The extension reaction to identify single nucleotide polymorphisms in the PCR products was performed in a total volume of 10 mL containing 2 mL purified PCR product, 1 mL primer (the concentration was 0.8 mM), 5 mL SNaPshot Multiplex Kit (Applied Biosystems, Foster City, CA, USA), and 2 mL ultrapure water. The cycling conditions for extension were 96uC for 1 min, 28 cycles of 96uC for 10 s, 52uC for 5 s, and 60uC for 30 s, and kept at 4uC. Then each extended product was added to 1 U shrimp alkaline phosphatase, incubated at 37uC for 1 hour, and the enzyme inactivated at 75uC for 15 min. Then, 0.5 mL was added to 0.5 mL Liz120 SIZE STANDARD (Applied Biosystems, Foster City, CA, USA), 9 mL Hi-Di (Applied Biosystems, Foster City, CA, USA), and sequenced by ABI3130XL Sequencer (Applied Biosystems, Foster City, CA, USA). Finally, the primary data was analyzed by GeneMapper 4.0 (Applied Biosystems, Foster City, CA, USA). Genotypes were determined by the type of nucleotide presented at SNP site, which was visualized by one or two different color peaks on the figures.