Inheritance of bacterial blight resistance in the rice cultivar
Ajaya and high-resolution mapping of a major QTL
associated with resistance
K. SUJATHA1#, P. NATARAJKUMAR1#, G. S. LAHA1#, B. MISHRA1, 2,
K. SRINIVASA RAO1#, B. C. VIRAKTAMATH1, P. B. KIRTI3, Y. HARI1,
S. M. BALACHANDRAN1, P. RAJENDRAKUMAR1, T. RAM1, S. K. HAJIRA1,
M. SHESHU MADHAV1, C. N. NEERAJA1AND R. M. SUNDARAM1*#
1Directorate of Rice Research, Rajendranagar, Hyderabad 500 030, AP, India
2Sher-E-Kashmir University of Agricultural Sciences and Technology, Jammu 180 012, India
3Department of Plant Sciences, School of Life Sciences, University of Hyderabad, AP, Hyderabad 500 046, India
(Received 17 June 2011; revised 3 November 2011; accepted 8 November 2011)
The cultivar Ajaya (IET 8585) exhibits durable broad-spectrum resistance to bacterial blight (BB) disease of rice
and is widely used as a resistance donor. The present study was carried out to decipher the genetics of BB
resistance in Ajaya and map the gene(s) conferring resistance. Genetic analysis in the F2indicated a quantitative/
additive nature of resistance governed by two loci with equal effects. Linked marker analysis and allelic tests
revealed that one of the resistance genes is xa5. Sequence analysis of a 244 bp region of the second exon of the
gene-encoding Transcription factor IIAc (the candidate gene for xa5) confirmed the presence of xa5. Bulked-
segregant analysis (BSA) revealed the putative location of the two quantitative trait loci (QTLs)/genes associated
with resistance on chromosomes 5 and 8. Composite interval mapping located the first locus on Chr. 5S exactly
in the genomic region spanned by xa5 and the second locus (qtl BBR 8.1) on Chr. 8L. Owing to its differential
disease reaction with a set of seven hyper-virulent isolates of Xanthomonas oryzae, a map location on Chr. 8L,
which was distinct from xa13 and data from allelism tests, the second resistance locus in Ajaya was determined
to be novel and was designated as xaAj. A contig map spanning xaAj was constructed in silico and the genomic
region was delimited to a 13.5 kb physical interval. In silico analysis of the genomic region spanning xaAj
identified four putatively expressed candidate genes, one of which could be involved in imparting BB resistance
in Ajaya along with xa5.
Bacterial blight (BB) disease of rice caused by
X. oryzae pv. oryzae (Xoo) limits rice yield in all major
rice-growing regions of the world especially in irri-
gated lowland and rainfed conditions. The estimated
yield loss due to BB under severe infection varies from
50 to 80% in tropical Asia (Khush & Ogawa, 1989).
Deployment of resistant cultivars is the most eco-
nomical and effective method to control the disease
due to the non-availability of effective bactericidal
agents. Two types of resistance, vertical and horizon-
tal resistance, have been recognized to be in operation
in rice against Xoo. Vertical resistance, governed by
single major genes, is race specific and can be broken
down easily unless two or more genes are deployed in
conjunction as gene-pyramids. Through classical
genetic analysis about 34 major genes conferring re-
sistance to various strains of Xoo (Chen et al., 2011)
have been identified and most of these have been
mapped to various chromosomes (Jin et al., 2007). So
far, six BB resistance genes, Xa21, Xa1, Xa26, Xa27,
xa5 and xa13 have been cloned by map-based cloning
strategy (Chu et al., 2006b). Horizontal resistance on
the other hand is quantitative, presumably non-race
specific and controlled by quantitative trait loci
(QTLs). QTL mapping provides an effective approach
to study complex and polygenic forms of disease re-
sistance. Recent progress in the QTL analysis using
DNA markers especially simple sequence repeats
* Corresponding author: Crop Improvement section, Directorate
of Rice Research, Rajendranagar, Hyderabad, AP, India. E-mail:
# These authors contributed equally.
Genet. Res., Camb. (2011), 93, pp. 397–408.
f Cambridge University Press 2011
(SSRs) allows a better understanding of the traits that
are controlled by multiple genes. QTLs responsible
for the BB resistance have been identified in earlier
studies (Li et al., 1999).
Large-scale cultivation of varieties with single re-
sistance genes like Xa3 and Xa4 resulted in develop-
ment of more virulent races making them ineffective
(Shi et al., 2001). Evolution of virulent populations
of Xoo against the major resistance gene, Xa21 has
been reported in some parts of India and Korea
(Sirisha et al., 2004). Widespread and repeated use of
a few resistance genes has accelerated the selection of
new pathogenic races at a rate equivalent to 1.64 times
the increase in specific virulence of the isolate follow-
ing host-plant selection pressure after a single crop
cycle (Nayak, 1986b). One of the strategies to delay
breakdown of resistance is to identify new resistance
genes from diverse sources especially identified
broad spectrum durable resistant cultivars and to in-
trogress them into susceptible but otherwise popular
Several varieties with resistance to BB have
been released under the auspices of the All India
Coordinated Rice improvement project (AICRIP) in
India. The variety, Ajaya (IET 8585) was released by
AICRIP in 1992 and possesses high level of resistance
against most of the pathotypes in India (DRR
Progress Report 1996–2009). Ajaya is commonly used
as a differential cultivar and as a resistant check in
AICRIP trials. However, information regarding the
inheritance of BB resistance in Ajaya is not clear with
one study indicating the action of two independently
segregating dominant genes (Saini et al., 1996), while
another reported the action of a single recessive
gene (Kameswara Rao et al., 2003). Hence, an effort
was made through the present study to clarify the
inheritance of resistance to BB in Ajaya, tag and map
the gene(s) conferring BB resistance with the help of
SSR markers and identify putative candidate genes
responsible for BB resistance through an in silico ap-
2. Materials and methods
The plant materials analysed in the present study
consisted of the BB resistant cultivar Ajaya, one of its
parents – BJ1, the BB susceptible varieties TN1, BPT
5204 (Samba Mahsuri), near isogenic lines (NILs)
of IR24 possessing single BB resistance genes-xa5
(IRBB5), xa13 (IRBB13), Xa21 (IRBB21), NILs
possessing combinations of Xa4, xa5, xa13 and Xa21
and the BB differential BJ1. Crosses were made with
Ajaya as donor and TN1, BPT 5204, IRBB5 and
IRBB13 as recipient parents to develop F2and F3
mapping populations. A population consisting of 800
F2individuals derived from the cross Ajaya/TN1 was
used to study the inheritance of resistance to BB and
for mapping the resistance gene(s) in Ajaya. An
alternate population consisting of 592 F2individuals
obtained from the cross Ajaya/BPT 5204 was used for
validation of genetic distances of the linked markers.
Two sets of populations, each consisting of 177 F2
individuals derived from Ajaya/IRBB5 and 326 F2
individuals of Ajaya/IRBB13 were used for allelism
test of xa5 and xa13, respectively. Seven strains of
Xoo, collected from different hotspot locations of
India were used to study the level and spectrum of BB
resistance of Ajaya along with NILs possessing single
or combination of BB resistance genes and susceptible
parents (Table 1).
Table 1. Disease reaction of Ajaya along with NILs of IR24 with seven hypervirulent isolates of Xoo
GenotypeDX-066DX-002 DX-018 DX-084DX-109 DX-119DX-139
Note: DX-066 collected from Raipur (Chattisgarh); DX-002 collected from Faizabad (Uttar Pradesh); DX-018 collected
from Kapurtala (Punjab); DX-084 collected from Kaul (Haryana); DX-109 collected from Kerala; DX-119 collected from
Hyderabad (Andhra Pradesh); DX-139 collected from Sambalpur (Orissa).
K. Sujatha et al.398
(a) Disease evaluation
The parents, F1, F2and F3populations of the above
crosses were phenotyped for their reaction to BB
at 55 days after sowing which coincides with the
active tillering phase of the crop. The bacterial
culture was grown on modified Wakimoto’s medium
(sucrose 20.0 g; peptone 5.0 g; Ca(NO3)2, 4H2O 0.5 g;
Na2HPO40.82 g; FeSO4, 7H2O 0.05 g; agar 20.0 g;
distilled water 1000 ml; pH 6.8–7.0) for 3 days at
28 xC. The bacterial cells were suspended in sterile
distilled water such that approximately a concen-
tration of 108–109cfu/ml was maintained. The parents
and the mapping populations were inoculated with
the bacterial suspension by the leaf clipping method
(Kauffman et al., 1973). Plant reaction to the BB
pathogen was recorded 14 days after inoculation.
Plants were scored based on the per cent diseased
leaf area (DLA) following the conditions described
by Chen et al. (2000) and IRRI–SES system of
scoring (Anonymous, 2002). The scores from 5 to
6 inoculated leaves from a single F2 plant were
averaged and categorized as highly resistant (score 1,
% DLA<5), moderately resistant (score 3, %
DLA=5–11.9), moderately susceptible (score 5, %
DLA=12–24.9), susceptible (score 7, % DLA=
25–49.9) and highly susceptible (score 9, % DLA=
(b) PCR analysis
Total genomic DNA was extracted from the parents
and mapping populations derived from the crosses
Ajaya/TN1 and Ajaya/BPT 5204 using the pro-
cedure of Zheng et al. (1995). PCR was performed
in a thermal cycler (Perkin–Elmer 480, USA) as
per the conditions described by Chen et al. (1997).
SSR marker resolution was done on 4% agarose
gels stained with ethidium bromide and photo-
graphed under ultraviolet light. In order to test the
presence of xa5 and xa13 in Ajaya, the xa5-linked
CAPS marker RG556 (Huang et al., 1997) and the
xa13-linked PCR-based marker xa13-prom (Chu
et al., 2006b) were analysed in Ajaya along with
IR24, IRBB5, IRBB13, TN1, BPT 5204 and the
parental line BJ1. Restriction digestion for the
CAPS marker RG556 was performed in a reaction
mixture consisting of 3.2 ml sterile distilled water,
1.5 ml restriction buffer (10r), 0.3 ml restriction en-
zyme Dra 1 (3U/ml) and 20 ml PCR product by in-
cubating at 37 xC for 1 h. The samples were then
resolved in a 2% agarose gel and visualized
after staining with ethidium bromide. PCR analysis
of xa13prom was done following conditions similar
to SSRs and the amplicons were resolved in 2%
(c) Analysis of functional nucleotide polymorphism
with respect to xa5 in Ajaya
A primer pair was designed based on a region on
chromosome 5 of Nipponbare rice sequence (Pseudo
molecule5) from 415 388 to 415 630 bp, which en-
compasses the 2 bp functional polymorphism TC/AG
in the exon II of the transcription factor IIAc (Iyer &
McCouch, 2004). The primers were used to amplify
the resistant and susceptible genotypes (IRBB5,
Ajaya, TN1 and BPT 5204). The primer sequences
were as follows: xa5F: TCC CCC CAC CCC AAA
AAG, xa5R: ACA AAC AAA ATG AGG AGT CG.
PCR amplification was performed using a high-
fidelity DNA polymerase–Pfu polymerase (Promega,
USA) to avoid errors as per the conditions described
earlier. The PCR products were resolved on a 0.8%
agarose gel and eluted using Wizard1SV Gel and
PCR clean-up system (Promega, USA). The products
were cloned using Qiagen1PCR cloning kit (Qiagen,
Germany) as per the manufacturer’s instructions.
Plasmids were isolated from the transformed colonies
using Qiaquick plasmid
Germany) and sequenced in both directions in an
ABI prism 3700 automated DNA sequencer using the
T7 and T13 primers of the vector. Vector sequences
were removed from the sequenced fragments and the
cleaned-up fragments were then aligned using Clustal
W (available at the website http://www.ebi.ac.uk/
clustalw) program of the European Bioinformatics
Institute (Thompson et al., 1994).
(d) Molecular mapping using SSR markers
Bulked segregant analysis (BSA). In order to map the BB
resistance genes in Ajaya and to identify linked SSR
markers, a set of 480 SSR markers spread across the
rice genome were used. BSA was carried out by am-
plification of the resistant and susceptible parents
along with the RB and SB with the polymorphic SSR
markers. Markers that displayed bulk-specific ampli-
fication were analysed in the entire mapping popu-
lation to study their linkage distance with respect to
the resistance genes.
Fine mapping. Once the tentative chromosomal lo-
cation of the resistance loci was known through BSA,
all the SSR markers in the vicinity, possessing a repeat
length of more than 20 bp, were selected for geno-
typing the F2population of Ajaya/TN1 for coarse
mapping. Fine-mapping of the two putative resist-
ance-associated loci was carried out using a separate
vicinity of xa5) and 45 SSR markers specific to the
long arm of chromosome 8. Of the 45 markers, 39
were from the RM series (available at the website
www.gramene.org) and six markers viz., RMAFM1 to
RMAFM6 (see Supplementary Table S1 available at
http://journals.cambridge.org/grh) were specifically
Mapping of bacterial blight resistance in Ajaya399
designed by FASTPCR software using the flanking
sequence data of the repeat motifs identified in-
between the flanking markers by SSRIT tool of
Gramene (available at the website http://www.gra-
mene.org). Linkage analysis and map construction
were performed using MAPMAKER/EXP, version 3
(Lander et al., 1987). QTL analysis was done using
Composite interval mapping function of Windows
QTL Cartographer ver 2.5 (Zeng, 1994) using per cent
DLA as trait phenotype.
(e) In silico physical mapping of BB resistance gene
The physical map of the resistance gene was con-
structed by bioinformatics analysis using bacterial
artificial chromosome (BAC) and P1-derived artificial
chromosome (PAC) clones of cv. Nipponbare re-
leased by the International Rice Genome Sequencing
Project (IRGSP, 2005). The flanking markers were
assigned to the respective BAC or PAC clones
through BLASTN analysis. The PAC/BAC clones
were aligned using the software tool Pairwise BLAST
(available at the website http://www.ncbi.nlm.nih.
(f) Candidate gene annotation
Based on the targeted region of the resistance gene,
the publicly available BAC or PAC sequences of
Oryza sativa cv. Nipponbare were analysed by gene
prediction programmes FGENESH (available at
the website http://genomic.sanger.ac.uk), Rice GAAS
(available at the website http://ricegaas.dna.affrc.
go.jp/) and Gene Scan (available at the website http://
were analysed through BLAST (available at the web-
site http://www.ncbi.nlm.nih.gov/blast/) and con-
firmed by the TIGR Rice Genome Annotation
Version 6.0 (available at the website http://rice.
The candidate genes
(i) Genetic analysis of BB resistance in Ajaya
When screened with multiple isolates of Xoo, Ajaya
displayed a broader spectrum of resistance as com-
pared with the susceptible checks (TN1 and BPT
5204) and the NILs possessing the single resistance
genes, Xa4, xa5, xa13 and Xa21 (Table 1). The re-
sistant parent Ajaya and the susceptible parents TN1
and BPT 5204 showed clear distinction in terms of
disease score with Ajaya displaying a score of 1 and
the susceptible parents, TN1 and BPT 5204 displaying
a score of 9. The F1s were moderately susceptible with
a disease score of 5. Out of the 800 F2plants screened,
48 were highly resistant, 210 were moderately resist-
ant, 274 were moderately susceptible, 212 were sus-
ceptible and 56 were highly susceptible (Fig. 1(A), see
Supplementary Table S2 available at http://journals.
cambridge.org/grh). This fitted well in a segregation
ratio of 1:4:6:4:1 (x2=4.27, P=0.37) with respect
to high resistance, moderate resistance, moderate
susceptibility, susceptibility and high susceptibility.
Similar segregation pattern was observed in the F2
population derived from the cross Ajaya/BPT 5204
(Fig. 1(B), see Supplementary Table S3 available
at http://journals.cambridge.org/grh). The F1s were
moderately susceptible (score 5) and among the 592
F2plants, 32 were resistant, 139 were moderately re-
sistant, 235 were moderately susceptible, 146 were
susceptible and 40 were highly susceptible (x2=2.23,
P=0.69). This indicated that BB resistance in Ajaya is
probably controlled by two additively interacting loci
with equal phenotypic effects.
(ii) Analysis with gene-linked markers
In order to test the presence of xa5 and xa13 in
Ajaya (as predicted from its pedigree) it was analysed
with linked markers RG556 (xa5) and xa13-prom
(xa13) along with the NILs of IR24 carrying
Fig. 1. Frequency distribution of the F2population derived from Ajaya/TN1(A) and Ajaya/BPT 5204 (B). A segregation
ratio of 1:4:6:4:1 with respect to resistance, moderate resistance, moderate susceptibility, susceptibility and high
susceptibility was noticed when the F2plants derived from the crosses Ajaya/TN1(A) and Ajaya/BPT 5204 (B) were
screened for BB resistance.
K. Sujatha et al. 400
xa5 (IRBB5), xa13 (IRBB13), the two BB sus-
ceptible varieties TN1, BPT 5204 and the parental
line BJ1. The CAPS marker RG556 amplified two
fragments of sizes 1000 and 1500 bp in all the geno-
types. Restriction digestion with the enzyme Dra
1 produced two fragments of sizes 390 and 410 bp
in Ajaya, IRBB5 and BJ1 and two fragments of sizes
350 and 450 bp in TN1 and BPT 5204 (Fig. 2(A)).
The PCR-based marker xa-13 prom, a functional
marker for xa13 amplified the resistance specific
allele, a 1.4 kb fragment in IRBB13 and BJ1,
whereas Ajaya amplified a 650 bp fragment as in
the other susceptible genotypes TN1 and BPT 5204
(iii) Allelism tests
In order to confirm the presence/absence of xa5 and
xa13 genes in Ajaya, it was crossed with IRBB5 and
IRBB13. The F1s and the F2population derived from
the cross IRBB5/Ajaya was screened with Xoo strain
DX-066, which showed resistance reaction with
IRBB5 and Ajaya. The F1was resistant (score 1) and
all the 177 F2plants were uniformly resistant (score 1)
without any segregation for susceptibility, indicating
that one of the two resistance genes in Ajaya is xa5.
The F1and the F2population obtained from IRBB13/
Ajaya were screened with Xoo strain DX-084, which
showed resistance reaction with IRBB13 and Ajaya
(score 1). The F1was highly susceptible (score 9) and
out of 326 F2 plants screened, 157 were resistant
(score 1) and 169 were highly susceptible.
(iv) Sequence analysis for xa5 candidate gene
A 244 bp fragment was amplified from the second
exon of the xa5 gene candidate gene (i.e. TFIIAc)
encompassing the functional nucleotide polymorph-
ism consisting of the 2 bp substitution GTC/GAG in
Ajaya, IRBB5 and two susceptible genotypes IR24
and TN1 and sequenced as explained in Materials and
methods. Ajaya possessed the 2 bp polymorphism
(TCpAG substitution) specific for xa5 gene, similar
to that of IRBB5 at the 17th and 18th nucleotide
position, while TN1 and BPT 5204 showed suscepti-
bility-specific TC sequence at the 17th and 18th
nucleotides (Fig. 3).
(v) Mapping of the second BB resistance locus in
Genetic analysis in the F2plants derived from the
crosses Ajaya/TN1 and Ajaya/BPT 5204 indicated
that the action of two loci are involved in conferring
BB resistance in Ajaya. Based on the results of
analyses with the xa5 linked marker, RG556, allelism
test with IRBB 5 and sequence analysis of xa5 candi-
date gene, the first locus was confirmed to be xa5. The
second locus was found to be non-allelic to xa13
(based on linked marker analysis), but linked to it
based on allelism test with IRBB13. In order to map
the second resistance locus in Ajaya, an analysis was
conducted to identify SSR markers polymorphic be-
tween Ajaya and TN1. Out of 480 SSR markers tes-
ted, 112 (23.3%) were polymorphic and 368 (76.6%)
Fig. 2. Analysis of xa5 (RG556) and xa13 (xa13 prom) linked markers in Ajaya. Ajaya displayed CAPS polymorphism
similar to IRBB5, indicating presence of xa5 (2A) when amplified with RG556 and restricted with Dra1. However, it did
not show an amplification pattern specific for the resistant allele of xa13, when amplified with the marker xa13 prom (2B).
M – Molecular weight marker (100 bp ladder), I5 – IRBB5, I13 – IRBB13, A – Ajaya, BJ – BJ1, T – TN1 and B – BPT
Fig. 3. Sequence alignment of the functional polymorphic region in second exon of xa5. Ajaya possessed the 2 bp
polymorphism (TCpAG substitution), which was similar to that of IRBB5 at the 17th and 18th nucleotide position
(highlighted in box), while TN1 and BPT 5204 showed susceptible specific TC sequence.
Mapping of bacterial blight resistance in Ajaya 401
were monomorphic. BSA revealed linkage of the SSR
marker RM17745, located on Chr. 5 and RM23535
on chromosome 8 with BB resistance. Out of 22 SSR
markers tested in the vicinity of RM 17745 on the
short arm of Chr. 5, 10 were polymorphic between
Ajaya and TN1. Out of 45 SSR markers tested in
the vicinity of RM23535 on the long arm of Chr. 8,
nine were polymorphic between Ajaya and TN1.
Linkage analysis was performed by genotyping the
800 F2plants of Ajaya/TN1 with nine SSR markers on
Chr. 5, RG556 and nine SSR markers on Chr. 8. The
nine SSR markers could be integrated into the linkage
map of Chr. 5 and covered a span of 30.5 cM, with
mean distance between the markers being 3.38 cM.
The nine markers on Chr. 8 spanned a genetic distance
of 29.8 cM with a mean distance of 3.31 cM. The
marker order on the linkage maps of chromosomes
5 and 8 was consistent with the SSR linkage maps of
Temnykh et al. (2000) and McCouch et al. (2002).
(vi) QTL analysis
Composite interval mapping (CIM) performed with a
set of ten SSR markers on the short arm of chromo-
some 5 and nine markers on the long arm of Chr. 8
mapped the first resistance locus (tentatively named as
qtl BBR 5.1) at a genetic distance of 0.3 cM from
RM17751 and 0.5 cM from RM17752 with both the
markers flanking the gene at an LOD score of 6.2.
RG556 co-segregated with resistance without any re-
combination indicating that qtlBBR 5.1 is xa5. The
second resistance locus (tentatively named as qtl BBR
8.1) was mapped at a genetic distance of 0.2 cM from
RM23499 and 0.4 cM from RMAFM1 with both of
them flanking the gene (Fig. 4(A)) at an LOD score
of 5.7 (Table 2). Marker-trait co-segregation analysis
combinants in a population of 800 individuals with
one recombinant from the moderately susceptible
Table 2. Details of the resistance genes/QTLs in Ajaya identified by composite interval mapping
Cross LocusMarker interval ChromosomeLOD
variance (%) A(cM)B(cM)C(cM)P(0.05)
Ajaya/BPT 5204 F2
A, additive effect of Ajaya allele; B, lesion length of plants with genotype of Ajaya and C, lesion length of plants with
genotype of TN1/BPT 5204.
Fig. 4. Genetic linkage map of the genomic region in the vicinity of qtl BBR 5.1 (xa5) and qtl BBR8.1 (xaAj) in the F2
mapping population derived from the cross Ajaya/TN1(A) and Ajaya/BPT 5204 (B). Two major QTLs/genes were
identified to be controlling BB resistance in Ajaya. One was xa5 located on Chr. 5 cosegregating with RG556 and the
other was xaAj located on Chr. 8 and flanked by the SSR markers RM23499 and RMAFM1.
K. Sujatha et al. 402
class and two from the susceptible class while with
respect to the marker RMAFM1, four recombinants
(i.e. two from the moderately susceptible class and
two from the susceptible class) were observed. All the
48 highly resistant plants showed amplification of
homozygous-resistant parent-specific allele and the 56
highly susceptible plants showed the homozygous
susceptible allele of RG 556 and RM23499 without
any recombination. The above two loci explained
97.5% of the total variance observed. Since the se-
cond resistance locus qBBR8.1 was observed to be
non-allelic but linked to xa13 based on allelic testing
with IRBB13, it can be considered as a new gene/QTL
for BB resistance.
(vii) Physical mapping
To physically map the locus qBBR8.1, the anchor
markers RMAFM1 and RM23499 were used to
land on the reference sequence of cv. Nipponbare
by BLASTN analysis. The matching sequences
showed that there were four PAC clones and three
BAC clones, which covered the target gene i.e.
OJ1113_A10, P0700D12, OJ1191_A10, P0702E04,
P0665C04, OJ1211_G06 and P0623F08. The marker
RM23327 was observed to be located on the BAC
clone OJ1113_A10 (AP004643), RM23450 on PAC
clone P0700D12 (AP004708), RM23468 on BAC
RMAFM1 and RM23499 on PAC clone P0702E04
(AP005529), RMAFM5 on PAC clone P0665C04
(AP004464), RM23535 on BAC clone OJ1211_G06
(AP003948) and RM23553 on PAC clone P0623F08
(AP004632). The BAC/PAC clones identified from
the IRGSP website were aligned as a contig map
via pairwise BLAST analysis. The marker AFM2
aligned at position 60.574 kb on the PAC clone
P0702E04, while the flanking markers RMAFM1
and RM23499 aligned at positions 77.895 and
91.330 kb, respectively. Consequently, a physical map
covering the qtlBBR8.1 was generated in a 13.5 kb
physical interval based on the RGP BAC/PAC con-
tigs (Fig. 5).
(viii) Validation of the linked markers in the
alternate mapping population
The utility of the linked SSR markers to predict re-
sistance in Ajaya was validated in an alternate F2
5204. The xa5 linked markers on chromosome 5 i.e.
RG556, RM17720, RM17738, RM17745, RM17751,
RM17760, RM17771 and RM17785 were polymor-
phic while RM17725 and RM17752 were monomor-
phic between Ajaya and BPT 5204. The markers
RM23499, RMAFM5, RM23535 and RM23553 on
chromosome 8 were polymorphic while RMAFM2
marker RG556 displayed perfect co-segregation with
Fig. 5. Physical map of xaAj. Nine SSR markers were used in this study. The long horizontal lines indicate the region
containing the xaAj. The short horizontal lines represent the BAC/PAC clones of cv. Nipponbare released by IRGSP and
assembled by the corresponding markers linked to the xaAj. The numbers at the top above the long horizontal line are
the recombination events in the mapping population. The digits between markers are physical distances in kilobase (kb).
The vertical and dashed lines denote the relative positions of the corresponding markers.
Mapping of bacterial blight resistance in Ajaya403
resistance. The seven polymorphic SSR markers
spanned a genetic distance of 29.2 cM on the linkage
map of Chr. 5 with a mean distance of 4.17 cM be-
tween the markers, whereas the eight SSR markers on
Chr. 8 spanned a genetic distance of 30.55 cM with a
mean of 3.81 cM between markers. Composite inter-
val mapping with these markers mapped the first
resistance locus (qtl BBR5.1) in the marker interval
of RM17751 and RM17760 with an LOD score of 7
and the second resistance locus (qtl BBR8.1/xaAj)
in the marker interval of RM23499 and RMAFM1
(Fig. 4(B)) with an LOD score of 4.5 (Table 2). The
gene was mapped at a distance of 0.1 cM from
RM23499 with a single recombinant (in the moder-
ately resistant class) in a population of 592 individuals
while the other flanking marker RMAFM1 showed
0.65 cM distance with five recombinants (one from
moderately resistant class, three from moderately
susceptible and one from the susceptible class). The 32
highly resistant plants showed homozygous resistant
allele and 40 highly susceptible plants showed homo-
zygous susceptible allele with respect to RG556,
RM23499 and RMAFM1. The two loci xa5 and xaAj
explained 92% of the total phenotypic variance ob-
served. The marker order and the linkage distances
between the markers were consistent with that ob-
served in the F2population derived from Ajaya/TN1.
Although there were few recombinants, no re-
combinants were observed in the highly resistant class
with respect to the flanking markers RM23499 and
RMAFM1 and so the marker combination of
RG556+RM23499 and RMAFM1, could predict
complete resistance in Ajaya even in the alternate
background of BPT 5204.
(ix) In silico identification of putative candidate genes
for qtl BBR8.1
In silico analysis of the intervening genomic region
of 13.5 kb flanked by the closest markers RM23499
and RMAFM1 revealed the presence of four putative
candidate genes (Table 3), which could be associated
with xa5 in imparting complete resistance in Ajaya.
Losses due to BB are significantly higher in tropical
Asia than in temperate regions because of the preva-
lence of virulent populations of Xoo. The Indian iso-
lates of Xoo are reported to be more diverse and
possess high level of virulence when compared with
isolates from other geographic regions (Seshu, 1989).
Thus, the resistance genes identified in other geo-
graphical regions may not be of significant value for
resistance breeding in India. In India, where rice is
grown under diverse agro-climatic conditions, a sig-
nificant diversity in the Xoo pathogen was reported
through an analysis of 67 isolates from 18 locations
(Yoshitola et al., 1997; Shanti et al., 2001). The cul-
tivar, Ajaya possesses high levels of resistance against
most of the Indian isolates and has been observed
to be durably resistant since its release in 1992 (Laha
et al., 2009).
The choice of durably resistant elite varieties like
Ajaya as donors of BB resistance shortens the time
required for developing a resistant variety through
traditional/marker assisted selection, since it is an al-
ready released cultivar with some desirable agronomic
features. However, in order to devise a breeding pro-
gramme for transfer of BB resistance from Ajaya,
knowledge about the genetics/mode of inheritance
of resistance is necessary. Results from two earlier
studies on inheritance of resistance to BB in Ajaya are
conflicting. Hence, the present study was devised to
study the genetics of BB resistance, tag and map the
resistance gene(s)/QTLs in order to facilitate marker
assisted introgression. In addition, the study also at-
tempted to identify putative candidates for BB resis-
tance in Ajaya for their possible map-based cloning in
Genetic analysis of the F2population derived from
the crosses involving Ajaya with TN1 and BPT 5204
revealed quantitative inheritance of resistance, gov-
erned by two loci with equal effects. The quantitative
nature of disease resistance involving 2–3 genes has
been reported in many species against a variety of
pathogens (Leonard, 1993). The popular IRRI bred
Table 3. Putative candidate genes annotated in the genomic region spanned by xaAj
Accession no. Gene no. Location Gene description
NP_001062339Os08g053230026624938-26625234 Nuclear transport factor 2 (NTF2) domain plays
an important role in the trafficking of
macromolecules, ions and small molecules between
the cytoplasm and nucleus
Hypothetical protein with a conserved nodulin domain
Unknown protein with a conserved domain of SUA7/
Transcription factor TFIIIB/Brf1 subunit of TFIIB
NP_001062342 Os08g0532700 26635260–26637736
K. Sujatha et al. 404
varieties IR28, IR26 and the differential cultivar
DV85 (Wang et al., 2005) were reported to possess
quantitative resistance against BB. In the present
study, the additive/quantitative interaction between
two genes/QTLs with equal effect was discovered and
one of these genes/QTLs was confirmed to be xa5 and
the same was validated in an alternate mapping
population. Based on these results, the durability of
resistance in Ajaya can be attributed to its quanti-
tative nature of inheritance of resistance.
Ajaya has a complex pedigree (see Supplementary
Figure S1 available at http://journals.cambridge.org/
grh) and was originally derived from a cross between
IET4141 and CR98-7216. The landrace BJ1, the pre-
dicted BB resistance donor of Ajaya has been re-
ported to possess the BB resistance genes xa5 and
xa13 (Ogawa et al., 1987a). Since genetic analysis of
F2populations in the present study revealed the ac-
tion of two genes/QTLs involved in conferring resis-
tance in Ajaya, its allelic status was tested with respect
to the xa5 linked marker RG556 (Blair & McCouch,
1997) and a functional marker for xa13, xa13-prom
along with its parent BJ1. Iyer & McCouch (2004)
reported that the functional polymorphism specific
for xa5 is a 2 bp substitution in the 17th and 18th
nucleotide positions of exon-2 (i.e. TCpAG) of
transcription factor IIAc (which is the candidate gene
for xa5 located on Chr. 5) and this results in the
change in a single amino acid (valinepglutamic acid)
at the 39th position in the encoded protein. We ob-
served presence of the TCpAG substitution in Ajaya,
which conclusively shows that xa5 is indeed one of the
genes governing BB resistance in Ajaya.
In an earlier study, Kameswara Rao et al. (2003)
reported that a gene non-allelic to xa5 controls BB
resistance in Ajaya and mapped it on the long arm of
Chr. 5 between the markers RM39 (14.5 cM from the
gene) and RM31 (17.7 cM from the gene). xa5 has
been conclusively mapped on the short arm of Chr. 5
(Blair & McCouch, 1997; Iyer & McCouch, 2004). In
our study, through all the four approaches i.e. marker
analysis with the closely linked CAPS marker RG556,
allelism test with IRBB5, sequence analysis of the
functional polymorphic region of xa5 and linkage
analysis in two populations segregating for xa5 we
have shown that xa5 is indeed one of the candidate
genes conferring resistance in Ajaya, which is in con-
trast to the observations of Kameswara Rao et al.
(2003) in terms of mapping of the recessive resistance
gene/QTL on Chr. 5.
The improved resistance of Ajaya to BB as com-
pared with IRBB5, both in terms of the level and
spectrum of resistance could be attributed to the ad-
ditive effect of the second resistance gene/QTL iden-
tified in the present study (xaAj) in addition to xa5.
Additive/dosage effects have been reported with re-
spect to the major BB resistance genes Xa1 and Xa3 in
Java 14 (Kaku, 1997) and between Xa1 and Xa4 in
IR20 (Kaku, 1999). The increased level of resistance
conferred by more than one gene governing resistance
to a single pathogen race has been described as
quantitative complementation and has been reported
in several studies involving gene-pyramiding studies
for BB resistance (Yoshimura et al., 1996).
Through QTL analysis in the F2populations de-
rived from the crosses Ajaya/TN1 and Ajaya/BPT
5204, we fine-mapped the second resistance locus qtl
BBR 8.1 which has been designated as xaAj, ap-
proximately at a distance of 100 kb from another BB
resistance gene xa13. Genetic analysis and QTL
analysis in the F2population revealed equal contri-
bution of resistance by the two loci in Ajaya. The BB
differential DV85 was reported to carry two QTLs for
BB resistance that were resolved to be xa5 and Xa7
(based on similar map location), with xa5 imparting a
larger effect than Xa7 (Wang et al., 2005).
When the resistance spectrum of Ajaya was studied
with seven hypervirulent isolates of Xoo, three isolates
could differentiate Ajaya and BJ1 (which possess xa5
and xa13), two isolates could differentiate Ajaya and
IRBB53 (which possesses xa5 and xa13) and five of
the seven isolates showed differential disease reaction
between Ajaya and IRBB13 (which possesses xa13;
Table 1). Based on these observations, the second re-
sistance locus identified in the present study, i.e. xaAj
can be considered as novel.
The gene density in the region flanking xa Aj is
about one gene every 4.3 kb, against the published
predictions of one gene every 9.9 kb (International
Rice Genome Sequencing Project, 2005). In the region
flanking xa13 (Chu et al., 2006a) and Xa30t (Cheema
et al., 2008), each centimorgan is roughly equivalent
to 96 and 60 kb, respectively, whereas in the region
flanking xaAj, 1 cM was calculated to be equivalent to
22.5 kb indicating a nearly 12-fold reduction in the
ratio of physical/genetic sizes as compared with the
average P/G ratio of 260–280 kb/cM estimated by Wu
& Tanksley (1993) and Yoshimura et al. (1996). This
reduction is apparently because of enhanced recom-
bination in this region (hotspot for crossing over). In
the xa5 region on chromosome 5, each cM is equiva-
lent to about 61 kb (Yang et al., 1998) and 130 kb in
the Xa1 region of chromosome 4 (Yoshimura et al.,
Plant disease resistance genes frequently occur in
the form of tightly linked clusters. Four dominant BB
resistance genes Xa3, Xa26t, Xa4 and Xa22 have been
mapped to the distal region of chromosome 11L
(Yang et al., 2003) and five genes, i.e. Xa1, Xa2, Xa12,
Xa14 and Xa30t in a 600 kb region on chromosome
4L (Cheema et al., 2008). Recessive resistance genes
generally do not cluster with other resistance genes in
rice (Richter & Ronald, 2000). However, in the pres-
ent study xaAj was mapped in a genomic region of
Mapping of bacterial blight resistance in Ajaya 405
approximately 100 kb to xa13. The close linkage of
xaAj with xa13 based on CIM analysis was consistent
with the results of genetic linkage between the two
genes as revealed by allelic testing of Ajaya with
One of the genes annotated through in silico
analysis in the genomic region spanned by xaAj
encodes a nuclear transport factor which is involved
in trafficking of ions and macromolecules between
nucleus and cytoplasm. The second gene encodes a
hypothetical protein with a conserved Nodulin do-
main. The candidate gene for xa13 has been identified
to be Medicago truncatulata, nodulin gene. Nodulin-
related genes are found in several species such as
nematodes, insects and animals, although the bio-
chemical functions of their proteins are unknown.
Yang et al., (2006) reported about 17 nodulin-related
genes distributed all over the rice genome. The third
gene predicted codes for a protein homologous to
SUA7, which codes for the general transcription fac-
tor TFIIIB or the BRF1 subunit of the transcription
factor TFIIB, involved in gene transcription in as-
sociation with the enzyme RNA Polymerase II. The
gene xaAj additively interacts with xa5 in conferring
complete resistance to BB in Ajaya based on the re-
sults on genetic analysis and CIM analysis. It should
be noted that xa5 is a general transcription factor but
serves as a resistance-associated protein under BB in-
fection (Iyer & McCouch, 2004). The fourth putative
candidate gene encodes a peroxidase. Peroxidases are
involved in lignin biosynthesis whose accumulation
was observed in the resistance mediated by xa5, Xa7
and Xa10 (Reimers & Leach, 1991). In rice, induction
of peroxidases has been correlated with resistance to
BB (Young et al., 1995; Hilaire et al., 2001). Based on
these, any of the four candidate genes mentioned
could be considered as a possible candidate for xaAj
and further analysis of these genes through cloning
and sequence analysis may reveal their functionality
with respect to xaAj. Transcriptional profiling in
Ajaya under BB infection revealed the differential
expression of defence-related and transcription fac-
tor genes, which is in agreement with our results on
in silico gene prediction (Kameswara Rao et al., 2007).
The present study has identified two genes/QTLs
governing BB resistance in Ajaya, one of which is xa5
and the other linked to, but non-allelic to xa13. The
second gene/QTL designated as xaAj was fine map-
ped on Chr. 8 in a physical interval of 13.5 kb close to
(approximately 100 kb) another major BB resistance
gene xa13. The gene can be deployed either singly or
in combination with other major BB resistance genes
xa5, xa13 and Xa21 to obtain broad-spectrum resist-
ance against BB. The tightly linked flanking markers
identified for xaAj, i.e. RM23499 and RMAFM1
can be used to track the introgression of the gene into
elite backgrounds. The putatively expressed genes,
which could be candidates for xaAj, can be validated
through map-based cloning. Information obtained
from the present study may be valuable in under-
standing the molecular mechanism(s) behind quanti-
tative resistance to Xanthomonas in rice.
One of the authors (K. Sujatha) gratefully acknowledges
the financial assistance in terms of a Junior Research
Fellowship and Senior Research Fellowship provided by the
Council for Scientific and Industrial Research (CSIR),
Government of India for her PhD study. The authors also
gratefully acknowledge the financial support provided by
the Department of Biotechnology, Government of India
and the Indian Council for Agricultural Research, Govern-
ment of India for carrying out the research study.
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