2.3 SSR analysis methods
Fresh leaf samples were used to extract the total genomic DNA following
the modified CTAB method. The quality of extracted DNA was determined
using electrophoresis in a 0.1 % agarose gel, and the concentration of
the extracted DNA was detected using an UV nucleic acid analyzer.
According to the SSR molecular markers from the EST (expressed sequence
tag) information of Liliaceae (Yang et al. 2008), eight pairs of primers
with clear amplification bands and good repeatability were screened out
from 18 SSR pairs of primers (Table 1). Subsequently, all collectedC. udensis individuals were amplified by PCR reactions using the
selected primers.
The PCR reaction volume was 10 μL, including mix 5 μL, DNA template 1
μL, SSR primers 0.6 μL, and ddH2O 3.4 μL. The reaction
procedure was as follows: pre-denaturation at 94℃ for 5min, denaturation
at 94℃ for 30 s, annealing at 45℃ - 60℃ for 30 s, extension at 72℃ for 2
min, 35 cycles in total, extension at 72℃ for 5 min, and the PCR
products were finally stored at -4℃. The amplified products were
separated using 10% denaturing polyacrylamide gel electrophoresis and
visualized using silver staining.