification has been necessary for the genus Epimedium.DNA barcoding is a novel classification that can be used for species identification, biodiversity assessment and ecological studies by a short gene sequence fragment. At present, the mitochondrial cytochrome oxidase subunit I (COI) is chosed as the standard barcode for animal identification with almost100%accuracy. For plants, COI has not been successfully used for specie identification due to slow evolutionary rate.Here, we selected four DNA regions (matK, rbcL, trnH-psbA, ITS) to explore the best candidate barcode for the genus Epimedium.1. Over the four tested DNA regions, trnH-psbA had showed considerable variation (35variable characters of533total base), ITS (18of687), matK.(11of799), rbcL (4of706) was the least. For regions, matK provided the longest amplification fragment and psbA-trnH was the shortest. The other two sequences had no significant length differences between species.2. Four tested loci showed high interspecific distance and low intraspecific distance. trnH-psbA indicated highest interspecific distance, and rbcL was the lowest. However, psbA-trnH indicated that average coalescent depth was a litter larger than the minimum interspecific distance. All four DNA regions exhibited low variation at intraspecific level and the lowest was rbcL.3. Wilcoxon signed rank tests on this four candidate DNA barcodes showed that psbA-trnH exhibited higher interspecific divergence than other three loci. The test indicated that trnH-psbA> matK> ITS> rbcL.4. An ideal DNA barcoding must exhibit a "gap" between inter-/intraspecific divergence. We evaluated the distribution of inter/intra specific divergence in classes of0.002distance units. The result did not show any large gap as COI in animal, although with trnH-psbA the distribution inter/intra specific divergence was relatively well separated. The data revealed obvious overlapping when the divergence distributions were calculated using matK, rbcL and ITS.5. The NJ phylogenetic tree of Epimedium based on the four loci showed that the rate of successful identification was trnH-psbA, followed by ITS and rbcL. Finally, some samples could not be identified by matK.