Study Of Ultrastructure And Molecular Identification Of The Related Species Of Cuscuta

To provide a morphology characteristics evidence for the identification of the seeds of Cuscuta, scanning electron microscope(SEM)were used to identify the fourteen species of seeds.Results showed that there were significant differences in these characteristics such as the shape of the hilum,cell range of hilum-rim,the surface structural characteristics and accessories of episperm among the different species of seeds.These characteristics provided a new evidence for identifying species of the seeds and the taxonomy of Cuscuta.Four methods of DNA extraction of the five related species weeds of Cuscuta were used respectively, including CTAB,SDS,Moller and TaKaRa DNA extraction kit.These four methods were compared and analysed by the DNA purity and concentration,extraction ratios of DNA of the materials.Results showed that SDS was the best method to extract the genomic DNA in weeds of the five species.Results of the ILPs-PCR analysis of the five related species in Cuscuta were as follows:(1)There was a 250bp ILPs-PCR product in Cuscuta engelmanii using the primer IP9.(2)There was a 240bp ILPs-PCR product in Cuscuta japonica using the primer IP4.(3)ILPs marker could be used to identify the related species in Cuscuta.This was a new method of taxonomy in Cuscuta.It also provided new science evidence,fast and exact detection method for weed quarantine.The result of this experiment also showed that there were differences in gene polymorphisms of intron length polymorphism of different genotype weeds.Firstly,a 275bp product was amplified from Cuscuta japonica NO.1 using the primer IP9,but no product was amplified from Cuscuta japonica NO.2. Secondly there was a 180bp product in Cuscuta japonica NO.1 using the primer IP4,but no product in Cuscuta japonica NO.2.The genomic DNA of Cuscuta australis were extracted and used to optimizing the amplifying reaction system.Different concentrations were compared with template DNA,MgCl₂,primer,dNTP and Taq DNA polymerase.The results indicated that the best reaction system was that in 25μl total reaction volume of RAPD-PCR contained 20 ng template DNA,3.0 mmol/L MgCl₂,10μmol/L primer,400μmol/L dNTP,1 unit Taq DNA polymerase,10×buffer 2.5μl.The thermal cycle condition was:One initial cycle denaturing at 94℃for 3 minutes.Followed by 40 cycles which involved denaturing at 94℃for 1 minute,annealing at 40℃for 1 minute,one cycle elongation at 72℃for 1 minutes.Finally elongation at 72℃for 10 minutes,and then kept at 4℃.