Feasibility Study Of DNA Barcoding Of Borers In Paddy Field Based On Mitochondrial COI Sequences

There are usually many different species of borers living in paddy field. Field survey on the populations of rice borers always runs into larvae and pupae of various species, which make it very difficult to identify the species of borers. Anyway, molecular identification provides a new way for overcoming this difficulty. Especially for DNA barcoding technique, any group of organisms could be easily identified by analysis of a piece of DNA fragment cloned from them with a couple of special primers designed. However, the key for this technique is the selection of the DNA fragment suitable for DNA barcoding. In this work, borers of 7 species, Chilo suppressalis from different geographic populations, and Sesamia inferens feeding on various host plants were collected as present samples for feasibility study of the DNA barcoding technique used for identification of the borers in paddy field. The results were summarized as follows.Firstly, a couple of special primers were designed based on the data reported, and the DNA fragments of mitochondrial COI gene were cloned from 55 C. suppressalis larvae of 6 geographic populations (Jilin, Anhui, Zhejiang, Hunan, Hubei, and Sichuan). Then, the sequences of these COI gene fragments were compared for analysis of the variation among populations. The results proved that the COI gene fragments of 709 bp could be easily cloned from all the larvae tested with the designed primers. The fragments contained 51 base substitution sites, but no insertion and deletion. By constructing Neighbor-joining tree, the 55 fragments were analized and no obvious branch was found. The genetic distance was calculated and not much difference was found between populations (0.006-0.012), which was similar as those between individuals of the same population. These results indicated that the cloned fragment of C. suppressalis COI contains suitable information of genetic variation. Though population within species varied, but not very much and could act as barcoding sequence for species identification.With the same method, COI gene fragments of 709 bp from 33 S. inferens larvae feeding on Triticum aestivum, Beckmannia syzigachne, Oryza sativa and Vetiver zizanioides were also easily cloned. Sequence analysis found that there was no insertion and deletion in these fragments. However, S. inferens COI gene contains more genetic variation information, and 125 base substitution sites were found among these fragment sequences. The calculated genetic distance revealed that no much variation between individuals (0-0.027), and the S. inferens feeding on Triticum aestivum, Beckmannia syzigachne and Oryza sativa (0.026-0.029). However, the borers feeding on Vetiver zizanioides were found much different from the others. The genetic distance was as much as 0.128～0.140. Neighbor-joining tree also showed that the borers feeding on Vetiver zizanioides formed a single branch, separating from the other branch formed by all other S. inferens. These results indicated that the borers feeding on Vetiver zizanioides might belong to different host types, and even cryptic species. On the other hand, these results also indicated that the cloned fragment of S. inferens COI contains suitable information of genetic variation and could act as barcoding sequence for borers in paddy field, even there may be a cryptic species.By the same way, COI gene fragments of 709 bp from 37 larvae of 7 borer species (C. suppressalis, C. auricilius, C. luteellus, C. sacchariphagus, S. inferens, Tryporyza incertulas, Ostrinia furnacalis) were all easily cloned. Sequence analysis showed that these fragments showed 199 base substitution sites and all had no insertion and deletion. Between species the genetic distances were calculated as 0.86～1.54, which were much higher than those between individuals of the same species (0.000～0.047). The constructed Neighbor-joining tree showed that every species formed a separate branch, and the phylogenic relationship could be found among the branches. These results proved that the primers designed were suitable for cloning the fragment of COI from borers. The cloned fragment contains suitable information of genetic variation. It conserves within species, and varys well between species. Thus, it should be a suitable barcoding DNA fragment for identification of the borers in peddy field.