Optimization of an AFLP protocol with four Phoebe species

To meet the needs of an amplified-fragment-length-polymorphism（AFLP） protocol，high quality genome DNA was extracted from Phoebe chekiangensis，Phoebe sheareri，Phoebe zhennan，and Phoebe bournei seedlings with improved cetyltrimethylammonium bromide（CTAB）methods. For optimization of restriction system（30.0 L），the genome DNA，300 ng；was buffered with 0.3 L；bovine serum albumin （BSA），0.3 L；EcoR，50 nkat；and Mse，25 nkat；and incubated at 37 C for 4 h；then the restriction enzyme was killed with 65 C for 15 min. The best ligation system（25.0 L） was with 20.0 L of the digestion products；T4 buffer，2.5 L；T4 ligase，666.8 nkat；EcoR adapter，5 pmol；and Mse adapter, 10 pmol；and left at 16 C overnight （10-14 h）. The optimal preamplification system （20.0 L） included 2.0 L ligation products；10 buffer，2.0 L；Mg2+，31.25 nmol；Taq DNA polymerase；16.67 nkat；dNTP，4 pmol； pre-amplification primers（E+A），6 pmol；and pre-amplification primers（M+C），6 pmol. Also，the best selective amplification system （20.0 L） was 2.0 L，1/40 pre-amplification products；10 buffer，2.0 L； Mg2+，31.25 nmol；dNTP，4 pmol；Taq DNA polymerase，16.67 nkat；amplification primers（M+CNN），15 pmol；and amplification primers（E+ANN），12 pmol. Moreover，screening with the optimized AFLP reaction system resulted in 13 pairs of suitable amplification primers for AFLP analysis of Phoebe chekiangensis. ［Ch，5 fig. 3 tab. 24 ref.］