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.