The Molecular Identification Study Of Pheretima By DNA Barcoding

Objective:To establish a rapids accurate and standardized method for identifying Pheretima aspergillum with DNA barcoding, analyze the genetic relationship of Pheretima aspergillum and other earthworms from3families,5genera and9species base on the COI and16SrRNA, and explore the feasibility of molecular identificetion of the12SrRNA and Cyt b for providing the necessary data to earthworms classification system. This study is aim to search DNA barcoding for identifying Pheretima aspergillum. At last, identify Chinese medicine and unknown earthworm using DNA barcoding technology, in addition, verify the, feasibility of this method.Method:1. Measured the COI and16SrRNA gene fragments from Pheretima aspergillum of5different populations5individuals, and12SrRNA and Cyt b gene fragments from2different populations2individuals. Assemble sequences with CodonCode Aligner, in addition, the other earthworm species were downloaded fromGenBank, obtaining genetic distance and NJ\MP molecular phylogenetic based on Kimura2-parameter.2. Download all sequences of COI and16SrRNA gene from medicinal earthworms, and testing barcoding gap by TAXON DNA.3. We obtain the COI sequences by extracting DNA、PCR and DNA sequencing, comparative analysis together with the standard COI of Pheretima aspergillum, detemine whether the identified samples are Pheretima aspergillum or not.Result:l.This research obtained the optimal DNA extract program from earthworms of Chinese medicine.-Soaking contained high concentrations of EDTA(0.1M), playing in soaking24h. increasing proK (20mg/mL) volume to50μL, during extract, joining DTT and Chelex, incubation time is4h, precipitated12h at-20℃, using DNA purification kit purificate before PCR can reduce the PCR reaction conditions of time and efforts to explore.2.COI16SrRNA、12SrRNA and Cyt b gene retain638bp、450bp、370bp and307bp. The number of variable sites and informative sites from COI were higher than16SrRNA and12SrRNA. There is no insertions and deletions in COI, but4numbers in16SrRNA and20in12SrRNA. The use of base frequency have significant A> T bias.3. Using MEGA4.1calculate K2P distance of earthworms. The intraspecific genetic distance of COI gene range from0.000to0.029, interspecific genetic distance range from0.1483to0.3278, the average genetic distance is0.149, R is1.31. The intraspecific genetic distance of16SrRNA gene range from0.000to0.009, interspecific genetic distance range from0.063to0.238, the average genetic distance is0.149, R is2.158.The MP tree of CI is0.962and RI is0.786from COI gene, The MP tree of CI is0.839and RI is0.588from16SrRNA. The MP tree of CI is0.718and RI is0.727from12SrRNA. The NJ tree and MP tree of COI、16SrRNA and12SrRNA show every species had high levels support and support for monophyletic.4. COI gene had obvious barcoding gap, but not16SrRNA. The intra distance concentrated in the1.5%, inter distance concentrated after14%. COI is more suitable for DNA barcoding of Pheretima aspergillum than16SrRNA.Conclusion:1. Facing the difficulty of extracting genomic DNA from the dry herbs, in this study, the improved method of DNA extraction was from tangerine peel old Zhang, which can extract DNA from the earthworm of Chinese medicine successfully, and meeting the requirements of downstream PCR.2. The application of COI、16SrRNA and12SrRNA gene were used to discuss classification of earthworms, the results were consistent with traditional taxonomic classification. COI、16SrRNA and12SrRNA gene may be good molecular marker for species identification as Pheretima aspergillum.3. Conduct barcoding gap test based on COI and16SrRNA, considering the genetic variation information sites、insertion or deletion sites, all datas show that COI gene is more suitable as DNA barcoding of Pheretima aspergillum.4. In the study, Pheretima aspergillum and Amynthas aspergillus was verified to be the same specie with different classification, besides, these two species had close genetic relationship, may be co-evolution, but not be single origin groups.