DNA Barcoding System Construction For The Main Invasive Plants In Liaoning Province

Biological invasion is an increasingly severe environment problem which is difficult to prevent and manage like environmental pollution. Conventional methods may not identify invasive species when their morphological information loss by squeezing and abrading propagules in spreading periods or their incompleteness, damage or degradation for certain reasons. DNA barcoding is a technique for characterizing species of organisms using a short DNA sequence from a standard and agreed-upon position in the genome. As a powerful supplement to conventional morphological identification, it is not affected by morphological traits of samples. This study used rbcL, matK and ITS to analyze DNA barcoding’s potential application in invasive species identification and established an identification system of 32 invasive plants in Liaoning Province. The results are as follows;1. Establishment of the DNA barcoding system of invasive plants. DNA extraction of invasive plant used modified CTAB method. Primers used for PCR were 390F/1326R for matK and 1F/724R for rbcL, respectively. [L9(34)] orthogonal experimental design was used for screening optimal system by a 4 factors 3 levels analysis of dNTPs, primers, rTaq DNA polymerase and templates. The optimal system was composed of 0.2 mmol·L-1 dNTPs,0.5 μmol·L-1 primers,0.3 U rTaq DNA polymerase,40 ng templates,2.5 μL buffer and filling up with ddH2O to 25 μL.2. The success rate of amplification and sequencing for rbcL was 97% and 100% respectively and 60 sequences were obtained. For matK, the success rate were 88% and 84% respectively and 39 sequences were obtained. The success rate of amplification for ITS was 84% and 48 sequences of invasive plants and their allied species were obtained. Consequently, the optimal system and primers were advisable.3.8 seriously invasive species of Liaoning were selected for DNA barcoding analysis. 178 rbcL sequences,144 matK sequences and 190 ITS sequences were found in GenBank databases as replicates. The feasibility of these 3 sequences and their pair combinations as DNA barcodes of invasive plants was accessed by K2P genetic distance analysis, NJ phylogenetic trees analysis and Barcode Gap discovery.4. Invasive plants of Ambrosia, Conyza, Erigeron, Subg. Solanum and Cenchrus are used to test higher identifying accuracy and cluster effect of DNA barcoding based on ITS sequences. DNA barcode of Xanthium is matK whereas matK+ITS barcodes are identifying efficient for Amaranthus which needs further researches. Thus ITS may act as core barcode with support of matK in the future research of DNA barcoding of invasive plants.