Application Study Of Quarantine Method For Six Species Of Lolium With Molecular Technology

Darnel(Lolium temulentum L.),belonging to gramineae(poaceae),distributes over 140 countries and regions in the world.There was no original Lolium temulentum in our coutry.It was first time turned up in the imported wheat from Bulgaria in 1954,and has been distributed to the 26 provinces,cities and autonomous regions through food imports or introduction of carring seeds.In China and the other countries Lolium temulentum is an important quarantine weeds,and has been the target of quarantine since 1957.Lolium remotum Schrank.,Lolium rigidum Gaud and Lolium multiflorum Lam,the three kinds of Darnel,which rank the highest rate of interception in the imported grain,are very difficult for the laymen to distinguish due to the high similarity of the seed morphology.Identification of Lolium temulentum mainly based morphology of the seeds at home and abroad.However,this method needs strong professional knowledge and skills.The establishment of an effective,accurate and rapid molecular detection means is very important for port weed quarantine.In this study genomic DNAs were extracted from 6 species of Lolium and the sequences of the rDNA ITS region(using ITS-1,5.8 S rDNA and ITS-2)of the 6 samples were determined by using the PCR products directly or the corresponding cloned plasmids.The results showed that the full length of Lolium ITS is about 647 bp,the length of ITS-1 regions in the six species is 235bp and content of G+C in ITS-1 region ranges from 54.64%to 55.67%.The length of 5.8S and ITS-2 regions in the 6 species is 267 bp and 145 bp respectively;the G+C content in 5.8S region ranges from 62.55%to 62.93%and that in ITS-2 region ranges from 55.86%to 57.93%. The totally numbers of variable sites are 16,7 and 7 in IT!-1,5.8S and ITS-2 regions respectively.There are 22 informative sites and 8 specific identification sites in these variable sites.These variable sites,which are different distinctively among the six species of Lolium,can be used to identify the six species.The fact that the phylogenetic tree based on NJ method is corresponding to the one based on morphological classification indicates the study laid down the molecular basis for the classification and determination of Lolium genus.The studies showed that the characteristic of rDNA ITS sequence can perfectively reflect the difference of the intra-species and inter-species of Lolium,thus provided a good molecular marker system for Lolium variety identification,and therefore provide an economic,high-speed and accurate identification method for.port weed quarantine.