Direct Sequencing of PCR Products or Sequencing by Cloning PCR Products—Method of Determining Internal Transcribed Spacer Sequences of Nuclear Ribosomal DNA inAthrotaxis
Abstract:The results of direct sequencing of PCR products or sequencing by cloning PCR products of the internal transcribed spacers and the 5.8S coding region of nuclear ribosomal DNA in Athrotaxis were compared and analyzed. The rDNA repeat units of A. selaginoides have been homogenized, so its ITS region sequences were determined by direct sequencing of PCR products, but the rDNA repeat units of A.
laxifolia and A. cupressoides have not been homogenized, there are many polymorphic sites and insersion/deletions in the ITS1 regions, so their ITS1 sequences were determined by cloning PCR products and then sequencing. There are polymorphic sites but no insersion/deletions in ITS2 region of A. laxifolia and A. cupressoides, so the direct sequencing of PCR products can also be used to display variable nucletides at one site. The results in our studies show that the different ITS regions of Athrotaxis have been homogenized at different extent, so the methods of direct sequencing of PCR products or sequencing by cloning PCR products can be selected to determine their sequences.