Establishment and optimization of AFLP molecular marker technology system for S. spontanuem L.

【Objective】The AFLP molecular marker technology system for Saccharum spontaneum L. was established and optimized to provide support for the analysis of genetic diversity and genetic map construction. 【Method】The DNA of Saccharum spontaneum L. materials viz., GXS85-30, GXS87-16, GXS79-9 , GXS96, GXS112 and GXS212 collected from Guangxi were extracted by modified SDS method, and completely digested by enzyme EcoRⅠand MseⅠ.The digested fragments were legated by the EcoRⅠand MseⅠadaptors. Then, on the basis of the orthogonal experimental design, the in-fluencing components on preamplification and selective amplification reactions, namely Mg2 +, DNA template, primer,dNTP, and Taq DNA polymerase, were optimized. 【Result】The DNA were completely digested by 3.0 U enzyme EcoRⅠand MseⅠ. By orthogonal design optimization, favorable preamplification system included 0.4 μL of dNTPs (20 mmol/mL),1.6 μL of Mg2+(25 mmol/mL), 2.0 μL of EcoR-P (5 pmol/mL), 2.0 μL of Mse-P(5 pmol/mL), 1 U Taq poly-merase (1 U/μL),and 10 times diluted DNA template. Selective amplification system included 0.4 μL of dNTPs(20 mmol/mL),0.8 μL of Mg2+(25 mmol/mL), 1.0 μL of EcoRⅠ-AAG (6 pmol/mL), 1.0 μL of MseⅠ-CAG(6 pmol/mL), 3 U of Taq polymerase (1 U/μL) and 20 times diluted DNA template. The clear polymorphic fingerprint maps were obtained after PCR product from optimized reaction system was undergone 5% denatured polyacrylamide gel electrophoresis and silver stain.【Conclusion】The high efficient and stable AFLP technical system could provide strong technical support for constructing molecular genetic linkage map of Saccharum spontaneum L.