2.5. EST-SSR Polymorphism Examination Analysis

There were 46 EST-SSR markers used to analyze the genetic structure and genetic diversity of the inbred and control populations. The sequences of the eight primers and microsatellite core sequences are shown in Table 1, and the other 38 EST-SSR markers are from Wang et al.'s study [30]. Primers were designed to flank the SSRs using Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA) and then synthesized by Shanghai Sangon Biological Engineering Technology (Shanghai, China). Polymerase chain reactions (PCRs) were performed in 20 μl reaction volumes containing 50 ng of template DNA, 1 μl of 10 μM each primer, 10 μl of 2× Reaction Mix, 0.2 μl Golden DNA Polymerase (2.5 U/μl), and 6.8 μl sterile distilled water. The following PCR program included an initial step at 95°C for 4 min followed by 35 cycles of 95°C for 40 s, 55°C-60°C (depending on the Tm of the primer set used) for 40 s, 72°C for 1 min, and a final extension for 10 min at 72°C. Finally, the amplified products were detected by 3730XL sequencing test platform.

Eight polymorphic EST-SSR markers and estimated polymorphisms.