To obtain 7 factors (DNA template dosage, Taq DNA polymerase dosage, primer concentration, dNTP concentration, Mg2+ concentration, annealing temperature and PCR cycles ) of ISSR-PCR Reaction System in Pyrus pyrifolia ‘Cuiguan’, a precocious cultivar of southern China was been used as template. An optimum reaction system was been established. The final total volume was 20 μL, contains 2 μL 10×PCR buffer (Mg2+ free), 60 ng DNA template, 0.5 U Taq DNA polymerase, 1 μmol/L of primer concentration, 90 μmol/L of dNTP concentration, 2.25 mmol/L of Mg2+ concentration. Amplication procedure was as follows: initial-denaturing of 5 min at 94 ℃, followed by 42 cycles of 45 s for denaturing at 94 ℃, at annealing temperature for 45 s and 1 min at 72 ℃, after that 10 min extending at 72 ℃, and then keep at 4 ℃. Use 1.5% of agarose gel electrophoresis to test the genetic polymorphism.
SONG Yan-chun
,
LI Yong-yu
,
CHEN Jian-yan
,
HUANG Tian-yi
,
GONG Li
,
WU Shao-hua
. Establishment and Optimization of ISSR-PCR Reaction System in Precocious Pyrus pyrifolia[J]. Subtropical Plant Science, 2013
, 42(02)
: 91
-96
.
DOI: 10.3969/j.issn.1009-7791.2013.02.001
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