| Fruit flies, a kind of the important pest insects economically, make directly a seriousdamage and have a significant effects on the production and trade of import and export ofmany fruits. There are more than 150 species of fruit flies which damage fruits andvegetables in different country. In order to avoid the introduction of fruit flies, almost eachgovernment adopts a series of serious quarantine methods. The identification of fruit flies wasmainly based on their externally morphological characters of adults in past many years, butmore larvae and ova rather than adults are found in trade of import and export of fruits.Generally, larvae and ova are very difficult to identify precisely and they have to be raised formore a long time so as to obtain their adults in laboratory. Both time of pass-customs isdelayed and more work is added. Therefore, it is important to identify fruit flies accuratelyand quickly in the pest quarantine and control. By means of RAPD and PCR, studies on themolecular identification of fruit flies were carried out. The results are as follows: 1. Genomic DNA in the fruit flies of dried specimen (stored generally more than a year)was extracted by CTAB and SDS etc. The results showed that genomic DNA of driedspecimen of most fruit flies could not be extracted, only a few can be extracted and beprocessed PCR. But the genomic DNA could well be extracted from dried specimen that werestored in super-low temperature refrigerator. 2. A reaction system with more stability and repeatability was established by many timesof screening in this paper. It could also be used in studies of other fruit flies. 3. In tested 69 primers, S361, S67 and S252 could be used to distinguish B. scutellaris,B. tau and D. trimacula. S112 could be used to distinguish B. tau, B. scutellaris and B. citri.S126 and S134 could be used to distinguish B. scutellaris and D. trimacula. S83 could beused to distinguish female and male of B. tau. 4. Genetic stability of 4 species of fruit flies was studied by means of 3 selected primerswhich amplify to get the stable and clear band. Average genetic similarity indexes of B. tau ,B. scutellaris, B. citri and D. trimacula are 0.551, 0.538, 0.373, 0.778 respectively. On thebasis of the genetic similarity indexes, genetic variation of D. trimacula is the lest, B. tautakes second place, the third is B. scutellaris, B. citri had the biggest genetic variation. 5. The genetic polymorphism of 4 species was studied by 12 primers and 5 primersrespectively. An individual was assessed for each species when 12 primers were used. Thephylogenetic tree of 4 species was constructed by UPGMA. The results showed that B. tauand B. scutellaris were clustered firstly, they and D. trimacula were clustered. Finally threespecies and B. tau were clustered. 3 individual was assessed for each species when 5 primerswere used. Phylogenetic tree constructed by Dice and Nei distance was different with theabove. B. tau and B. scutellaris were clustered firstly, they and B. citri were clustered. Finallythree species and D. trimacula were clustered. The latter was consistent with the conventionalphylogenetic relationship among 4 species. 6. 4 species were distinguished by amplified ITS1 fragment. Amplified special band of B.tau, B. scutellaris, B. citri and D. trimacula was 650bp, 640bp, 1010bp, 773bp respectively.ITS1 amplification needed less time than RAPD. Generally ITS1 amplification only needed30h or so and had better repeatability. It is the first time that the fruit flies was identified by two kinds of the molecular markermethods in China. Two molecular markers were more accurate and quicker than conventionalmethod based on morphological characters. In theory, they were not affected by insectdevelopment stage and could also be used to identify larvae and pupae of 4 species. At thesame time, these methods could be consulted in the identification of other fruit flies. |
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