Real-Time PCR For The Identification Of Frankliniella Occidentalis And Sequencing And Analysis Of The Complete Mitochondrial Genome Of Frankliniella Intonsa

The western flower thrip is a kind of worldwide greenhouse pest and important quarantine object, firstly found in Beijing in the mainland of China, in 2003. Morphological identification of thrips is difficult because of their small size, high degree of similarity to other types of thrips. In this study, we investigated the occurrence of western flower thrips in Jiangsu Province and established a standard curve with plasmids containing the target DNA fragment, designed a pair of specific primers which were based on the COI gene of mtDNA in F.occidentalis, to rapidly determine the western flower thrips using real-time fluorescent quantitative PCR, and compared with normal PCR. And we designed two couples of specific primers of Frankliniella intonsa using long PCR, then sequenced, analyzed and researched the complete mitochondrial genome of Frankliniella intonsa, all results showed as following:1. The western flower thrips have a wide range of host plants, including flowers and vegetables, and a wide distribution in Jiangsu Province, including Nanjing, Changzhou, Taizhou, Lianyungang and so on.2. We established a standard curve with plasmids containing the target DNA fragment, designed a pair of specific primers, to rapidly determine the western flower thrips using real-time fluorescent quantitative PCR. Then we can get the size of 158 bp specific fragment.3. And the detection system has high specificity and the primers can only detect the fluorescence signal of western flower thrips, and there are no cross reactions with other thrips.4. The sensitivity of traditional PCR is low,1/120 of the adults can be detected, when the diluted concentration is lower than it, we can not detect any signals. Real-time fluorescent quantitative PCR has high sensitivity, when the template concentration is diluted 104 times, the fluorescence signal can still be detected, then the content of the target DNA fragment is 11.63 copies/?L.5. We designed two couples of specific primers of Frankliniella intonsa using long PCR, then sequenced and analyzed the complete mitochondrial genome of Frankliniella intonsa. The results indicated that complete mitochondrial genome of F. intonsa is a circular molecule of 15213 nucleotides. And the gene content includes 13 protein-coding genes,22 tRNA genes,2 rRNA genes and 3 control regions. We predicted the secondary structures of 22 tRNA genes. Compared with Thrips imaginis, eight of 22 tRNA genes of mitochondrial genome of F. intonsa had changed, compared with F.occidentalis, three of that had changed. The areas of these changes are distributed in control regions, so the control region is an important factor in the evolution of mitochondrial genes.