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Rice Genomics and Genetics 2012, Vol.3, No.4, 19
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pollen particles in each visual field, and then calculated
the average number for verifying the seed-setting rate.
According staining the reaction differentia of the
pollen grains to 1% I-KI, the pollen were divided into
4 kinds, the deformity without staining was typical
abortive, circular no staining was round defeats,
circular shallow staining was stained abortion and
circular deep staining was normal fertile, respectively.
Figure 4 Process of the restorer selection by MAS
3.3 DNA extraction
The experimental materials were cultivated in Rice
Research Institute of Liaoning Province experimental
field, after transplanting for 30 days, DNA from
leaves were extracted by using the CTAB method.
Taking 10 parents, the F
2
generation for each
combination a total of 163 plants in 2008, as well as a
total of 1265 plants from the F
4
group of each
combination in 2009, we hang tags on each plant
sequentially.
3.4 PCR amplification and electrophoresis validation
Two pairs of primers used in SSR analysis, named as
RM10353, RM6100 from http://www.gramene.org/,
were synthesized from Shanghai Biological Engineering
Company. RM10353 used for identification of
Rf3
is
located on the first chromosome and linkage distance
with the target gene is 6.79 kb. RM6100 used for
identification of
Rf4
is located on the tenth
chromosome, and linkage distance with the target
gene is 2.4 cM. Primer sequence and their name were
shown in Table 4.
The volume of PCR reaction system was 20 μL
reaction, including Mg
2+
(25 mmol/L) 2.0 μL, PCR
Buffer (no Mg
2+
) 2.0 μL, dNTP (10 mmol/L) 0.3 μL,
each primer (15 ng/μL) 2.0 μL; template DNA (about
15 ng/μL) 1.4 μL,
Taq
enzyme (10 U/μL) 0.15 μL,
double-distilled water sterilization 12.15 μL.
PCR reaction were carried out in the Biometra PCR
instrument, and reaction conditions are as follows:
94
℃
degeneration for 5 min, and then 94
℃
1 min,
55
℃
1 min, 72
℃
1.5 min, a total of 32 cycle, at last,
72
℃
extension for 10 min. the PCR products were
stored at 4
℃
.
Acknowledgment
This project was supported by Liaoning Province Science and
Technology Reserch (2006201002) and Liaoning Province
Science and Technology Fund (20052129).
Table 4 Primer sequence and their name
Primers
Chromosome
Genes
Primers
References
RM10353
1
Rf3
F: GGACACTTTGAATGAAGGCAACC
R: TTGTTAGTTGGCGAAAGGAAGC
Qi et al., 2008
RM6100
10
Rf4
F: TCCTCTACCAGTACCGCACC
R: GCTGGATCACAGATCATTGC
Li et al., 2006
References
Cao L.Y., Zhan X.D., Zhuang J.Y., and Cheng S.H., 2005, Breeding of
Indica
hybrid rice Guodao 1 with good quality, high yield and
resistance to bacterial leaf blight by molecular marker-assisted
selection technique, Zajiao Shuidao (Hybrid Rice), 20(3): 16-18
Chen J.M., Fu Z.Y., Quan B.Q., Tian D.G., Li G., Wang F., 2009, Breeding
hybrid rice restoring line with double resistance to rice blast and
bacterial blight by marker-assisted selection, Fenzi Zhiwu Yuzhong
(Molecular Plant Breeding), 7(3): 465-470
Chen Y.J., Ding F., Wang X.J., Zhang C., Shao G.J., and Qiu F.L., 2008,
Selection and study on Yinshui Type
Japonica
sterile rice(I), Beifang
Shuidao (North Rice), 38(1): 15-19
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