Optimization and validation of SSR-PCR reaction system for Asparagus officinalis L.
Based on DNA of asparagus young leaves,a single factor screening test combined with a L16(43)orthogonal de-sign method was employed to optimize the factors of 2 ×Taq Master Mix,template DNA,and primer combination applied for SSR-PCR and followed by gradient annealing temperature test and gradient cycle times test to screening the optimum annealing temperature and cycle numbers.The results showed that the optimal SSR-PCR system for asparagus included 50 ng·μL-1 DNA template 0.125 μL,10 μmol·L-1 primers 0.5 μL,2×Taq PCR Mix 3 μL,sterilized ddH2O 5.875 μL,with a total reaction volume of 10 μL.The PCR amplification procedure for asparagus was:94 ℃ pre-denaturation for 4 min,94 ℃ denaturation for 30 s,60 ℃ annealing 30 s,72 ℃ renaturation for 35 s,25 cycles,72 ℃ extension for 10 min,4 ℃ for storage.This reaction system was proved to be stable and reliable by PCR amplification with 5 pairs of primer combi-nations and DNA templates of five different asparagus cultivars.The optimized SSR-PCR reaction system could be satis-factorily used for genetic diversity analysis,seed purity identification,marker-assisted breeding and map-based cloning of important agronomic traits of asparagus.
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