Studies On The Molecular Identification Technology For Lolium And Its Similar Species

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 tunred 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 carting 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 L.mainly based morphology of the seeds at home and abroad.However,this method needs strong professional knowledge and skills,and morphological identification of the need to establish whether the seeds in the integrity of the characteristics of discrimination,for those who completely lost or in part,determine the loss of the characteristics of the seed is not used to determine the morphology,and the practice of such cases in quarantine occupies a very large proportion.Therefore the establishment of an effective, accurate and practical,rapid identification of the molecular biology testing methods is very important.In this study we used PCR-sequencing,PCR-RFLP,Single Nucleotide Polymorphisms (SNP) detection technology identification Lolium temulentum and five of the approximates,with the best selection test combination,main contents include:1.PCR-sequencingIn 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.The length of 5.8S and ITS-2 regions in the 6 species is 267 bp and 145 bp respectively; The totally numbers of variable sites are 16,7 and 7 in ITS-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.2.PCR-RFLPThis is the first application of PCR-RFLP,according to district ITS sequencing results,the Lolium temulentum and five similar species rDNA ITS PCR-RFLP analysis.Lolium temulentum and five of the approximates were be effective digested by three restriction enzyme(BssSI,TfiI, NheI),three restriction enzyme digestion maps by PCR-RFLP analysis technology show these results:TfiI identification restriction sites G↓AWTC(56,301),Lolium temulentum and Lolium rigidum Gaud.can be completely separated and identify by it;BssSI identification restriction sites C↓ACGAG(29),can be combined with TfiI identified Lolium remotum Schrank.;NheI identification restriction sites G↓CTAGC(185),can be combined with TfiI identified Lolium multiflorum Lam.Therefore,the application of PCR-RFLP technology can identify the four similar species,Lolium temulentum L.,Lolium remotum Schrank.,Lolium rigidum Gaud.and Lolium multiflorum Lam.,so as to establish the PCR-RFLP molecular detection method of Lolium temulentum L.and the approximates.3.Single nucleotide polymorphism(SNP) technologyWith Single nucleotide polymorphism(SNP) technology,the first design and selection of the four SNP TaqMan-MGB probes(Du-57,Long-555,Ri-302,Tian-31) to detect and analyze the single-base variations of Lolium temulentum L.and its similar species,the analyse results of allele identification test using SDS 2.3 software showed that:Du-57 probe can separate Lolium temulentum L.from other specise;Long-555 probe can identify Lolium temulentum var. longiaristatum Parnell;Ri-302 probe can identify Lolium rigidum Gaud.;Combining Du-57 with Tian-31 probes can distinguish Lolium remotum Schrank.from other similar species.Single nucleotide polymorphism(SNP) technology has a high stability,easy reading,while test hundreds of data in the identification of approximately,with high flux features of gene chip technology,the technology is applicable to the large-scale sample screening,shorten the identification of the quarantine time and provided strong evidence for the study of single nucleotide polymorphism of Lolium.The three methods of molecular biology 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 provide an economic,high-speed and accurate identification of the quarantine method for China's quarantine pests.