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45
leaves from the two parents and their generations by
using cetyl trimethyl ammonium bromide (CTAB)
method. The resistant bulk and the susceptible bulk
were prepared by separately pooling equal amount of
genomic DNA (about 1 μg) from 15 resistant and 15
susceptible plants randomly selected from the F
2
population derived fromQihuang22×Nannong1138
-
2.
3.4 Simple sequence repeats analysis
The primers whose sequences were obtained from the
Soybase website (http://129.186.26.94/ssr.html) were
synthesized by Invitrogen Biotech Ltd. Co. (Shanghai,
China).
When the SSR markers were used for screening and
MAS, PCR amplification was performed in a total
volume of 10 μL containing 50 ng genomic DNA, 1.5 μL
10×PCR buffer, 2 mmol/L MgCl
2
, 0.3 μmol/L of each
primer, 0.24 mmol/L dNTP, 0.6 U
Taq
polymerase.
Each reaction mixture was covered with paraffin oil.
Amplifications were carried out following these PCR
cycling conditions: 94
℃
for 3 min, followed by 30
cycles of 95
℃
for 30 s, 55
℃
for 30 s, and 72
℃
for
40 s, with final incubation at 72
℃
for 8 min.
Each PCR product mixed with 2 μL loading buffer was
separated on 8% non-denaturing polyacrylamide gels,
and then viewed by silver staining.
3.5 Linkage analysis
The linkage between SSR markers and the resistance
gene was calculated under MAPMAKER
/
EXP ver-
sion 3.0b (Lander et al., 1987) and transformed into
cM according to Kosambi’s function (Kosambi 1944).
The logarithm of likelihood ratio (LOD) 3.0 was used
as a criterion to test the linkage. The linkage map was
drawn by a Microsoft Excel macro called MapDraw
(Liu and Meng, 2003).
Acknowledgements
This work was supported by the National Natural Science Foundation of
China (Grant No. 30971815), the Key Technologies R&D Program of China
(Grant No. 2006BAD01A04), the State Key Basic Research and Develop-
ment Plan of China (Grant No. 2006A10A111).
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