Optimization for ISSR- PCR Reaction System on Leptosphaeria biglobosa

doi:10.3969/j.issn.1000-7091.2013.04.037

Abstract

Abstract: An orthogonal design was used to optimize ISSR reaction system for Leptosphaeria biglobosa on oil- seed rape in five factors(Taq polymerase，Mg^{2 +}，dNTPs and the concentration of primer or template) at four levels． A suitable ISSR- PCR reaction system for Leptosphaeria biglobosa was established．The optimized reaction system consists of 1 U Taq DNA polymerase，2． 0 mmol /L Mg^{2 +}，0． 2 mmol /L dNTPs，0． 6 mol /L primer，40 ng template DNA，and 2． 5 L 10 × Buffer．24 primers were selected for the abundant and clear band from 110 random primers and their optimal annealing temperature was decided．This optimization of ISSR- PCR reaction system can be used to study the genetic diversity of the pathogen which was confirmed by testing on the stability of 20 strains of Leptospha- eria biglobosa．

Key words: Leptosphaeria biglobosa, ISSR- PCR, Optimization reaction system

CLC Number:

S435.654

HAO Li-fen, SONG Pei-ling, LI Zi-qin, LI Xin-zhou, ZHANG Jian, Bao Yu-ying. Optimization for ISSR- PCR Reaction System on Leptosphaeria biglobosa[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2013, 28(4): 204-207. doi: 10.3969/j.issn.1000-7091.2013.04.037.

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