High-resolution mapping of two rice brown planthopper resistance genes, Bph20(t) and Bph21(t), originating from Oryza minuta

Abstract

Brown planthopper (BPH) is one of the most destructive insect pests of rice. Wild species of rice are a valuable source of
resistance genes for developing resistant cultivars. A molecular marker-based genetic analysis of BPH resistance was conducted
using an F2 population derived from a cross between an introgression line, ‘IR71033-121-15’, from Oryza minuta (Accession number 101141) and a susceptible Korean japonica variety, ‘Junambyeo’. Resistance to BPH (biotype 1) was evaluated using 190 F3 families. Two major quantitative trait loci (QTLs) and two significant digenic epistatic interactions between marker intervals
were identified for BPH resistance. One QTL was mapped to 193.4-kb region located on the short arm of chromosome 4, and the
other QTL was mapped to a 194.0-kb region on the long arm of chromosome 12. The two QTLs additively increased the resistance
to BPH. Markers co-segregating with the two resistance QTLs were developed at each locus. Comparing the physical map positions
of the two QTLs with previously reported BPH resistance genes, we conclude that these major QTLs are new BPH resistance loci
and have designated them as Bph20(t) on chromosome 4 and Bph21(t) on chromosome 12. This is the first report of BPH resistance genes from the wild species O. minuta. These two new genes and markers reported here will be useful to rice breeding programs interested in new sources of BPH
resistance.

Full text

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Theor Appl Genet
DOI 10.1007/s00122-009-1125-z
123
ORIGINAL PAPER
High-resolution mapping of two rice brown planthopper
resistance genes, Bph20(t) and Bph21(t), originating
from Oryza minuta
Md Lutfor Rahman · Wenzhu Jiang · Sang Ho Chu · Yongli Qiao · Tae-Ho Ham ·
Mi-Ok Woo · Joohyun Lee · M. Sakina Khanam · Joong-Hyoun Chin ·
Ji-Ung Jeung · D. S. Brar · K. K. Jena · Hee-Jong Koh
Received: 28 December 2008 / Accepted: 21 July 2009
© Springer-Verlag 2009
Abstract
destructive insect pests of rice. Wild species of rice are a
valuable source of resistance genes for developing resistant
cultivars. A molecular marker-based genetic analysis of
BPH resistance was conducted using an F2 population
derived from a cross between an introgression line,
‘IR71033-121-15’, from Oryza minuta (Accession number
101141) and a susceptible Korean japonica variety,
‘Junambyeo’. Resistance to BPH (biotype 1) was evaluated
using 190 F3 families. Two major quantitative trait loci
(QTLs) and two signiWcant digenic epistatic interactions
Brown planthopper (BPH) is one of the most
between marker intervals were identiWed for BPH resis-
tance. One QTL was mapped to 193.4-kb region located on
the short arm of chromosome 4, and the other QTL was
mapped to a 194.0-kb region on the long arm of chromo-
some 12. The two QTLs additively increased the resistance
to BPH. Markers co-segregating with the two resistance
QTLs were developed at each locus. Comparing the physi-
cal map positions of the two QTLs with previously reported
BPH resistance genes, we conclude that these major QTLs
are new BPH resistance loci and have designated them as
Bph20(t) on chromosome 4 and Bph21(t) on chromosome
12. This is the Wrst report of BPH resistance genes from the
wild species O. minuta. These two new genes and markers
reported here will be useful to rice breeding programs inter-
ested in new sources of BPH resistance.
Introduction
Brown planthopper (BPH), Nilaparvata lugens Stål
(Homoptera: Delphacidae), is one of the most destructive
insect pests of rice (Oryza sativa L.), especially of temper-
ate japonica rice cultivars where few resistance genes
against BPH have been incorporated (Jena et al. 2006).
BPH extracts phloem saps of rice plants using its stylet-
type mouthparts, resulting in a severe damage symptom
known as ‘hopper-burn’. BPH also transmits rice grassy
stunt virus and ragged stunt virus as vectors (Rivera et al.
1966; Heinrichs 1979). On the basis of diVerential reac-
tions, BPH populations in diVerent countries have been
categorized into four biotypes (Khush et al. 1985). The
original populations in East and Southeast Asia belonged to
biotype 1. Biotype 2 originated in Phillippines, Indonesia
and Vietnam (Khush 1979) and is the dominant biotype in
these countries. Biotype 3 was produced in the laboratory at
Communicated by T. Tai.
M. L. Rahman · W. Jiang · S. H. Chu · Y. Qiao · T.-H. Ham ·
M.-O. Woo · J. Lee · M. S. Khanam · H.-J. Koh (&)
Department of Plant Science, Plant Genomics and Breeding
Institute, and Research Institute of Agriculture and Life Sciences,
Seoul National University, Seoul 151-921, Korea
e-mail: heejkoh@snu.ac.kr
M. S. Khanam
Department of Crop Physiology,
Bangladesh Institute of Nuclear Agriculture,
Mymensingh 2200, Bangladesh
J.-H. Chin · D. S. Brar
Plant Breeding, Genetics and Biotechnology Division,
International Rice Research Institute, Los Baños,
DAPO BOX 7777, Manila, Laguna, Philippines
K. K. Jena
Plant Breeding, Genetics and Biotechnology Division,
International Rice Research Institute, C/o IRRI-Korea OYce,
National Institute of Crop Science, RDA, Suwon 441-857, Korea
J.-U. Jeung
Rice Research Division, National Institute of Crop Science,
RDA, Suwon 441-857, Korea

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Theor Appl Genet
123
the International Rice Research Institute (IRRI) (Pathak
and Khush 1979) and in Japan (Ikeda and Vaughan 1991).
Biotype 4 is found only in South Asia (Khush 1984). In
Southeast Asia, the BPH populations shifted from biotype 1
to biotype 2 in the 1970s (Feuer 1976; Mochida et al. 1977;
Stapley et al. 1979), and at present comprise mostly a com-
plex of biotypes 2 and 3 (Medrano and Heinrichs 1985;
Sogawa et al. 1987; Huynh and Nhung 1988). On the other
hand, Chelliah and Bharathi (1993) categorized BPH popu-
lations into Wve biotypes on the basis of their diVerential
reactions to a set of reference cultivars. Of the biotypes (1,
2, and 3) known to be present in Korea, biotype 1 is the
most prevalent (Park and Song 1988). Conventional meth-
ods of controlling BPH are highly dependent on spraying
poisonous chemicals, which is expensive in terms of labor,
money, and the environment. In addition, the occurrence of
resurgence, a phenomenon of pest population increase after
application of insecticides (Heinrichs et al. 1982), is prob-
lematic. Thus introduction of resistant cultivars is beneWcial
economically and environmentally (Huang et al. 2001).
Incorporating resistance gene(s) from wild species into
cultivated species can be an alternative approach to develop
BPH resistance in susceptible commercial cultivars. In gen-
eral, wild species are poor in important agronomic traits
such as yield, plant type, grain type, eating quality, seed
shattering habit, etc. However, the wild species of Oryza
are regarded as a treasure trove of novel genes for resis-
tance to disease or insect pests and tolerance against envi-
ronmental stresses. Since few useful genes from wild
germplasm accessions have been explored, there is still
great potential for exploring novel genes. Several wild spe-
cies including O. minuta, O. latifolia, O. nivara, O. oYci-
nalis and O. punctata were reported to possess resistance
genes to diVerent BPH biotypes (Wu et al. 1986). Nine
resistance loci among a total of 19 BPH resistance loci
reported so far in rice have been identiWed from wild spe-
cies. They are Bph10 on the long arm of chromosome 12
from O. australiensis (Ishii et al. 1994), Bph12(t) on the
short arm of chromosome 4 from O. latifolia (Yang et al.
2002), Bph13(t) on the long arm of chromosome 2 from
O. eichingeri (Liu et al. 2001), another Bph13(t) against
BPH biotype 4 on the short arm of chromosome 3 from
O. oYcinalis (Renganayaki et al. 2002), Qbp1 and Qbp2
(later named as Bph14 and Bph15, respectively) on the long
arm of chromosome 3 and the short arm of chromosome 4,
respectively, from O. oYcinalis (Huang et al. 2001),
Bph18(t) on the long arm of chromosome 12 from O. aus-
traliensis (Jena et al. 2006), and bph11(t) and bph12(t) on
the long arm of chromosome 3 and chromosome 4 respec-
tively, from O. oYcinalis (Hirabayashi et al. 1999).
O. minuta (2n = 48, BBCC genome, Acc No.101141), an
allotetraploid wild species, belonging to the O. oYcinalis
complex, is endemic to Philippines and Papua New Guinea.
It is known to have useful genes for resistance to bacterial
blight (BLB), rice blast, and BPH (Brar and Khush 1997).
However, few studies have been conducted to transfer the
resistance genes from O. minuta to commercial rice culti-
vars. Amante et al. (1998) evaluated the resistance of
advanced progenies introgressed from O. minuta for resis-
tance against BLB and rice blast disease. The introgression
line IR71033-121-15 derived from the cross of O. sativa/
O. minuta showed resistance to BPH biotypes of Korea
(Jena KK, unpublished). However, molecular mapping of
these resistance gene(s) has not been reported.
The present study was conducted to identify the BPH
resistance genes in IR71033-121-15 and to develop the
markers for use in breeding BPH resistance in rice.
Materials and methods
Plant materials
The introgression line, IR71033-121-15, derived from an
interspeciWc cross between IR31917-45-3-2 and a wild spe-
cies O. minuta (Acc. No. 101141) showed strong resistance
to the Korean BPH biotype 1. IR71033-121-15 and
Junambyeo (a Korean japonica cultivar susceptible to BPH)
were used as parents to develop an F2 population of 190
plants for genetic analysis. The 190 F3 lines harvested from
each of the F2 plants were bioassayed for BPH resistance.
IR31917-45-3-2 (progenitor of IR71033-121-15) and two
Tongil-type rice cultivars, Taebakbyeo and Andabyeo,
were used as check cultivars in the bioassays. IR31917-45-
3-2, Taebakbyeo, and Junambyeo are susceptible to Korean
BPH whereas IR71033-121-15 and Andabyeo are resistant.
Seeds of IR71033-121-15, IR31917-45-3-2, and O. minuta
were obtained from the Plant Breeding and Genetics and
Biotechnology Division of IRRI, Los Baños, Philippines.
Seeds of Junambyeo, Taebakbyeo and Andabyeo were
obtained from Rice Research Division of National Institute
of Crop Science (NICS), Rural Development Administra-
tion (RDA), Korea.
Bioassay for BPH resistance
A pure BPH population developed from Korean BPH bio-
type 1 was used for bioassay of parents and F2/F3 popula-
tion. Bioassays were conducted at the greenhouse facility of
the Rice Research Division of NICS, RDA. The bioassay
was performed with a modiWed bulk seedling test following
the method of Pathak et al. (1969). Fifteen seedlings from
each of 190 F3 lines were planted in a row per line with
three replications. Junambyeo and Taebakbyeo were used
as susceptible (S) checks, and IR71033-121-15-2 and
Andabyeo were used as resistant (R) checks. Seedlings at

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Theor Appl Genet
123
the two and a half leaf stage were infested with second or
third-instar nymphs at a density of 10–12 nymphs per seed-
ling. The reaction against the BPH was scored following
the guidelines of Standard Evaluation Systems for Rice
(IRRI, 1988): 0, no damage; 1, very slight damage; 3, Wrst
and second leaves of most plants partially yellowing; 5,
pronounced yellowing and stunting or about 10 to 25% of
the plants wilting; 7, more than half of the plants wilting or
dead and remaining plants severely stunted or dying; 9, all
plants dead.
PCR ampliWcation and marker detection
Plant DNA was extracted from the frozen leaves of rice
plants using the CTAB method (Murray and Thompson
1980). For PCR ampliWcation of markers, each 20 ?L reac-
tion mixture contained 50 ng DNA, 5 pmol of each primer,
10£ PCR buVer [100 mM Tris (pH 8.3), 500 mM KCl,
15 mM MgCl2, 2 ?g gelatin], 250 ?M of each dNTP and
0.5 U of Taq polymerase. PCR was performed in a PTC-
100 Programmable Thermal Controller thermocycler (MJ
Research Inc, Waltham, MA). The thermocycler proWle
was: 5 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at
48°C (for STS) or 55°C (for SSR), and 2 min at 72°C, with
a Wnal extension of 5 min at 72°C. AmpliWed PCR products
were resolved by electrophoresis in 3% agarose gels with
ethidium bromide staining or 8% polyacrylamide denatur-
ing gels with silver-staining for SSR markers (Panaud et al.
1996).
Construction of a framework map, detection of QTLs
for BPH resistance and high-resolution mapping
Linkage analysis was conducted using MAPMAKER ver-
sion 3.0 software (Lander et al. 1987). Map distances were
estimated by the Kosambi function (Kosambi 1944). The
linkage map in this study was basically the same as previ-
ously reported (Rahman et al. 2007). In this study, 46 more
F2 plants were added to construct a framework map. The
M-QTL and E-QTLs between random marker intervals
were determined using QTLMapper version 2.0 with the
mixed linear model approach (Wang et al. 1999). After
detecting QTL positions for BPH resistance based on LOD
and R2 values, Wne mapping of the resistance loci was per-
formed with SSR and STS markers. Graphical data of the
recombinants between markers Xanking the target regions
were analyzed to clarify QTL positions, and introgressed
segments from O. minuta were checked at the QTL regions
for conWrmation of the QTLs. SSR markers located within
the two Xanking markers of the resistance QTL regions
were adopted from the public database released by the
International Rice Microsatellite Initiative (IRMI, http://
www.gramene.org/microsat), and PCR-based STS markers
were developed according to the sequence information
which is available at http://www.ncbi.nlm.nih.gov/ (for
indica) and at http://www.rgp.dna.affrc.go.jp/ (for japon-
ica) as described in Chin et al. (2007). For developing new
SSR markers, the putative simple sequence repeat motifs
were searched and identiWed using the Simple Sequence
Repeat IdentiWcation Tool (SSRIT, http://www.gram-
ene.org/db/searches/ssrtool) and for new STS markers, the
putative sequences which were highly divergent between
the two reference genomes were targeted. All PCR primers
were designed using Primer3 version 4.0 (http://frodo.wi.
mit.edu/). The markers used for Wne mapping of the target
regions are listed in Table 1.
Physical map construction
The physical map of the target QTLs was constructed by
bioinformatics analysis using bacterial artiWcial chromo-
some (BAC) and P1-derived artiWcial chromosome (PAC)
clones of cv. Nipponbare released by the International Rice
Genome Sequencing project (IRGSP, http://rgp.dna.affrc.
go.jp/IRGSP/index.html). The clones were anchored with
the target gene-linked markers and then alignment of
sequences was carried out using pairwise BLAST (http://
www.ncbi.nlm.nih.gov/blast/bl2seq/b12.html).
Results
Inheritance of BPH resistance in “IR71033-121-15”
The introgression line IR71033-121-15 expressed strong
resistance to the Korean BPH biotype 1, while the recurrent
parent, Junambyeo, was completely susceptible to BPH
(Fig. 1). The F1 plants from the cross between Junambyeo
and IR71033-121-15 showed partial resistance to BPH,
indicating that BPH resistance of IR71033-121-15 was con-
trolled by more than one dominant gene and the dominance
was not complete. The resistance scores of the 190 F3 lines
infested with BPH ranged from 0.0 to 9.0 and showed a
continuous variation with two peaks around 1.5 and 3.5 in
the segregation curve (Fig. 1), demonstrating the involve-
ment of major genes or QTLs controlling the segregation of
resistance.
Linkage map construction and identiWcation
of QTLs for BPH resistance
A linkage map was constructed by genotyping 190 F2 indi-
viduals using 143 markers. Using the QTLMapper version
2.0, two chromosomal loci for the resistance were identiWed
on the short arm of chromosome 4 delimited by MS10 and
RM5953 (3.5 cM distance) and the long arm of chromosome

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Theor Appl Genet
123
12, delimited by RM3726 and RM5479 (4.1 cM distance)
(Table 2; Figs. 2, 3). At both loci, alleles from the resistant
parent IR71033-121-15 signiWcantly increased BPH resis-
tance. These two QTLs were tentatively designated as QTL-
4 on chromosome 4 and QTL-12 on chromosome 12. The
R2 values of QTL-4 and QTL-12 were fairly high represent-
ing 26.6% and 14.5% of phenotypic variation, respectively,
and the summed R2 of the two QTLs was 41.1% (Table 2).
In addition, a total of two signiWcant epistatic interactions
between random marker intervals were identiWed which
included a signiWcant interaction between the two QTLs
(Table 2). Total phenotypic variation explained by these
QTLs and signiWcant interactions was 47.2%. However,
there was no dominance eVect in the two major QTLs and
other marker loci, explaining the intermediate resistance
phenotype of F1 plants shown in Fig. 1. The allele eVect of
each major QTL on BPH resistance was greater in QTL-4
than in QTL-12.
Position of QTLs on the physical map
All the anchor markers used for Wne mapping of the two
resistance loci were landed on the reference sequences of
cv. Nipponbare by bioinformatics analysis using a software
tool BLASTN. The 1.31 Mb virtual contig map of chromo-
some 4 composed of 14 overlapping BAC/PAC clones was
constructed to locate the physical regions for markers
MS10 and RM5953 and thus delimit the QTL-4 locus. Sim-
ilarly, the 1.13 Mb virtual contig map of chromosome 12
composed of 13 overlapping BAC/PAC clones was con-
structed to locate the physical regions for markers RM3726
and RM5479 and delimit the QTL-12 locus. Additional
markers were developed using publicly available sequence
databases aided by bioinformatics analysis to narrow down
the physical distance between the two Xanking markers
(Table 1). Finally, two saturated genetic maps and subse-
quent contig maps were constructed for QTL-4 on the short
arm of chromosome 4 (Fig. 2) and QTL-12 on the long arm
of chromosome 12 (Fig. 3).
Table 1 Summary of PCR-based markers developed for two speciWc QTLs
aC61009, R288 (http://rgp.dna.affrc.go.jp/publicdata/caps/chr4.html); MS5, MS10 (Yang et al. 2004); B- and S- markers were developed in this
study
Markera
ForwardReverseExpected
amplicon size (bp)
C61009
MS5
MS10
B40
R288
B41
B42
B43
S4019A
S4019B
B44
S12091A
S12091B
S12094A
B120
S12094B
B121
B122
5?-ggccagcaaggtgtagtaag
5?-ttgtgggtcctcatctcctc-3?
5?-caatacgagaagcccctcac-3?
5?-caataccggatatcttgactcc
5?-cctcatcgccagcaaga
5?-gctggtcttgaccaacgatt
5?-ctgggctgcatacctagctt
5?-actccaattggttcctgtgg-3?
5?-ccaccgtttgatcattcatct-3?
5?-ttctcggtttcttcgggtta-3?
5?-tctcaaaccggctctaccag-3?
5?-tggggttaaatgttgcctct
5?-ggctttcttcctcacactgc
5?-tgcaatgctgtggcaataat
5?-ttgaaaactacggggtgagg
5?-tgcaacatggtaagcgattt-3?
5?-cgtcgtacattctgaaatggag-3?
5?-tcgtcaccaaacagcactaca-3?
5?-acaaaccccagcaccctaag
5?-tgacaacttgtgcaagatcaaa-3?
5?-ctgaaggaacacgcggtagt-3?
5?-cgaccacgctgcctatattc
5?-atagcagacttagcagcact
5?-gaagttgccggagtcgag
5?-agggtgtgttcggtagatgg
5?-tggactaaaagccgatgagc-3?
5?-aacaaatttgagggcaaaaa-3?
5?-atctttggcttgctccacac-3?
5?-ttactggtatggcaggagca-3?
5?-catatgtgggagcagactagca
5?-cgaggacgagatgagacga
5?-tgcaatgctgtggcaataat
5?-tacccgcaggatgagatac
5?-gcaaggtccttttcatggttt-3?
5?-ggacatggagatggtggaga-3?
5?-gtgacgactccccaattgtc-3?
205
215
168
102
2,453
154
117
764
231
198
207
167
204
116
1,496
234
101
214
Fig. 1 Frequency distribution of the brown planthopper (BPH) resis-
tance scores among F3 families from the cross Junambyeo/IR71033-
121-15

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Theor Appl Genet
123
For QTL-4, the markers B43, S4019A, and S4019B
derived from OSJBa0028M15, Xanked by B42 and B44,
co-segregated with the BPH resistance phenotypes.
Association of marker genotypes with phenotypes (BPH
resistance) of F3 lines that contained the crossover event
on the QTL-4 region was analyzed (Fig. 2b). Based on
comparisons of the genotypes at both QTL-4 and QTL-
12 together with the phenotypes, the lines 73 and 91
delimited the left margin of the QTL-4 position, and sim-
ilarly, the lines 76 and 115 delimited the right margin of
the QTL-4 position. From these results, it was concluded
that QTL-4 resides between B42 and B44, which spans
1.2 cM and corresponds to a 193.4-kb region in the ref-
erence physical map (http://www.rgp.dna.affrc.go.jp/)
(Fig. 2a).
For QTL-12, the markers B120, S12094B and B121
derived from OJ1310_CO3, Xanked by S12094A and
B122, co-segregated with BPH resistance. Using the same
procedure as for QTL-4, the association of marker geno-
types with BPH resistance of F3 lines containing the cross-
over event in the QTL-12 region was analyzed (Fig. 3b).
The lines 13 and 28 delimited the left margin of the QTL-12
position, and the lines 43 and 99 delimited the right margin
of the QTL-12 position. Therefore, it was concluded that
QTL-12 resides between 12094A and B122, which spans
1.0 cM and corresponds to a 194.0 kb region in the refer-
ence physical map (Fig. 3a).
Evidence of O. minuta chromosome segment integration
in two putative QTLs
To conWrm that the two chromosomal regions containing
resistance QTLs were introgressed from the wild species
O. minuta, the alleles of markers B43, S4019A, S4019B,
and B44 on chromosome 4, and B120, S12094B, B121, and
B122 on chromosome 12 were compared between
IR71033-121-15 and O. minuta. In the QTL-4 region, the
same PCR amplicon (¸200 bp) was produced for markers
S4019A and S4019B in both O. minuta and IR71033,
which was diVerent from the amplicon generated in the
recurrent parent IR31917-45-3-2 and Junambyeo (Fig. 4a).
However, B43 at the upper position and B44 at the lower
position were negative for introgression. Similarly, analysis
of markers in the QTL-12 region revealed that the same
PCR amplicons were produced for markers S12094B and
B121 in IR71033-121-15 and O. minuta, which were diVer-
ent from the amplicons generated in the recurrent parent
IR31917-45-3-2 and Junambyeo (Fig. 4b). However, B120
at the upper position and B122 at the lower position were
not positive for introgression. These results indicate that the
two resistance QTLs correspond to the introgression of
O. minuta DNA into chromosome 4 (S4019A to S4019B)
and 12 (S12094B to B121) of IR31917-45-3-2.
Table 2 Major QTLs and signiWcant digenic epistatic interactions identiWed for BPH resistance in F2:3 from a cross between Junambyeo and IR71033-121-15
h2 is the percentages of the phenotypic variations explained by the QTL
a* indicate p < 0.05; ns = non signiWcant
bCh-i and Ch-j represent the chromosome number to which the marker interval belongs
caaij, adij, daij, and ddij are the eVects of additive by additive interaction, additive by dominant interaction, dominant by additive interaction, and dominant by dominant interaction between the
marker interval i and j
QTL
Chromosome
Marker interval
LOD
Add.(a)
Dom.(d)
a/d
h2 (%)
Major QTLs
QTL4
4
MS10-RM5953
16.69
1.94*a
¡0.14 ns
¡0.07
26.6
QTL12
12
RM3726-RM5479
11.28
1.09*
¡0.32 ns
¡0.29
14.5
Ch-ib
Marker interval
Ch-jb
Marker interval
LOD
aaijc
adij
daij
ddij
h2 (%)
Digenic epistatic interactions
4
MS10-RM5953
12
RM3726-RM5479
17.23
0.56*
¡0.14 ns
0.17 ns
¡0.33 ns
2.6
8
S80036-S80016
12
S120712-RM3726
9.12
0.71*
¡0.36 ns
0.27 ns
¡0.21 ns
3.5

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Theor Appl Genet
123
Discussion
Nineteen major BPH resistance loci have been reported in
indica cultivars along with four wild species, O. australien-
sis, O. eichingeri, O. latifolia, and O. oYcinalis. Of these,
17 resistance loci (Bph1, bph2, Bph3, bph4, Bph6, Bph9,
Bph10, bph11, bph12(t), Bph12, Bph13(t), Bph13, Bph14,
Bph15, Bph17, Bph18, and bph19) have been assigned to
rice chromosomes (Ikeda 1985; Ishii et al. 1994; Hirabay-
ashi and Ogawa 1995; Hirabayashi et al. 1999; Murata
et al. 1998, 2001; Kawaguchi et al. 2001; Liu et al. 2001;
Jena et al. 2002, 2006; Renganayaki et al. 2002; Sharma
et al. 2003, 2004; Yang et al. 2004; Kim and Sohn 2005;
Sun et al. 2005; Chen et al. 2006; Jairin et al. 2007). QTL
studies involving the BPH-resistant cultivars IR64, Kasa-
lath, DV85, Teqing, Col.5, and B5 introgression line cre-
ated through the introgression of wild rice O. oYcinalis
have also been carried out (Alam and Cohen 1998; Huang
et al. 2001; Su et al. 2002, 2005; Xu et al. 2002; Ren et al.
2004; Soundararajan et al. 2004). Candidate genes for BPH
resistance have been reported for the indica cv. Samgangb-
yeo (Park et al. 2007).
The main goal of this study was to identify new BPH resis-
tance loci originating from the wild species, O. minuta, which
has the BBCC genomes. Our analysis resulted in the identiW-
cation of two major BPH resistance QTLs on chromosomes 4
and 12. These QTLs together explained over 40% of the
observed phenotypic variance. Studies involving BPH resis-
tance introgressed from other wild species have also resulted
in the identiWcation of loci mapping to chromosome 4. Previ-
ously, Huang et al. (2001) reported that two QTLs for BPH
resistance, introgressed from O. oYcinalis (CC), were located
on the short arm of chromosome 4 and long arm of chromo-
some 3. Later, Yang et al. (2004) identiWed one BPH resis-
tance locus on chromosome 4 (Bph15) using the same
introgressed line from O. oYcinalis. Yang et al. (2002) also
reported that a BPH-resistant gene, Bph12(t) from O. latifolia
(CCDD), was located on the short arm of chromosome 4. Of
Fig. 2 a Genetic and physical
map of the Bph20(t) locus on the
short arm of chromosome 4.
Positions of QTLs (blue vertical
bars) previously reported near
the Bph20(t) locus were esti-
mated based on the sequence
analysis of markers Xanking the
QTLs using Gramene DB
(http://www.gramene.org/):
Bph12 from O. latifolia (Yang
et al. 2002); Bph15 from O. oY-
cinalis (Huang et al. 2001; Yang
et al. 2004); Bph17 from Rathu
Heenati (Sun et al. 2005). b Phe-
notype (BPH resistance) and
graphical genotype of selected
F3 lines showing crossovers near
by the Bph20(t) locus. I IR71033
allele, J Junambyeo allele, H
heterozygous

Page 7

Theor Appl Genet
123
the Bph genes that have been mapped to chromosome 4,
Bph17 from the Sri Lankan cultivar Rathu Heenati is the clos-
est to QTL-4 described in our study (Fig. 2a). Bph17 is report-
edly located between two SSR markers RM8213 and
RM5953 with map distances of 3.6 cM and 3.2 cM, respec-
tively (Sun et al. 2005). Part of this region covers the QTL-4
locus; however, the fact that QTL-4 originates from the wild
species O. minuta suggests Bph17 is a diVerent gene. Further
studies such as the cloning of these genes will clarify their
relationship.
For the long arm of chromosome 12, six resistance loci
have been reported: Bph1 from ‘Mudgo’, ‘MTU15’,
Fig. 3 a Genetic and physical
map of the Bph21(t) locus on the
long arm of chromosome 12.
Positions of QTLs (blue vertical
bars) previously reported near
the Bph21(t) locus were esti-
mated based on the sequence
analysis of markers Xanking the
QTLs using Gramene DB:
Bph10 from O. australiensis
(Ishii et al. 1994), Bph18(t) from
O. australiensis (Jena et al.
2006). b Phenotype (BPH resis-
tance) and graphical genotype of
selected F3 lines showing cross-
overs near by the Bph21(t) locus.
I IR71033 allele, J Junambyeo
allele, H heterozygous
Fig. 4 Introgression test of O.
minuta segments into IR71033-
121-15 a at the QTL4 locus on
chromosome 4, and b at the
QTL-12 locus on chromosome
12. S Size marker, I IR71033-
121-15, M O. minuta (accession
# 101141), J Junambyeo, R
IR31917-45-3-2. An arrow indi-
cates the B120 marker

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Theor Appl Genet
123
‘Co22’, ‘MGL2’, ‘Samgangbyeo’ (Kim and Sohn 2005)
and ‘Gayabyeo’ (Jeon et al. 1999); bph2 from ‘Karsamba
Red, ASD7’ (Murata et al. 1998); Bph9 from ‘Pokkali’
(Murata et al. 2001) and Kaharamana (Su et al. 2006);
Bph10 (Ishii et al. 1994),and Bph18(t) (Jena et al. 2006)
from O. australiensis. Of these loci, Bph10 and Bph18(t)
are located nearby QTL-12. However, as with QTL-4, com-
parative analysis of marker loci revealed that these resis-
tance genes actually map apart from QTL-12 (Fig. 3a).
Given that the two major QTLs in this study diVer in chro-
mosomal location and/or origin (O. minuta) from BPH
resistance loci reported previously, we have designated
these loci as Bph20(t) and Bph21(t) on chromosome 4 and
12, respectively. This is the Wrst report on BPH resistance
loci introduced from a BBCC genome wild species.
It is notable that BPH resistance genes are clustered in
the same regions of chromosome 4 and 12 despite their
diVerent origins. Similar Wndings were also observed in the
case of BPH resistance loci Bph13(t) (Renganayaki et al.
2002) and the recessive bph19(t) clustered on the short arm
of chromosome 3 (Chen et al. 2006), and Bph10 (Ishii et al.
1994) with Bph18(t) (Jena et al. 2006) on the long arm of
chromosome 12.
Besides the two major QTLs, signiWcant epistatic inter-
actions between two random markers were found to play a
certain role in the expression of resistance to BPH
(Table 2). This suggests that epistatic interactions as well as
major QTLs should be taken into consideration for breed-
ing BPH-resistant cultivars through MAS, although this
might complicate the MAS process (Qiao et al. 2008). The
existence of an additive eVect for the two major QTLs
reported here indicates that lines with stronger BPH resis-
tance can be developed by incorporating multiple QTLs.
We are now developing resistant japonica lines by back-
crossing the resistant progenies with the recurrent parent
Junambyeo using MAS for Bph20(t) and Bph21(t).
Of the Wve biotypes reported (Chelliah and Bharathi
1993), resistance genes have been identiWed against four of
them (Panda and Khush 1995). Bph1 confers resistance
against biotypes 1 and 3, bph2 is closely linked with Bph1
and provides resistance against biotypes 1 and 2, Bph3 and
bph4, which are closely linked, confer resistance against
four biotypes, and Bph5, Bph6, and Bph7 provide resistance
against only biotype 4. The relationship between other
resistance genes and corresponding BPH biotypes has not
been comprehensively documented. Considering the poten-
tial changes of BPH biotypes, due to the extensive cultiva-
tion of high yielding rice cultivars and diVerent
environmental factors, new resistance genes need to be
identiWed to ensure the durability of host resistance. Wild
species of rice are important genetic resources for breeding
insect resistant cultivars. The parental line, IR71033-121-
15, used in this study carries two new resistance genes,
Bph20(t) and Bph21(t), introgressed from O. minuta, and is
expected to be a valuable source of BPH resistance.
Acknowledgments
CG3111) from the Crop Functional Genomics Center of the 21st
Century Frontier Research Program funded by the Ministry of Science
and Technology, Republic of Korea. We are also thankful to Rural
Development Administration, Korea for providing necessary green-
house facilities for BPH bioassay.
This research was supported by a grant (code#
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