The Research Of Molecular Biology Method To Identify Xiphinema

Xiphinema spp. belongs to Nematoda, Dorylaimida, Dorylaimoidea, Longidoridae. This group of nematodes are large size nematodes, which are provided with long spear,with that they pierce the host's roots. These nematodes are not only directly damaging but also of considerable importance as vectors of virus.Now, the X. americanum group and Xiphinema index have reported in China. Because of their similar form,they are hard to indentify except experts.In our study,we want to find a simple and accurate molecular biology method to identy Xiphinema spp..Microsatelite markers containing simple sequence repeats (SSR) are a valuable tool for nematodc genetic analysis. Our objective is to find the special microsatellite markers in order to identify Xiphinema americanum group nematodes. In this study, we experiment a new approach that used for isolation of new microsatellite markers. In order to overcome the low amount of DNA, DOP-PCR was applied and it successfully increased the yield of primary DNA fragment of X. americanum sensu stricto. Before cloning, DOP-PCR products were ligated into cloningpGEM(?)-T vectors and selectively amplified by a simple repeats (CA)9 primed PCR. The additional processes highly improve the efficiency of cloning of fragments containing (CA)9. There are nine sequences that carrying (CA)n repeat have been obtained. Three primer pairs have been designed according to the nucleic acid sequence that carrying (CA)n simple repeat. After testing the validity of primer pairs in some species of Xiphinema, Longidorus, Paratrichodorus, Pratylenchus and X. americanum group, we found a specific diagnostic tool for X. americanum subgroup and a specific primer pair for identify of X. americanum sensu stricto.DOP-PCR was applied to increase the yield of primary DNA fragment of X.index.Selecting from the DOP-PCR products,we fina seven fragment which contain (CA)n.They can used for specific diagnostic tool for X.index.