全 文 ：作物学报 ACTA AGRONOMICA SINICA 2012, 38(11): 1960−1968 http://www.chinacrops.org/zwxb/
ISSN 0496-3490; CODEN TSHPA9 E-mail: xbzw@chinajournal.net.cn

This work was supported by the National High Technology Research and Development Program of China (Grant No. 2011AA10A101 and
2012AA101102) and the Ministry of Finance, China (Grant No. 2012RG002-4).
* Correspondence author: WU Jian-Li, E-mail: beishangd@163.com, Tel: +86-571-63370326
** These authors contributed equally to this work.
Received(收稿日期): 2012-04-17; Accepted(接受日期): 2012-07-05; Published online(网络出版日期): 2012-09-10.
URL: http://www.cnki.net/kcms/detail/11.1809.S.20120910.1404.023.html
DOI: 10.3724/SP.J.1006.2012.01960
Development and Validation of CAPS Markers for Marker-Assisted Selection
of Rice Blast Resistance Gene Pi25
WANG Hui-Mei1,**, CHEN Jie1,**, SHI Yong-Feng1, PAN Gang2, SHEN Hai-Chao1, and WU Jian-Li1,*
1 State Key Laboratory of Rice Biology / China National Rice Research Institute, Hangzhou 310006, China; 2 College of Agriculture and Biotechno-
logy, Zhejiang University, Hangzhou 310058, China
Abstract: To promote the application of rice blast resistance gene Pi25 in rice breeding programs, we developed four sets of
gene-specific CAPS markers (CAP1/Hinc II, CAP3/Bgl II, CAP3/Nde I, and CAP3/Hpy 99I) based on the coding sequences of the
locus. One hundred and sixty-nine rice accessions, 98 recombinant inbred lines (RILs) and 217 transgenic plants were used for the
validation of the markers. The results showed that all the four sets of markers were able to accurately and efficiently detect the
Pi25/pi25 locus, CAP1/Hinc II and CAP3/Hpy 99I could digest specifically the dominant allele Pi25 while CAP3/Bgl II and
CAP3/Nde I were able to digest specifically the recessive allele pi25. RILs and transgenic lines carrying Pi25 allele were resistant
to the blast isolate JS001-20 while the lines carrying pi25 allele were susceptible, indicating a perfect detection of the target locus
by the CAPS markers. In addition, a low frequency (1.2%) of the dominant allele was detected in the germplasm collections, in-
dicating this gene has not been fully utilized in rice breeding programs in China. Markers CAP1/Hinc II and CAP3/Hpy 99I are
recommended and will be useful for the improvement of blast resistance, especially for the early-season indica rice.
Keywords: Oryza sativa; Blast resistance; Cleaved amplified polymorphic sequence (CAPS); Marker-assisted selection (MAS);
Single nucleotide polymorphism (SNP)
稻瘟病抗性基因 Pi25特异性 CAPS标记的开发与验证
王惠梅 1,** 陈 洁 1,** 施勇烽 1 潘 刚 2 沈海超 1 吴建利 1,*
1中国水稻研究所 / 水稻生物学国家重点实验室, 浙江杭州 310006; 2浙江大学农业与生物技术学院, 浙江杭州 310058
摘 要: 为在水稻育种中快速与高效利用稻瘟病抗性基因 Pi25, 本文利用该基因不同等位基因编码区序列差异开发
了 4套 CAPS标记(CAP1/Hinc II、CAP3/Bgl II、CAP3/Nde I和 CAP3/Hpy 99I), 并利用 169份稻种资源、98个重组
自交系(RIL)以及 217个水稻转基因后代, 对 4套标记的准确性和选择效果进行了验证。结果表明, 4套标记均能准确
地检测 Pi25/pi25座位。其中, 标记 CAP1/Hinc II和 CAP3/Hpy 99I特异性识别并酶切显性等位基因, 而标记 CAP3/Bgl
II和 CAP3/Nde I特异性识别并酶切隐性等位基因。利用稻瘟病菌株 JS001-20接种 RIL与转基因材料, 抗性表现与标
记检测的结果完全一致, 表明该 CAPS标记准确可靠。分析稻种资源后发现, Pi25基因频率较低(1.2%), 说明该基因
在我国水稻稻瘟病抗性育种中还没有被充分利用。本文的研究结果特别是开发的 2对识别并酶切显性等位基因的
CAPS标记可用于分子标记辅助选择, 改良我国早籼稻的稻瘟病抗性。
关键词: 水稻; 稻瘟病抗性; 酶切扩增多态性序列; 标记辅助选择; 单核苷酸多态性
More than 50 major rice blast resistance genes have
been indentified since late 1980s because of the deve-
lopment and utilization of molecular markers [1-3]. Among
them, 15 blast resistance genes have been successively
cloned through the map-based cloning strategy [4-12]. Al-
though a serious of effort has been attempted in elucidat-
ing the structure and function of the genes while there is
no fundamental breakthrough in application of the genes
to improve rice blast resistance in rice breeding programs.
With the development of molecular markers, the selec-
tion for targeted blast resistance genes using specific
molecular markers is commonly practiced and some in-
termediate breeding materials carrying the target genes
have been bred [13]. One of the quick and effective ways
第 11期 王惠梅等: 稻瘟病抗性基因 Pi25特异性 CAPS标记的开发与验证 1961


for improving blast resistance in new varieties is to
backcross the intermediate breeding materials with
commercial elite lines. However, the accuracy of marker-
assisted selection (MAS) for the target genes largely de-
pends on the strength of linkage between the markers and
the target genes. Fortunately, functional markers for those
cloned genes have been developed and could provide the
accurate and efficient selection [14-15].
Gumei 2 is the only early-season semi-dwarf indica
rice cultivar with stable and broad-spectrum resistance to
Magnaporthe oryzae, the causal pathogen of blast dis-
ease[16]. It is considered as a valuable donor for rice blast
improvement in China because of its elite agronomic
traits. Previous studies have identified and located at
least four blast resistance genes and a number of partial
resistance QTLs in Gumei 2 [17-18]. Among them, Pi25 is
a single copy intronless CC-NBS-LRR type of blast re-
sistance gene located in chromosome 6 [19].
This work aims at developing and validating the Pi25
functional markers based on the coding sequences of the
locus using three categories of rice materials. Two sets of
markers, CAP1/Hinc II and CAP3/Hpy 99I, are able to
specifically digest the dominant allele Pi25 and recom-
mended for MAS in rice breeding programs.
1 Materials and Methods
1.1 Plant materials
A total of 169 rice germplasm accessions including
129 indica rice and 40 japonica rice, 98 recombinant
inbred lines (RILs, F9) derived from the cross Zhongjian
100/Gumei 2 (donor of Pi25) were grown in the paddy
field at the Fuyang Experimental Station, China National
Rice Research Institute (CNRRI) and 217 transgenic
plants derived from Zhongjian 100 transformed with
Pi25 were grown in the greenhouse at CNRRI.
1.2 Development of CAPS markers
There are six single nucleotide substitutions (g775a,
t1197c, t2444a, c2566g, g2680a, and g2687a) in the cod-
ing sequences between Pi25, the resistant allele from
Gumei 2, and pi25, the susceptible allele from Zhongjian
100, respectively, leading to the substitution of five
amino acids (V259I, F815Y, H856D, V894I, and R896Q).
One of the single nucleotide polymorphisms (SNPs),
t1197c, is a synonymous mutation in both alleles (Fig. 3).
Among the other five SNPs, four SNPs (g775a, t2444a,
c2566g, and g2687a) corresponding to four restriction
endonuclease (RE) recognition sites were chosen for the
development of gene specific markers. Hinc II (g775a)
and Hpy 99I (g2687a) detect and digest specifically for
the resistant allele Pi25 while Nde I (t2444a) and Bgl II
(c2566g) detect and digest specifically for the susceptible
allele pi25 (Fig. 1). On the base of these SNPs, we deve-
loped four pairs of CAPS markers (CAP1/Hinc II, CAP3/Bgl
II, CAP3/Nde I, and CAP3/Hpy 99I) for the detection of the
Pi25/pi25 locus. The PRIMER3 online program (http://
frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) was
used for primer design and the primers were synthesized
by Invitrogen (Shanghai) (Table 1).

Fig. 1 Four SNPs and the corresponding RE recognition sites at
the Pi25/pi25 locus
SNPs are underlined; RE recognition sites are boxed. R: resistant allele;
S: susceptible allele.

Table 1 Pi25 specific primers and the corresponding restriction endonucleases
Primer Sequence (5′–3′) Product size (bp) Enzyme Pi25 pi25
CAP1F TGAAATGGGTGAAAGATGAG
CAP1R GCCACATCATAATTCCTTGA
406 Hinc II + –
Nde I – + CAP3F CCTCACGTTTCTACGTCTTG
Bgl II – +
CAP3R CACACCATTTCTGATGAACC
409
Hpy 99I + –
+: Digestion; –: Non-digestion.

1.3 PCR and RE digestion
The PCR was carried out as previously described [20].
The PCR products were digested with Hinc II (Fermentas,
ER0491), Nde I (Fermentas, ER0585), Bgl II (Fermentas,
ER0082) and Hpy 99I (NEB, R0615L) independently.
For Hinc II and Hpy 99I, 4 hours incubation at 37°C, 20
min at 65°C for inactivation in 20 µL reaction mixture
containing 5 µL of PCR products (0.25 µg), 5 U RE and
1×Tango buffer (33 mmol L–1 Tris-acetate, pH 7.9, 10
mmol L–1 magnesium acetate, 66 mmol L–1 potassium
acetate, 0.1 mg L–1 BSA) for Hinc II, and 1×NEB buffer
4 (20 mmol L–1 Tris-acetate, 50 mmol L–1 potassium
acetate, 10 mmol L–1 magnesium acetate, 1 mmol L–1
dithiothreitol, 0.1 mg L–1 bovine serum albumin) for Hpy
99I. For Nde I and Bgl II, incubation at 37°C overnight in
30 µL reaction mixture containing 8 µL of PCR products
(0.4 µg), 10 U RE and 1× buffer O (50 mmol L–1
Tris-HCl pH 7.5, 10 mmol L–1 MgCl2, 100 mmol L–1
1962 作 物 学 报 第 38卷

NaCl, 0.1 mg mL–1 BSA). Inactivation of Nde I was
achieved at 65°C for 20 min while inactivation of Bgl II
was achieved at a final concentration of 20 mmol L–1
EDTA, respectively. The digested products were frac-
tionated on 2% agarose gel, and stained with 2×GelRed
(Biotium, USA) for visualization.
1.4 Cloning of Pi25 alleles
For the development and validation of the CAPS
markers, we isolated the coding sequences of Pi25/pi25
alleles from Gumei 2, Zhongjian 100, G19, C101A51,
Gumei 4, and Tetep as previously reported [19]. The frag-
ments were purified and inserted into the pGEM-T Easy
vector (Promega, USA) and sequenced by Invitrogen
(Shanghai). The coding sequences of the alleles from
Gumei 2 (accession number HM448480), Zhongjian 100
(accession number JQ838019), and Tetep (accession
number JQ838018) have been deposited in the GenBank.
1.5 Blast evaluation
The blast isolate JS001-20 was used for inoculation of
98 RILs, 217 T3 transgenic plants and the recipient
Zhongjian 100, the donor Gumei 2 and the susceptible con-
trol Lijiangxintuanheigu (LTH) as previously reported [19].
Disease evaluation was carried out following the method
described by Bonman et al. [21]
2 Results
2.1 CAPS analysis of germplasm accessions
To identify the allelic presence/absence of Pi25/pi25
gene in the germplasm collection, we tested all four pairs
of CAPS markers CAP1/Hinc II, CAP3/Nde I, CAP3/Bgl
II and CAP3/Hpy 99I. The results showed that three out
of 169 rice accessions including C101A51, G19 (Gumei
2/Zhong 156) and Gumei 4 were specifically digested by
CAP1/Hinc II and CAP3/Hpy 99I and showed the same
band pattern as Gumei 2 (Fig. 2 and Table 2). Further-
more, the coding sequences of them were identical.
Therefore, C101A51, G19 and Gumei 4 possessed the
dominant Pi25 allele as Gumei 2. The other accessions
except Tetep were detected and digested by the CAPS
markers CAP3/Nde I and CAP3/Bgl II and showed the
same band pattern as Zhongjian 100 (Table 2). From the
comparison of the coding sequences among Gumei 2,
Zhongjian 100, and Tetep, we found that Tetep was con-
sistent with Zhongjian 100 at two SNP sites (a775a and
a2687a) while consistent with Gumei 2 at the other two
SNP sites (t2444t, c2566c) (Fig. 1 and Fig. 3). Because
the nucleotide substitutions at positions 2 444 and 2 566
between Zhongjian 100 and Tetep did not result in amino-
acid substitutions, therefore, Tetep was predicted to have
the identical amino-acid residues at the four SNP sites the
same as Zhongjian 100, but different from the donor
Gumei 2 at positions 775 and 2687, where a valine and
an arginine in the Pi25 were substituted by an isoleucine
and a glutamine in pi25, respectively. Although Tetep
showed the same amino-acid residues at these four sites
as Zhongjian 100, it possessed a new allele because its
full coding sequence was 312 nucleotides shorter than
that of Gumei 2 and Zhongjian 100 (Fig. 3). Nevertheless,
our results indicated that the two markers, CAP1/Hinc II
and CAP3/Hpy 99I, could specifically recognize and di-
gest the dominant allele Pi25.
In addition, the Pi25 allele presented in G19 was
originated from Gumei 2 since G19 was derived from the
cross Zhongjian 100/Gumei 2. Gumei 4 is a sister line of
Gumei 2 with unknown progenitor. The Pi25 allele pre-
sented in C101A51 is derived from 5173 [22]. Thus, ex-
cept G19 and Gumei 4, only Gumei 2 and C101A51 pos-
sessed the dominant Pi25 allele in 167 rice germplasm
accessions tested (Table 2). The results indicated that the
frequency of Pi25 was low at 1.2% (2/167) and it has not
been fully utilized in rice breeding programs in China.
2.2 CAPS analysis of RILs
To validate the accuracy of the four CAPS markers, we
genotyped 98 RILs derived from the cross Zhongjian
100/Gumei 2. The results showed that Zhongjian 100
possessed the pi25 allele while Gumei 2 contained the
Pi25 allele as expected. Among 98 RILs, 56 RILs
showed the same band pattern as Gumei 2 while 42 RILs
exhibited the same band pattern as Zhongjian 100 (Fig.
4). The ratio of Pi25 to pi25 in the RILs was consistent
with the expected 1:1 (χ2=2.00<χ20.05=3.84), indicating a
free segregation of the locus and could be easily applied

Fig. 2 CAPS marker analysis of rice germplasm accessions
1: Gumei 2; 2: Zhongjian 100; 3: LTH; 4: C101A51; 5: Tetep; 6: CDR22; 7: G19; 8: Gumei 4; 9–21: Other accessions showing the same band pattern.

第 11期 王惠梅等: 稻瘟病抗性基因 Pi25特异性 CAPS标记的开发与验证 1963


Table 2 Distribution of Pi25/pi25 locus in the germplasm based on the CAPS marker analysis
Variety Type Pi25/pi25) Variety Type Pi25/pi25 Variety Type Pi25/pi25
II-32B I – Lucaihao I – Zhonghan 1 I –
Guangzhan 63S I – Mianhui 725 I – T97 I –
9311 I – Minkezao 1 I – IRBB 14 I –
AUS373 I – Minghui 63 I – Baxiludao I –
Basmati 370 I – Nantehao I – H19 I –
CDR22 I – Minghui 77 I – Vandana I –
CO39 I – Minghui 86 I – G19 I +
Zhongyoudao 1 I – Minghui 70 I – T657 I –
C101LAC I – Pei’ai 64 I – C Bao J –
H161 I – Nipponbare J – Han9 J –
C101A51 I + Shouguangsimiao I – IR68 I –
H333 I – Shuhui 517 I – Wazushandao J –
H593 I – Shuhui 85 I – IRBB13 I –
C101PKT I – Shuhui 881 I – Zhongjian 2 I –
IR24 I – Shuanggui 1 I – C57 J –
IR30 I – Shuangqizhan I – IRBB3 I –
IR36 I – Taizhong 65 I – Java14 J –
IR64 I – Teqing I – IRBB5 I –
Morobereken J – Texianzhan 25 I – Nanjing 42 J –
H811 I – Tianjiqing 776 J – IRBB8 I –
R402 I – Chunjiang 06 J – Wuyujing 20 J –
R752 I – Wuyoudao 1 J – IRBB11 I –
Tetep I +/– Wufujing J – 300 hao J –
To974 I – Xiushui 11 J – Gumei 4 I +
V20B I – Wuyujing 14 J – IRBB21 I –
ZDZ057 I – Wuyujing 3 J – IR26 I –
Aimeizao 3 I – Wuyujing 7 J – IRBB4 I –
Aijiaonante I – Reyan 1 J – Nanjing 15 J –
Aizaizhan I – Xianfeng 1 I – Jingang 30 I –
BoB I – Xiangzaoxian 31 I – IRBB7 I –
Suifuruanzhan I – Xieqingzao B I – THZ I –
Ce46 I – Xiushui 04 J – Jefferson I –
Daonuzhong 58 J – Xiushui 110 J – Zhongguang B I –
Duoxi 1 I – Yanhui 559 I – R9308 I –
Enhui 58 I – Yujing 6 J – CPSLO17 J –
Gang 46B I – Yuexiangzhan I – Lunhui 422 I –
Fuhui 838 I – H1 I 　 IRBB10 I –
Gumei 2 I + Zhangyouzhan I – MXZ2 I –
Guanghui 128 I – Zhenlong 13 I – Zhejing 29 J –
Guangluai 4 I – Zhenhui 084 I – Quanzhen 10 I –
Gui99 I – Zhenzhan 97B I – Donglian 5 I –
Guichao 2 I – Zhong 156 I – Jiayu 253 I –
Jiahezhan I – R8006 I – Youzhan I –
Jiayu 948 J – Zhou 903 I – Zhongzao 39 I –
Jianghui 151 I – Zhonghan 4 I – Guixiaozhan I –
Jin23B I – Zhongjian 100 I – Zaoxian 276 I –
Kendao10 J – Zhong 9B I – Hefengzhan I –
Yueguang J – Zuke 2 I – Yongxian 15 I –
Kongyu 131 J – Milyang 46 I – Shanxiaozhan I –
LTH J – PKTX I – Zaoxian 213 I –
Liantangzao I – Zachaodao I – Xiangzaoxian 7 I –
Liaojing 294 J – IRAT13X J – Longjing 21 J –
Longtepu B I – Zhonghua 11 I – Daohuaxiang 2 J –
Luhui 17 I – Hongjiaozhan I – Songjing 11 J –
Lujiangzao 1 I – 02428 J – Changlixiang J –
Mianhui 501 I – Zhonghan 3 J – H17 I –
Han 2 J – –
+: present for Pi25; –: present for pi25; +/–: Tetep shows the same amino acid residues at the 4 SNPs sites as “Zhongjian 100” but a shorter
coding sequence and is considered as a new allele; I: indica rice; J: japonica rice.
1964 作 物 学 报 第 38卷




(to be continued)
第 11期 王惠梅等: 稻瘟病抗性基因 Pi25特异性 CAPS标记的开发与验证 1965



Fig. 3 Comparison of the coding sequences in three rice accessions
1966 作 物 学 报 第 38卷


Fig. 4 CAPS marker analysis of RILs
1: Gumei 2; 2: Zhongjian 100; 3: LTH; 4–21: 18 RILs derived from the cross Zhongjian 100/Gumei 2. R: resistant; S: susceptible.

in breeding practice. The results indicated that the CAPS
markers were able to detect accurately the Pi25/pi25 lo-
cus in a breeding population, and the markers CAP1/
Hinc II and CAP3/Hpy 99I specifically digesting for the
dominant allele were more practical with relatively
shorter digestion time.
2.3 CAPS analysis of transgenic lines
To further validate the accuracy of the CAPS markers,
we genotyped 217 T3 transgenic progenies (balk seed
from 10 independent T2 lines) originally derived from
Zhongjian 100 transformed with Pi25 using the four
CAPS markers. The results indicated that four out of 217
individual plants possessed the same band pattern as the
donor Gumei 2 while the remaining 213 plants showed
the same band pattern as the recipient Zhongjian 100 (Fig.
5) showing that the CAPS markers were also able to ac-
curately detect the Pi25/pi25 gene in the transgenic
progenies. Again, markers CAP1/Hinc II and CAP3/Hpy
99I specifically detecting and digesting for the dominant
allele were much easier to use.

Fig. 5 CAPS marker analysis of transgenic plants
1: Gumei 2; 2: Zhongjian 100; 3: LTH; 4–21: 18 T3 transgenic plants; R: resistant; S: susceptible.

2.4 Blast resistance evaluation
To confirm the RILs and transgenic plants containing
the Pi25 allele were truly resistant to M. oryzae, we
chose a blast isolate JS001-20 previously identified
compatible to pi25 but incompatible to Pi25 for inocu-
lating the 98 RILs and 217 transgenic plants. Our results
showed that the 56 RILs and four transgenic plants har-
boring Pi25 allele were resistant to JS001-20, the same as
the donor Gumei 2 while the remaining RILs and trans-
genic plants harboring pi25 allele were susceptible to the
isolate, the same as the recipient Zhongjian 100 and the
susceptible control LTH (Fig. 4 and Fig. 5). Thus, the
results from marker-assisted selection were consistent
with those of disease evaluation, indicating the four
CAPS markers could accurately and efficiently detect the
Pi25/pi25 locus. In addition, Tetep was likely to harbor a
new allele at the locus, and was also resistant to the
isolate JS001-20. However, it is unknown whether the
Tetep allele is incompatible to the isolate because Tetep
possesses a number of other blast resistance genes [23].
In practice, we recommend two markers CAP1/Hinc II
and CAP3/Hpy 99I that specifically detect and digest
the Pi25 allele in order to reduce the workload and the
cost.
第 11期 王惠梅等: 稻瘟病抗性基因 Pi25特异性 CAPS标记的开发与验证 1967


3 Discussion
Accuracy and low cost are two key factors affecting
marker-assisted selection. The accuracy of selection de-
pends on the strength of linkage between the markers and
the target genes. Previous marker-assisted selection usu-
ally results in the unsatisfactory outcome because the
markers are located far from the target genes [24-25]. In
addition, it requires a large sample size and consequently
increases the workload and the cost. One of the solutions
is to employ flanking markers closely linked to the target
gene. This approach is able to largely improve the accu-
racy of the selection but also increases the workload and
the cost because an additional marker is required [26].
With the progress of fine mapping and cloning of blast
resistance genes, the accuracy of MAS reaches a new
higher level. First, functional markers theoretically im-
prove the selection accuracy to 100%. Second, instead of
flanking markers, a single marker is generally enough
and the workload and the cost are reduced.
At present, a number of PCR-based markers for blast
resistance genes have been developed. These new gen-
eration/functional markers show a reliable accuracy and
efficiency in the selection of the target genes [14-15]. In this
study, the CAPS markers were developed based on the
nucleotide difference in the coding region between Pi25
and pi25 alleles. Using an extensively-used germplasm
collection, a breeding population and a balk of transgenic
rice lines we verified the accuracy and effectiveness of
the markers. The percent of accuracy reaches 100% and
the effectiveness of selection for the locus than for the
markers previously reported is hugely improved [18].
Breeding for improved blast resistant varieties has
been always an important criterion in Chinese rice
breeding programs. However, limited information is
available on the usage of known blast resistance genes
and on the gene frequency and distribution in Chinese
rice germplasm [27]. The blast resistance of Gumei 2 is
controlled by multiple genes, one of them Pi25 confer-
ring both leaf and neck resistance is considered valuable
for improving the resistant level especially for early-
season indica rice since the donor Gumei 2 belongs to the
early-season indica type of cultivars in China. Unex-
pectedly, the frequency of this gene is low for unknown
reasons in the extensively used germplasm resources. In
addition, at least a new allele of Pi25, with a shorter
length of coding sequence as well as base substitutions,
was identified in the traditional donor Tetep. Further
screening and sequencing of the coding sequence would
further clarify the number of alleles at the locus in Chi-
nese rice germplasm. This study not only provides four
sets of reliable markers for the selection of Pi25/pi25, but
also would facilitate the further application of the Pi25
gene to the improvement of blast resistance for the
early-season indica rice. In practice, we recommend two
markers CAP1/Hinc II and CAP3/Hpy 99I for the speci-
ficity to the dominant allele and for the simplicity and
cost effective as a whole.
4 Conclusions
On the base of the coding sequences of the blast resis-
tance gene Pi25 and its susceptible allele pi25, we de-
veloped four sets of CAPS markers in the present study.
Using various materials including germplasm accessions,
recombinant inbred lines and transgenic plants, the accu-
racy and efficiency of the CAPS markers are validated.
To make full utilization of the dominant allele, we rec-
ommend two sets of the markers CAP1/Hinc II and
CAP3/Hpy 99I for marker-assisted selection, especially
for the early-indica rice improvement of blast resistance
in rice breeding programs.
References
[1] Jeung J U, Kim B R, Cho Y C, Han S S, Moon H P, Lee Y T, Jena
K K. A novel gene, Pi40(t), linked to the DNA markers derived
from NBS-LRR motifs confers broad spectrum of blast resistance
in rice. Theor Appl Genet, 2007, 115: 1163–1177
[2] Terashima T, Fukuoka S, Saka N, Kudo S. Mapping of a blast
field resistance gene Pi39(t) of elite rice strain Chubu 111. Plant
Breed, 2008, 127: 485–489
[3] He X Y, Liu X Q, Wang L, Wang L, Lin F, Cheng Y S, Chen Z M,
Liao Y P, Pan Q H. Identification of the novel recessive gene
pi55(t) conferring resistance to Magnaporthe oryzae. Sci China
Life Sci, 2012, 55: 141–149
[4] Lin F, Chen S, Que Z Q, Wang L, Liu X Q, Pan Q H. The blast
resistance gene Pi37 encodes a nucleotide binding site-leucine-
rich repeat protein and is a member of a resistance gene cluster
on rice chromosome 1. Genetics, 2007, 177: 1871–1880
[5] Liu J L, Liu X L, Dai L Y, Wang G L. Recent progress in eluci-
dating the structure, function and evolution of disease resistance
genes in plants. J Genet Genomics, 2007, 34, 765–776
[6] Liu X Q, Lin F, Wang L, Pan Q H. The in silico map-based clon-
ing of Pi36, a rice coiled-coil-nucleotide-binding site-leucine-rich
repeat gene that confers race-specific resistance to the blast fun-
gus. Genetics, 2007, 176: 2541–2549
[7] Ashikawa I, Hayashi N, Yamane H, Kanamori H, Wu J, Matsu-
moto T, Ono K, Yano M. Two adjacent nucleotide-binding
site-leucine-rich repeat class genes are required to confer Pikm-
specific rice blast resistance. Genetics, 2008, 180: 2267–2276
[8] Fukuoka S, Saka N, Koga H, Ono K, Shimizu T, Ebana K, Ha-
yashi N, Takahashi A, Hirochika H, Okuno K, Yano M. Loss of
function of a proline-containing protein confers durable disease
1968 作 物 学 报 第 38卷

resistance in rice. Science, 2009, 325: 998–1001
[9] Hayashi K, Yoshida H. Refunctionalization of the ancient rice
blast disease resistance gene Pit by the recruitment of a retro-
transposon as a promoter. Plant J, 2009, 57: 413–425
[10] Lee S K, Song M Y, Seo Y S, Kim H K, Ko S, Cao P J, Suh J P,
Yi G, Roh J H, Lee S, An G, Hahn T R, Wang G L, Ronald P, Jeon
J S. Rice Pi5-mediated resistance to Magnaporthe oryzae re-
quires the presence of two CC-NB-LRR genes. Genetics, 2009,
181: 1627–1638
[11] Shang J J, Tao Y, Chen X W, Zhou Y, Lei C L, Wang J, Li X B,
Zhao X F, Zhang M J, Lu Z K, Xu J C, Cheng Z K, Wan J M, Zhu
L H. Identification of a new rice blast resistance gene, Pid3, by
genomewide comparison of paired nucleotide-binding site-
leucine-rich repeat genes and their pseudogene alleles between
the two sequenced rice genomes. Genetics, 2009, 182: 1303–1311
[12] Okuyama Y, Kanzaki H, Abe A, Yoshida K, Tamiru M, Saitoh H,
Fujibe T, Matsumura H, Shenton M, Galam D C, Undan J, Ito A,
Sone T, Terauchi R. A multi-faceted genomics approach allows
the isolation of rice Pia-blast resistance gene consisting of two
adjacent NBS-LRR protein genes. Plant J, 2011, DOI: 10.1111/
j.1365-313X.2011.04502.x
[13] Yin D-S(殷得所), Xia M-Y(夏明元), Li J-B(李进波), Wan
B-L(万丙良), Zha Z-P(査中萍), Du X-S(杜雪树), Qi H-X(戚华
雄). Development of STS marker linked to rice blast resistance
gene Pi9 in marker assisted selection breeding. Chin J Rice Sci
(中国水稻科学), 2011, 25: 25–30 (in Chinese with English ab-
stract)
[14] Jia Y, Wang Z, Singh P. Development of dominant rice blast Pi-ta
resistance gene markers. Crop Sci, 2002, 42: 2145–2149
[15] Hayashi K, Yasuda N, Fujita Y, Koizumi S. Identification of the
blast resistance gene Pit in rice cultivars using functional markers.
Theor Appl Genet, 2010, 121: 1357–1367
[16] Peng S-Q(彭绍裘), Huang F-Y(黄费元), Sun G-C(孙国昌), Liu
E-M(刘二明), Sun Y-J(孙永吉), Ai R-X(艾仁孝), Zhao J-X(赵
家秀), Bai S-Z(白世枝), Xiao F-H(肖放华). Studies on durable
resistance to blast disease in different latitudes for rice. Sci Agric
Sin (中国农业科学), 1996, 29: 52–58 (in Chinese with English
abstract)
[17] Wu J L, Chai R Y, Fan Y Y, Li D B, Zheng K L, Leung H, Zhuang J
Y. Clustering of major genes conferring blast resistance in blast re-
sistance rice cultivar Gumei 2. Rice Sci, 2004, 11: 161–164
[18] Wu J L, Fan Y Y, Li D B, Zheng K L, Leung H, Zhuang J Y. Ge-
netic control of rice blast resistance in the durably resistant culti-
var Gumei 2 against multiple isolates. Theor Appl Genet, 2005,
111: 50–56
[19] Chen J, Shi Y F, Liu W Z, Chai R Y, Fu Y P, Zhuang J Y, Wu J L.
A Pid3 allele from rice cultivar Gumei 2 confers resistance to
Magnaporthe oryzae. J Genet Genomics, 2011, 38: 209–216
[20] Shi Y F, Chen J, Liu W Q, Huang Q N, Shen B, Leung H, Wu J L.
Genetic analysis and gene mapping of a new rolled leaf gene in
rice (Oryza sativa L.). Sci China (Ser C: Life Sci), 2009, 52:
885–890
[21] Bonman J M, Vergel de Dios T I, Khin M M. Physiological spe-
cialization of Pyricularia oryzae in the Philippines. Plant Dis,
1986, 70: 767–769
[22] Mackill D J, Bonman J B. Inheritance of blast resistance in
near-iosgenic lines of rice. Phytopathlogoy, 1992, 82: 746–749
[23] Shen Z-T(申宗坦), Zhang W-G(张旺根), He Z-H(何祖华), Sun
S-Y(孙漱源), Tao R-X(陶荣祥), Shi D(施德). Genetic analysis
for blast resistance in some indica race varieties (Oryza sativa L.).
Chin J Rice Sci (中国水稻科学), 1986, 1: 1–7 (in Chinese with
English abstract)
[24] Hittalmani S, Foolad M R, Mew T, Rodriguez R L, Huang N.
Development of a PCR-based marker to identify rice blast resis-
tance gene, Pi-2(t), in a segregating population. Theor Appl Genet,
1995, 91: 9–14
[25] Naqvi N I, Bonman J M, Mackill D J, Nelson R J, Chatto B B.
Identification of RAPD markers linked to a major blast resistance
gene in rice. Mol Breed, 1995, 1: 341–348
[26] Zheng K L, Huang N, Bennett J, Khush G S. PCR-based
marker-assisted selection in rice breeding. IRRI Discussion Paper
Series 1995, No.12, International Rice Research Institute, Manila,
The Philippines
[27] Shi K(时克), Lei C-L(雷财林), Cheng Z-J(程治军), Xu X-T(许
兴涛), Wang J-L(王久林), Wan J-M(万建民). Distribution of two
blast resistance genes Pita and Pib in major rice cultivars in
China. J Plant Genet Resour (植物遗传资源学报), 2009, 10:
21–26 (in Chinese with English abstract)