Research In RAPD Marker To SCAR Marker For Rare Tea Plant Germplasm

Tea plant(camellia sinensis L.), having a cultivated history of several thousand years, is an important economic plant over the world. The germplasm resource is precious natural resource and important material base for research. As the cradle of tea plant, China has an abundant germplasm resource which distributes widely over the nation. And rare tea plant is the precious germplasm resource for genetic diversity analysis, cross breeding, and so on.In recent years, molecμlar marker technology is widely amplified in genetic analysis, cultivar identification and other research areas of crops. And it has important reference and actual using value for tea plant research. But no systematic research was taken out about these rare tea plant germplasm.In this research,8rare tea plant cultivars and2popular cultivars were extracted their genomic DNA by a modified SDS method. And then their DNA was amplified by50random primers with optimized PCR reaction system and amplification program. The specific fragment of298bp was amplified by primer S20in’Yujinxiang’, named Y20-And specific fragments of593bp,1091bp and788bp were amplified by primer Si, S12and S91in’Ziyan’, named Z1, Z12and Z91, respctively. We blasted these four fragments in NCBI and found no homologous sequence in the Database. And we got four GenBank numbers for these four sequences(JN250590、JN250592、JN250589、JN250591). The designed SCAR primer pairs, based on former RAPD primers, were used to amplify genomic DNA from these ten cultivars. In each case, a single band of the same size as the progenitor RAPD fragment was amplified confirming of the corresponding cultivar. In three cases, SCAR markers, SY20, SZi and SZ12, the RAPD polymorphism were retained as the presence or absence of bands when the corresponding SCAR primers were used at an annealing temperature of60℃,63℃and64℃respectively. We successfully converted Z91to a SCAR marker whose polymorphism was also presence or absence of band by using a Touchdown PCR whose PCR protocol consisted of the annealing temperature was set to67℃and, at each of the9subsequent cycles, the annealing temperature was decreased by0.5℃. Then these four bands were sequenced and showed the same sequence as the corresponding progenitor RAPD fragments. Therefore, these four RAPD markers were successfully converted to SCAR markers which could be scored as dominant markers(presence or absence of an amplified band). A triple PCR was built for iedntification of’Ziyan’based on a touchdown PCR. Successful conversion from RAPD makers to SCAR markers provides a good foundation for utilizing these rare cultivars for further research.