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mixture contained 1.5 µL 10x buffer (50 mmol KCl,
10 mmol Tris-HCl pH 8.3), 1.5 mmol/L MgCl2, 200 µmol/L
each dNTP, 250 nmol/L each primer, 1 unit Taq polymerase
and 50~100 ng template DNA. The reagents in PCR
reaction were obtained by Chengdu Feike BioTechno-
logy Co., Ltd (http://www.fkbio.com). The PCR conditio-
ns were as follows: denaturation at 94℃ for 4 min,
followed by 40 cycles of 94℃ for 1 min, 50~60℃
(depending on the individual primer) for 1 min, 72℃ for
1 min and a final extension at 72℃ for 10 min. PCR
products were mixed with 6 µL formamide loading
buffer (98% formamide, 10 mmol/LEDTA, 0.25%
bromophenol blue, 0.25% xylene cynol, pH 8.0). Each
sample of 8~10 µL was loaded on 6% denaturing
polyacrylamide gels and run at 400 V for approximately
1 h and then dyied by the silver staining method as
described by Bassam (1991).
3.4 Linkage analysis
The linkage map was generated using MAPMAKER/Exp
version 3.0b (Lincoln et al., 1992). An LOD score of 3.0
was used as a threshold for the declaration of linkage,
Genetic distances in centimorgans (cM) were calculated
by applying the Kosambi (Kosambi, 1944) mapping
function and. The genetic map was drawn with the
software Mapdraw V2.1 (Liu and Meng, 2003).
Authors’contributions
JL conducted the major part of this study including experimental design,
construction of the F2 population, SSR analysis, and manuscript preparation.
HTW participated SSR analysis and genetic linkage mapping. XRH
participated construction of the F2 population. BRL assisted in the
development of the project and manuscript preparation. WYY participated
in the development of the project, experimental design and manuscript
preparation. All authors read and approved the final manuscript.
Acknowledgements
The authors thank Jie Liao for her part experiments. This work was
supported by the National 863 program (No. 2006AA10Z1C6), and the
National Natural Science Foundation of China (No.30771338 and
30700495).
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