| Citrus is a perennial woody fruit tree, which has complicated genetic background. Its evolutionary relationship is much complicated after a long period hybridization and mutant selection. In order to distinguish and identify the citrus germplasm resources efficiently, scientists used methods such as DNA molecular markers to identify the Citrus germplasm resources. A specific random primer OPA08 which was sift out by RAPD method could distinguish the main germplasm resources including three genera and eight categories of citrus. In order to uncover the special characteristics of OPA08, we analyzed the relationship between the primer and genetic information of Citrus. In this study, we collected 22 samples which covered three genera of Citrus and then performed RAPD experiment by using primer OPA08. According to the results of RAPD analysis, 23 different specific amplified fragments which were separated and recovered from gel electrophoresis were performed DNA sequencing analysis. Furthermore, PCR-Southern hybridization method was employed for further analysis. The results of special fragments sequence alignment and cluster analysis not only identified the relationships among the different germplasm resources but also supported former research results about evolutionary relationships among Citrus species. Besides, the results revealed that there was a high homology between the specific fragments and retrotransposons which might play an important role in citrus evolution. The main results are as following:1. A suitable RAPD amplification system was established: 2.0μl 10×buffer; 2.0mmol/L Mg2+; 0.2mmol/L dNTPs; 0.5μmol/L OPA08 primer; 100ng DNA sample; 0.5 U Taq DNA polymerase.2. According to the results of PCR-Southern Blotting, there were strong hybridization signals at the same place in gel electrophoresis among Citrus aurantium, C. reticμlata Blanco, C. paradisi by using OPA08-280bp fragment as probe which was amplified from C. sinensis 'Newhall'. The OPA08-200bp fragment of C. reticulata, OPA08-280bp fragment of C. grandis 'Shatian', OPA08-400bp and OPA08-500bp fragment of C. limon had relatively strong hybridization signals by using OPA08-280bp fragment as probe which was amplified from C. ichangensis, and the large fragments had more hybridization signals than others. The results reveal that OPA08-280bp fragment from C. sinensis 'Newhall' contains relatively new genetic information in citrus. While OPA08-280bp fragment from C. ichangensis contains some common genetic information in all citrus species, which inferred it should be one of the primitive types.3. The results of special fragments sequence alignment and cluster analysis showed that OPA08-310bp fragments amplified from C. grandis, Poncirus trifoliate Raf., C. limon, and OPA08-200bp fragments amplified from C. reticulata shared high sequence identity. The OPA08-280bp fragments amplified from C. sinensis, C. aurantium, C. reticulata Blanco shared high sequence identity, and all of them shared partial sequence identity with OPA08-500bp fragment amplified from Fortunella crassifolia. OPA08-280bp fragment from C. ichangensis shared similarities with OPA08-500bp fragments from C. limon, C. jambhiri.4. According to bioinformatic analysis, we found that fragments amplified from different citrus species by using primer OPA08 all shared high identity with retrotransposons. The result showed that retrotransposons might play an important role in variability and evolution of citrus, and retrotransposons provided a valid evidence for the scientists to study and analyze evolutionary relationship of citrus species. |
