Molecular mapping of the Pi2/9 allelic gene Pi2-2 conferring broad-spectrum resistance to Magnaporthe oryzae in the rice cultivar Jefferson
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences
Beijing 100193, China
Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization, College of Agronomy, College of Bio-Safety Science and Technology, Hunan Agricultural University
, Changsha 410128,
State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University
, Wuhan 430070,
Department of Plant Pathology, Ohio State University
Columbus, Ohio 43210, USA
Background: Utilization of broad-spectrum resistance (R) genes is an effective and economical strategy to control the fungal pathogen Magnaporthe oryzae, the causal agent of the rice blast disease. Among the cloned blast resistance genes, Pi9, Pi2 and Piz-t confer broad-spectrum resistance to diverse M. oryzae isolates and were isolated from the Pi2/9 locus on chromosome 6. Identification and isolation of additional R genes with different resistance spectra from this locus will provide novel genetic resources for better control of this important rice disease. Results: In this study, we identified a dominant R gene, Pi2-2, at the Pi2/9 locus from Jefferson, an elite U.S. rice cultivar, through genetic and physical mapping. Inoculation tests showed that Jefferson has different resistant specificities to M. oryzae isolates compared rice lines with the Pi9, Pi2 and Piz-t genes. Fine mapping delimited Pi2-2 to a 270-kb interval between the markers AP5659-3 and RM19817, and this interval contains three nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes in the Nipponbare genome. Five bacterial artificial chromosome (BAC) clones spanning the region were identified, and a BAC contig covering the Pi2-2 locus was constructed. Conclusions: We identified a new allelic gene at the Pi2/9 locus and fine-mapped the gene within a 270-kb region. Our results provide essential information for the isolation of the Pi2-2 gene and tightly linked DNA markers for rice blast resistance breeding.
Rice is the staple food for more than half people of the
world, and the demand is increasing because of the
expanding rice-eating population, particularly in many
developing countries in Africa and Asia. However, rice
production is severely affected by various biotic and
abiotic stresses (Khush and Jena 2009). Rice blast, caused
by the fungal pathogen Magnaporthe oryzae, is one of
the major limitations, and usually causes 10-30% yield
loss in rice production when a rice blast epidemic occurs
(Talbot 2003; Skamnioti and Gurr 2009). Use of host
resistance is an effective and economical way to control
the blast disease (Khush and Jena 2009). To date, over
80 blast resistance genes have been identified, and are
distributed on 11 rice chromosomes except chromosome
3 (Liu et al. 2010; Yang et al. 2009). So far, 21 have been
cloned (Pib, Pita, Pi9, Pi2, Piz-t, Pid2, Pi36, Pi37, Pik-m,
Pit, Pi5, Pid3, pi21, Pb1, Pish, Pik, Pik-p, Pi54, Pia,
NLS1 and Pi25). Interestingly, most of them are
NBSLRR genes except Pi-d2 and pi21 (Wang et al. 1999;
Bryan et al. 2000; Qu et al. 2006; Zhou et al. 2006; Chen
et al. 2006; Liu et al. 2007; Lin et al. 2007; Ashikawa
et al. 2008; Hayashi and Yoshida. 2009; Lee et al. 2009;
Shang et al. 2009; Fukuoka et al. 2009; Hayashi et al.
2010; Takahashi et al. 2010; Zhai et al. 2011; Yuan et al.
2011; Sharma et al. 2005; Okuyama et al. 2011; Tang
et al. 2011; Chen et al. 2011). Pi-d2 encodes a
receptorlike kinase protein with a predicted extracellular domain
of a bulb-type mannose-specific binding lectin (B-lectin)
and an intracellular serine-threonine kinase domain
(Chen et al. 2006). Pi21 encodes a proline-rich protein
that includes a putative heavy metal-binding domain and
protein-protein interaction motifs. The resistant allele
pi21 carrying deletions in the proline-rich motif can
reduce blast infection rate (Fukuoka et al. 2009). Pik,
Pik-m and Pik-p are located at the locus of Pik on
chromosome 11, and interestingly, each of them requires
two independent NBS-LRR genes for the blast resistance
(Zhai et al. 2011; Ashikawa et al. 2008; Yuan et al. 2011).
Similarly, both Pi5 and Pia also require two NBS-LRR
members for their resistance function (Lee et al. 2009;
Okuyama et al. 2011).
At least eight blast resistance genes were identified
from the Pi2/9 locus, which is located on the short arm
and near the centromere of chromosome 6. Among
them, Pi9, Pi2 and Piz-t were successfully cloned
(Qu et al. 2006; Zhou et al. 2006). Pi26(t) (Wu et al.
2005), Pigm(t) (Deng et al. 2006), Piz(t) (Fjellstrom et al.
2006), Pi40(t) (Jeung et al. 2007) and Pi50(t) (Zhu et al.
2012) are in the process of being cloned by different
laboratories. Interestingly, most of them confer
broadspectrum resistance to diverse M. oryzae races or
isolates. The near isogenic line C101A51 carrying Pi2 is
resistant to 455 isolates collected from Philippines and
most of the 792 isolates from China (Chen et al. 1996,
1999). The Pi9-bearing line, 75-1-127, is resistant to 43
isolates collected from 13 different countries (Liu et al.
2002). Piz-t and Pigm from Toride and Gumei4,
respectively, are resistant to more than 90% of tested isolates
from China and Thailand (Shen et al. 2003). The
nearisogenic line containing Pi50(t) is incompatible to 97.7%
of the 523 isolates from different regions of China
(Zhu et al. 2012). However, the underlying mechanism of
broad-spectrum resistance of these genes is still not well
Jefferson, a long-grain tropical japonica cultivar grown
in the southern U.S., has retained its resistance to
blast since its first use in 1997 (McClung et al. 1997;
Skamnioti and Gurr 2009). It was reported that Jefferson
possesses three blast resistance genes, Piz(t), Pi-d(t) and
Pi-kh(t), based on its disease reactions (McClung et al.
1997). Our preliminary observation showed that Jefferson
was immune in the blast nursery of Taojiang County,
Hunan Province, China, which contained 11 major M.
oryzae races including ZC9, ZC11, ZE3, ZB29, ZG1,
ZB25, ZB31, ZB13, ZC7, ZA9, and ZF1 (unpublished).
To determine the genetic basis of broad-spectrum
resistance in Jefferson, we performed greenhouse
inoculations with individual isolates and genetic analysis using
an F2 population derived from a cross between Jefferson
and the susceptible cultivar CO39. We identified a
dominant R gene in Jefferson on chromosome 6 at the Pi2/9
locus, named Pi2-2. Allelism analysis indicated that Pi2-2
is tightly linked or allelic to Pi9. We constructed a BAC
contig in the genomic region and fine-mapped the gene
within a region approximately 270 kb. These data will
facilitate both the positional cloning of the R gene and
molecular breeding programs of rice blast resistance.
Resistance spectrum of Jefferson to 28 M. Oryzae isolates
To test the resistance spectrum of Jefferson, we inoculated
the cultivar with 28 M. oryzae isolates collected from six
countries, and the inoculation results are summarized in
Additional file 1: Table S1. Three known broad-spectrum
resistant cultivars, Tianye carrying Pi2-1 and Pi51
(Wang et al. 2012), XZ3150 carrying Pi47 and Pi48
(Huang et al. 2011), and 75-1-127 carrying Pi9 (Qu et al.
2006) were used as resistance controls and the highly
susceptible cultivar CO39 was used as a susceptible control.
Interestingly, Tianye was resistant to all the isolates and
Jefferson was only susceptible to the blast isolate RB11
from Japan. XZ3150 was susceptible to three isolates
(2361, RB6 and ROR1) and 75-1-127 was susceptible to
two isolates (ROR1 and X2007A-7). By contrast, the
susceptible control cultivar CO39 was susceptible to 27 of all
28 tested isolates. These results indicate that Jefferson
confers broad-spectrum resistance to M. oryzae.
Resistance to M. oryzae isolate 3182 is controlled by a
single dominant locus in Jefferson
The M. oryzae isolate 3182 from Hunan Province of
China was used for genetic analysis of the blast
resistance in Jefferson. We developed the F2 population
derived from a cross between Jefferson and CO39. All
the F1 plants were resistant to 3182 (32R:0S), indicating
that the dominant inheritance of the R gene in Jefferson.
The segregation of resistant and susceptible individuals
in the F2 population fitted a ratio of 3:1 (194R:60S,
2=0.257, 0.5<P<0.9 ), suggesting that the resistance
to 3182 is controlled by a single dominant R gene in
Jefferson. We designated this R gene in Jefferson as
Pi2-2 is tightly linked or allelic to Pi9 on chromosome 6
Previous research reported that there are three blast
resistance genes, Piz(t), Pi-d(t) and Pi-kh(t), in Jefferson
(McClung et al. 1997). Piz(t) is located on chromosome 6
near the Pi2/9 locus (Fjellstrom et al. 2006). Pi-d(t) and
Pi-kh(t) are located on chromosome 11. Therefore, we
selected 25 SSR markers around the Pi2/9 and Pi-kh loci
for linkage analysis. Twenty highly resistant and twenty
highly susceptible individuals from the F2 population of
the JeffersonCO39 cross were genotyped with the
polymorphic markers. No marker around the Pi-kh locus
cosegregated with the resistance to 3182. But two
polymorphic SSR markers around Pi2/9, RM7178 and
RM7311 (Table 1), were associated with the resistance,
indicating that Pi2-2 is located on chromosome 6.
Previous studies showed that Pi2 and Piz-t are tightly
linked to Pi9 (Zhou et al. 2006, 2007) and Piz(t) is allelic
or tightly linked to Piz-t (Hayashi et al. 2004). However,
the exact location of Piz(t) has not been determined yet.
To understand the linkage relationship between Pi2-2
and the R genes in the same region, we developed an F2
population from a cross between Jefferson and
Pi9carrying line 75-1-127 for allelism test. A total of 637 F2
individuals were inoculated with M. oryzae isolate
3182, which was incompatible to both Jefferson and
75-1-127, to observe the phenotype segregation. No
susceptible plant was found in 637 F2 individuals,
suggesting that Pi2-2 is tightly linked or allelic to the Pi9 gene.
Jefferson shows different resistance spectrum with the
cultivars carrying other R genes at the Pi2/9 locus
Previous research showed that the three cloned R genes
at Pi2/9 locus have different resistance spectra. 75-1-127
(Pi9) was susceptible to ROR1, a M. oryzae strain from
Korea. The isolate CHNOS60-2-3 from China could
distinguish C101A51 (Pi2) and Toride (Piz-t) resistance
specificities (Zhou et al. 2006). However, Jefferson was
immune to both of them (Table 2). In the inoculations
with 28 blast isolates (Additional file 1: Table S1),
Jefferson and 75-1-127 also have different resistance
spectra. In addition, another two isolates from Hunan
Province, China, showed different reactions to Jefferson
and Toride (Piz-t) or 5173 (Pi2). These results suggest
that Pi2-2 is a different R gene at the Pi2/9 locus.
However, isogenic lines with all the R genes at the Pi2/9
locus should be used in inoculations with different
isolates to confirm the conclusion.
Fine mapping and in silico mapping of the Pi2-2 gene
To finely map the Pi2-2 gene, another 14 SSR markers
were used, and four of them exhibited polymorphism
between the two parental lines (Table 1). A total of 583
susceptible individuals from the JeffersonCO39 F2
population were genotyped with these polymorphic markers.
Finally, the Pi2-2 gene was delimited by the closest
flanking markers RM19817 and AP5659-3, with one and three
recombinant events detected, respectively (Figure 1A).
The markers RM7178 and AP5659-5 co-segregated with
Pi2-2 in all 583 susceptible plants. The physical distance
between the closest flanking markers, RM19817 and
AP5659-3, was estimated to be about 270 kb according to
the Nipponbare genome information in this region. A
virtual contig map consisting of three overlapping Nipponbare
BAC clones (P0491D10, P0502B12 and P0649C11) was
constructed (Figure 1B). Annotation of the corresponding
genomic sequence indicates that there are three NBS-LRR
genes in this region, which are paralogs of the Pi9 gene
Construction of a BAC contig covering the Pi2-2 locus
For the cloning of the Pi2-2 gene, we constructed a
genomic BAC library of Jefferson with an average insert size of
140 kb. The tightly linked SSR markers spanning Pi2-2
were used for PCR screening of the BAC library pools. Six
positive clones were identified by four SSR markers and
were end-sequenced (Table 3). To confirm whether these
BAC clones overlapped, the end sequences were compared
with the corresponding sequences on chromosome 6 in the
Nipponbare genome. The results showed that BAC clones
BJ21-2-4-43 and BJ21-5-4-41 were the same. For clone
BJ24-1-13, only one end was anchored at Pi2/9 locus and no
homologous sequence was identified on this chromosome
compared with the Nipponbare genomic sequence for the
other end. The NIP (nitrite-induced protein) and PK
(protein kinase) genes are the 50 and 30 boundaries of the Pi2/9
locus, respectively, these are highly conserved in different
haplotypes (Zhou et al. 2007). Thus, the specific primer pairs
NIP-2F/R (NIP-2F, 50- TTTGGCGTGTCACATCGG-30;
Genomic position (bp)
Expected size (bp)
Table 1 Polymorphic SSR markers around the Pi2/9 locus used for linkage analysis
Forward primer (50-30)
Reverse primer (50-30)
a Previously reported markers in this region.
b SSR markers released by Gramene database (http://www.gramene.org/db/markers/).
Genomic position and expected PCR product size for each marker were determined based on the reference sequence of rice cultivar Nipponbare released by
International Rice Genome Sequencing Program (IRGSP).
Table 2 The disease reactions of Jefferson and donors of Pi2, Piz-t and Pi9
Zhu et al. 2012), and contains several NBS-LRR
type genes in both cultivated and wild rice lines
(Zhou et al. 2007; Dai et al. 2010). Three R genes at this
locus have been successfully isolated. The paralog
NBS2Pi9 is the Pi9 gene, and the paralogs NBS4-Pi2 and
NBS4-Piz-t are the Pi2 and Piz-t genes, respectively
(Zhou et al. 2006). In our study, three candidate NBS-LRR
genes (NBS-LRR1, NBS-LRR2 and NBS-LRR3) at the
Pi2/9 locus were identified for Pi2-2 according to
the sequence of Nipponbare genome. However, the
Nipponbare genome did not fully reflect the structure of
the Pi2-2 locus in Jefferson. Thus, sequence analysis of the
BAC clones of Jefferson covering Pi2-2 and
complementation test of candidate genes are necessary for determining
which NBS-LRR gene is Pi2-2.
Three blast resistance genes, Piz(t), Pi-d(t) and Pik-h(t),
were reported in Jefferson (McClung et al. 1997). Pi-d(t)
and Pik-h(t) are tightly linked on chromosome 11. Piz(t)
was originally reported in the U.S. rice cultivar Zenith
(Kiyosawa 1967), and has been widely introduced into
different cultivars by rice breeders (Conaway-Bormans
et al. 2003). Piz(t) was mapped on the short arm of
R and S denote resistant and susceptible reaction, respectively.
NIP-2R, 50-TGGAGCGGAGACAGAGTGG-30) and
50-CGTTCACTGACTTCCCTTTCCC30; PK-1R, 50-TCCGCATCGCCGTCTTCTG-30), designed
based on the NIP and PK sequences, were employed for
detecting the relative location of the five BAC clones at the
Pi2/9 locus. The PCR results showed BJ2-4-1-13 contained
the PK gene. A contig map consisting of 5 BAC clones
(BJ2-7-10-8, BJ21-2-3-10, BJ21-7-3-51, BJ21-2-4-43 and
BJ2-4-1-13) was constructed that covered both Pi2-2 and
the whole Pi2/9 locus in Jefferson (Figure 2).
Many plant disease resistance genes are located in
complex clusters in which multiple copies of closely related
sequences are formed through gene duplication and
uneven crossing over. Allelic genes in different genetic
backgrounds have evolved to carry diverse resistance
specificities due to exposure of these loci to different
pathogen populations. In rice, over half of the identified
blast resistance genes are clustered at different loci,
especially on chromosomes 6, 11 and 12. The Pi2/9 locus
is a region with at least eight R genes (Yang et al. 2009;
Table 3 PCR screening of positive BAC clones from the Jefferson BAC library
+, positive; , negative.
The physical locations were determined by comparing the end sequences of the BAC clones with the Nipponbare genomic sequence.
chromosome 6, close to the centromere, by several groups
using different cultivars (Hayashi et al. 2006; Fjellstrom
et al. 2006; Conaway-Bormans et al. 2003), but the exact
location has not been determined yet. Based on the fine
mapping results in this study, we speculate that Pi2-2 is
likely Piz(t). Our on-going cloning effort of the Pi2-2 gene
will provide us the answer in the near future.
This study demonstrated that the rice cultivar Jefferson
harbors the blast resistance gene Pi2-2 at the Pi2/9 locus
on chromosome 6. The gene was finely mapped to a 270
kb interval. A BAC contig covering Pi2-2 was constructed,
which provides essential foundation for the isolation of the
Seven rice cultivars, Jefferson, Tianye, XZ3150, 5173
(Pi2), Toride (Piz-t), 75-1-127 (Pi9) and CO39, were
used in this study. F1 and F2 populations from a cross
between Jefferson and highly susceptible cultivar CO39
were constructed for genetic analysis. The F2 population
derived from a cross between Jefferson and 75-1-127
was constructed for allelism tests.
Blast inoculation and disease evaluation
The 28 M. oryzae isolates used in the study are listed in
Additional file 1: Table S1. The collection sites and
providers are included in the table. Rice seedlings at 34
Figure 2 A BAC contig map spanning the Pi2-2 locus based on the end sequence. The physical position of each BAC clone is shown in
parentheses. NIP, nitrite-induced protein gene; PK, protein kinase gene.
leaf-stage were spray-inoculated with M. oryzae spore
suspensions (1.5105 spores/ml) and then kept in
darkness at 25C-27C and over 90% relative humidity for
24 h. The inoculated plants were subsequently kept
under a 12/12 (day/night) photoperiod at the same
temperature and relative humidity. Disease reaction
evaluation was carried out 7 days after inoculation
according to the 05 scoring system described by
(Bonman et al. 1986).
Genetic and allelism analysis
The JeffersonCO39 F2 population was inoculated
with the M. oryzae isolate 3182, which is avirulent to
Jefferson and virulent to CO39. 3182, which is also
avirulent to 75-1-127, was employed to inoculate the
Jefferson75-1-127 F2 population for allelism analysis.
Genotyping and genetic mapping
A total of 39 SSR markers spanning the Pi2/9 and Pik
loci were used for the polymorphism survey between
Jefferson and CO39. Six polymorphic SSR markers
spanning the Pi2/9 locus were used for preliminary and fine
mapping of the R gene in Jefferson (Table 1). The
genomic DNA of 20 highly resistant and 20 susceptible F2
individuals, which were phenotypically confirmed in the
F3 generation, were extracted from leaves for segregation
analysis (Saghai-Maroof et al. 1984). All PCRs began
with a denaturation step of 94C/4 min, followed by 35
cycles of (A) 94C/30 sec, 55C/30 sec, 72C/30 sec, with
a final extension step of 72C/7 min. Linkage analysis
was performed using the MAPMAKER/V3.0 using all
highly susceptible individuals.
Physical mapping of the Pi2-2 locus
The physical positions of the markers tightly linked to
Pi2-2 locus were determined based on the genome of
Nipponbare using the BLAST program on Gramene
(http://www.gramene.org/Multi/blastview) (Jaiswal et al.
2006). The genomic sequences flanked by the markers
RM19817 and AP5659-3 were annotated using the Rice
Genome Annotation Project (http://rice.plantbiology.
msu.edu/) (Ouyang et al. 2007) and Rice Genome
Automated Annotation System (http://ricegaas.dna.affrc.go.jp/)
(Sakata et al. 2002).
Construction of the BAC library of Jefferson
The genomic BAC library of Jefferson was constructed
using the method described by (Luo and Wing 2003).
The markers tightly linked to Pi2-2 were used for
screening of positive clones from the BAC pools. The contig
map spanning the Pi2-2 locus was constructed based on
the end sequencing results of the positive BAC clones.
Additional file 1: Table S1. Disease reaction of Jefferson and other 4
cultivars to 28 M. oryzaeisolates collected from different regions.
The authors declare that they have no competing interests.
NJ and ZL contributed equally to this work. NJ carried out resistance
spectrum analysis, allelism analysis, genetic analysis, molecular mapping,
construction of the BAC contig map and wrote the manuscript; ZL carried
out molecular mapping, construction of the BAC contig map; JW carried out
spectrum analysis and allelism analysis; YW participated in molecular
mapping. LW carried out resistance spectrum analysis; SW carried out
resistance spectrum analysis; DW carried out resistance spectrum analysis; TW
carried out resistance spectrum analysis; YL participated in molecular
mapping; PS carried out carried out genetic analysis; JL participated in the
design of the study; LD participated in the design of the study; ZW
participated in experimental designing; CW constructed the BAC library of
Jefferson; ML participated in the design of the study and constructed the
BAC library of Jefferson; XL designed the research and wrote the manuscript;
GW designed the research and wrote the manuscript. All authors read and
approved the final manuscript.
This work was financially supported by the National Natural Science
Foundation of China (31171526 and 30571063), Hunan Provincial Natural
Science Foundation (06JJ10006) and the US National Science Foundation to
GLW (IOS #1120949).