[Objective] The aim was to provide molecular basis for the identification of species in the moss family Bryaceae by the construction of inter-simple sequence repeats （ISSR） fingerprinting. [Method] In order to seek ...[Objective] The aim was to provide molecular basis for the identification of species in the moss family Bryaceae by the construction of inter-simple sequence repeats （ISSR） fingerprinting. [Method] In order to seek standardizing PCR reaction set-up, an orthogonal design was used to optimize ISSR-PCR amplification system of Bryaceae in five factors （Mg2＋, dNTPs, primer, DNA template, Taq DNA polymerase） at four levels respectively. [Result] A suitable ISSR reaction system was obtained, namely： 20 μl reaction system containing 5 ng of DNA template, 0.2 μmol/L primer, 2.25 mmol/L MgCl2, 0.6 U of Taq DNA polymerase, 0.4 mmol/L dNTPs. Proper annealing temperature was found at 48-50 ℃.The above system and six ISSR-PCR primers were used for the PCR amplification of 14 samples from Bryaceae and the related species in Mniaceae. A total of 86 bands were amplified, all showed polymorphism. NJ cluster analysis showed a star-shaped cladogram. [Conclusion] The results manifested that ISSR fingerprinting could provide the appropriate degree of polymorphism at low taxonomic level, so it would be a useful tool to provide additional evidence for resolving taxonomic relationships at the species level of Bryaceae.展开更多
基金Supported by Natural Science Foundation of Hebei Province(C2006000147)Zhengzhou Science and Technology Program(10PTGN449-6)~~
文摘[Objective] The aim was to provide molecular basis for the identification of species in the moss family Bryaceae by the construction of inter-simple sequence repeats （ISSR） fingerprinting. [Method] In order to seek standardizing PCR reaction set-up, an orthogonal design was used to optimize ISSR-PCR amplification system of Bryaceae in five factors （Mg2＋, dNTPs, primer, DNA template, Taq DNA polymerase） at four levels respectively. [Result] A suitable ISSR reaction system was obtained, namely： 20 μl reaction system containing 5 ng of DNA template, 0.2 μmol/L primer, 2.25 mmol/L MgCl2, 0.6 U of Taq DNA polymerase, 0.4 mmol/L dNTPs. Proper annealing temperature was found at 48-50 ℃.The above system and six ISSR-PCR primers were used for the PCR amplification of 14 samples from Bryaceae and the related species in Mniaceae. A total of 86 bands were amplified, all showed polymorphism. NJ cluster analysis showed a star-shaped cladogram. [Conclusion] The results manifested that ISSR fingerprinting could provide the appropriate degree of polymorphism at low taxonomic level, so it would be a useful tool to provide additional evidence for resolving taxonomic relationships at the species level of Bryaceae.
