Page 8 - Rice Genomics and Genetics

Rice Genomics and Genetics 2012, Vol.3, No.2, 8 -12
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17
Pi1 resistance gene is a broad-spectrum blast resistance
gene. In this study, molecular markers detection
results showed that 36.99% of the Yunnan landraces
contained the Pi1 gene and those landraces were
distributed in different ecosystem, the different
ecological types and sub-species provide more choices
for resistant material in breeding program, and the
blast resistance breeding is a crucial work in China.
Our results confirmed that the effectiveness of SSR
marker identification for Pi1 gene, which was an
effective method for identification the Pi1 gene in a
mass of resources.
3 Materials and Methods
3.1 Rice materials
One hundred and seventy-three rice landraces were
used for this study, and these landraces were randomly
selected from the Yunnan core collection of resources
library, which were conserved in Institute of Biotechnology
and Genetic Resources, Yunnan Academy of Agricultural
Sciences, Kunming, China. The selected 173 landraces
were collected from 46 counties of 13 prefectures in
Yunnan, China. Among them, 102 are japonica, 71 are
indica. All materials were identified by SSR marker
(MGR4766) of Pi1 gene. Based on the PCR detected
results, 30 landraces were selected for seedling
artificial inoculation, Lijiangxintuanheigu variety as
susceptible control.
3.2 Identification blast isolate
The identification blast isolate 96-2-2b was selected
base on the reaction on the monogenic lines in Plant
Pathology Laboratory of Agricultural Environment
and Resources Research Institute, Yunnan Academy of
Agricultural Sciences, China.
3.3 Rice genomic DNA extraction
DNAs were isolated from three to four leaf stage of
rice, genomic DNA was prepared by CTAB method
following instruction of Xu et al (1998).
3.4 SSR primers
SSR primers MGR4766 were designed according to
Chen et al (2005), and the forward primer: 5'-ATTGC
TGCAAAGTGGGAGAC-3', the reverse primer: 5'-AA
GTGGAGGCAGTTCACCAC-3'. The primers were
synthesized by Sangon Biotech. (Shanghai) Co., Ltd.
3.5 PCR amplification and polyacrylamide gel
electrophoresis
PCR reaction volume was 25 μL, containing 2.5 μL
10×Buffer, 2.0 μL dNTP, 3.0 μL 4 mol/L forward and
reverse primers, 0.2 μL 5 U/μL Taq enzyme, 3.0 μL
20 ng/μL template DNA, ddH2O 14.3 μL. PCR
program follows: 94 ℃ predenaturation 5 min,
followed by 35 cycles of 9℃ for 45 s, 55℃ for 45 s,
72℃ extension of 1 min, then 72℃ extension of 7 min.
PCR products were separated on 8% non-denaturing
polyacrylamide gel electrophoresis, and then silver
staining followed by Li et al., 2008.
3.6 Rice blast fungus inoculation in the greenhouse
The 173 landraces seeds are sown in a plastic tray (5
cm× 15 cm×10 cm) half-filled with sieved garden
soil. Each landrace are sown about 16 seeds and
placed in greenhouse. At 2.5-leaf stage applied 0.2 g
urea each plate, a total of 2~3 times.
Inoculation of blast isolates was carried out following
the method. The spore concentration was standardized
to 2~5×105 spores per ml. Plants were inoculated at
approximately the 3.5th~4.5th -leaf stages by spraying
spore suspension on each tray using a fine atomizer,
and then placed in charmber under 24~26 ℃ for
16~20 h, and then back to the greehouse. The degree
of disease of each seedling was evaluated at 6~7 days
after inoculated. The rice varieties resistant evaluation
as described by Ahn et al (1994).
Authors’ Contributions
JBL worked on the inoculation, resistance test, confirmation
and modified the manuscript. DL worked on PCR detection and
finished draft. YDS analyzed the data. MHX performed the
experiment designs, analyzed the data, wrote and modified the
manuscript. All authors have read and approved the final
manuscript.
Acknowledgements
This research was supported by National Natural Science Fund
of China (31160355) and Natural Science Fund of Yunnan of
China (2010ZC173).
Reference
Ahn S.W., 1994, International collaboration on breeding for resistance to
rice blast, In: Zeigler R.S., Leong S.A., and Teng P.S.(eds.), Rice blast
disease, CAB International, Wallingford, UK, pp.137-154
Chen H.L., ChenChen B.T., Zhang D.P., Xie XY.F., and Zhang Q.F., 2001,
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