202 Institute of Biotechnology, Acharya NG Ranga Agricultural University, Hyderabad 500 030 India

High Quality Genome Sequence of Rice

Accelerated Discovery of Genes for Blast
Resistance

Sharma TR, Rai AK, Gupta SK, Thakur S, Gupta YK, Singh PK. and
Singh NK

National Research Centre on Plant Biotechnology, Indian Agricultural Research

Institute, Pusa Campus, New Delhi-110012. E-mail: trsharma@nrcpb.org

ABSTRACT

The decoding of rice genome was completed in 2005. A very high quality genome sequence of rice is now made
available in public domain by the International Rice Genome Sequencing Consortium (IRGSP). In the post
genomics era most of the gene mapping and cloning activities are taking the help of rice genome sequence data along
with very well planned genetic experiments for the cloning and characterization of genes for blast resistance. The
rice blast disease caused by Magnaporthe oryzae is one of the most important biotic stresses which drastically affect
rice yield globally. Deployment of disease resistant varieties is one the best options for the management of rice blast.
Studies on the genetic basis of resistance to leaf blast began in Japan in 1917. During these past 93 years, extensive
genetic and genomics information are available in rice- M. oryzae system which is playing an important role in the
cloning and characterization of blast resistance genes and alleles from the rice germplasm lines. It has been reported
that rice blast resistance is mainly controlled by single dominant gene and more than 84 genes have already been
identified in different rice lines. Besides, 347 QTLs responsible for resistance to blast fungus have also been
identified in rice. Before the decoding of rice genome sequence in 2005, only two genes for blast resistance i.e. Pib
and Pita were cloned in Japanese and US laboratories, respectively. Then by using positional cloning approach in
conjunction with the draft sequence of rice genome, the third blast resistance gene Pi-kh (Pi54) was cloned and
functionally validated from rice line Tetep at NRCPB, New Delhi, India. This gene is now being transferred in
commercial varieties of rice using marker assisted selection. Till date, 14 blast resistance genes have been cloned and
characterized in different countries. Of these, 11 genes have been cloned after the release of rice genome sequence
data in public domain. It shows that more than 84% of the genes for blast resistance have been cloned within past 5
years. Once the genes are cloned and functionally validated, one can search for the better form of alleles of these
cloned genes in local land races and wild species of rice using allele mining approach. Allele mining involves PCR-
based amplification and isolation of different versions of genes found in germplasm (inbred lines or pure lines,
varieties, landraces and wild relatives). We have used 22 land races and 9 wild species of rice for mining alleles of
three blast resistant genes Pi-ta, Pi-kh(Pi54) and Pi-z(t). The Pi-kh(Pi54) and Pi-z(t) alleles were more variable than
Pi-ta alleles in Indian land races of rice. Coding region of Pi-ta allele was highly conserved in all the land races
whereas Pi-kh(Pi54) and Pi-z(t) alleles were more variable. In other studies, alleles for Pita and Pikm genes have
been reported from large number of rice accessions and varieties. In this paper a comprehensive analysis of the
studies published on rice blast genetics and genomics will be presented along with the studies conducted in our lab
on rice M. orygae system.

Key Words: Rice, Magnaporthe oryzae, resistance genes, blast, genome

Genomics and Crop Improvement: Relevance and Reservation
INTRODUCTION
Rice blast, caused by Magnaporthe oryzae, is a major
fungal disease of rice. Rapid changes in the
virulence structure of M. oryzae populations raise a
continuous threat to the effectiveness of existing
blast resistant varieties. Hence, there is an urgent
need to develop varieties with durable resistance
to the disease, exploiting both major and minor
genes. Therefore, molecular mapping and cloning
of blast resistance genes has been one of the
important areas of research in rice genetics and
genomics. More than 70 dominant blast resistance
genes have been mapped on various chromosomes
of rice with tightly linked DNA markers. Besides,
more than 300 QTLs have also been reported
which are being exploited for breeding durable
blast resistant varieties. The decoding of rice
genome was completed in 2005 and a very high
quality genome sequence of rice is now made
available in public domain by the International
Rice Genome Sequencing Consortium (IRGSP
2005). In the post genomics era most of the gene
mapping and cloning activities are taking the help
of rice genome sequence data along with very well
planned genetic experiments for the cloning and
characterization of genes for blast resistance. This
sequence information can also be used for mining
novel genes for blast resistance. Plant genetic
resources are considered as reservoirs of gene pool
which can be exploited for the improvement of
various cultivars across crop species. Genetic
resource collections available within the gene
banks can be effectively utilized for the discovery
of genes and useful alleles using allele mining
approach. In simple words, allele mining is an
approach used for the identification of allelic
variation of relevant traits within genetic resource
collections. It is based on the exploitation of the
sequence information of one genotype to isolate
corresponding alleles from related genotypes. For
the cloned and functionally validated genes,
genetic resource collections may be screened for
allelic variation by tilling strategy and by using a
PCR based approach. Allele mining involves PCR-
based amplification and isolation of different
versions of genes found in germplasm lines, local
203
land races and wild species. Variation in gene
sequence is then correlated with the phenotype of
the target traits in these lines. Large collection of
germplasm lines can be grouped into core
collection based on their phenotypic evaluation
across the environments.
Development of new crop varieties is
dependent on the allelic variation present within
the breeding lines. The breeders make use of this
variation by combining useful alleles by different
breeding methods. Recent molecular tools can play
an important role in exploiting and using
potentially new alleles in genetic recombination.
The rice blast resistance genes cloned and
characterized at molecular level, can be the best
source of allele mining. The genetic diversity at
these loci over a wide range of germplasm can be
rapidly analyzed based on the sequence
information of these alleles. The rapid analysis of
allelic diversity in the gene pool of rice and its
relatives is facilitated by the availability of genetic
diversity at particular loci. This in turn allows the
molecular isolation of broad spectrum new blast
resistance alleles. The objectives of present paper
were to review all the published information on
genetics, mapping and cloning of rice blast
resistance genes and discuss the potential
applications of mining blast resistance
genes/alleles and high throughput genotyping
approaches in rice improvement programmes.
GENETICS OF RICE BLAST RESISTANCE
Studies on understanding the genetic basis of
resistance to rice leaf blast started in Japan in 1917
by Sasaki. For about 40 years many workers tried
different methods of genetic analysis using
different strains of the fungus. Hence, these studies
could not be compared among each other. After
1960, systematic work was started on the analysis
of the genetics of blast resistance across the world
(Kiyosawa 1967b; Kiyosawa and Cho 1980;
Kiyosawa and Ling 1984; Yamada et al 1976; Purba
et al 1994). Monogenic rice lines containing
individual resistance genes were developed by
introgression from original donor lines, which set
204 Institute of Biotechnology, Acharya NG Ranga Agricultural University, Hyderabad 500 030 India
the platform to analyze the genetic basis of
resistance to a great extent (Koide et al 2009). The
resistance to rice blast has been genetically
characterized as monogenic against different
strains of the pathogen, which basically provide
complete resistance to the targeted strains. More
than 73 single dominant resistance genes have
been reported for blast resistance in rice (Ballini et
al 2008). However, this type of resistance is mostly
broken down by the highly variable strains of M.
oryzae pathogen. In many cases, field resistance to
rice blast has been reported to be controlled by
quantitative trait loci (QTLs). The study of genetics
of blast resistance has already completed more
than 90 years. During this period tremendous
progress has been made in the identification and
characterization of blast resistance genes. In the
beginning, the focus of blast resistance research
was more on identification and characterization of
strains of M. oryzae and varieties/cultivars across
the world. Studies on QTL identification for blast
resistance in rice are relatively new. The first
analysis for QTLs associated with blast resistance
was published in 1994 (Wang et al 1994). Within
less than two decades, 347 QTLs have already been
identified and mapped in rice. This advancement
in QTL identification is because of the availability
of high quality genome sequence of rice (IRGSP
2005). Analysis of these QTLs, showed only 165
detectable Meta QTLs (Ballini et al 2008). These
loci have an average size of 3.3 Mbp. About 11% of
which have been finely mapped with DNA
markers. These QTLs have been identified based
on several independent experiments under
different environments and hence considered more
robust than others and were more effective against
a range of M. oryzae strains. The work on QTLs
reported for rice blast resistance has already been
reviewed (Ballini et al 2008). The cumulative effect
of OTLs on the spectrum of resistance is very high.
However, it is relatively difficult to use these QTLs
in breeding programmes in the absence of
diagnostic markers for selection of the
corresponding chromosomal regions. Besides, the
partial nature of resistance imparted by the
cultivars containing QTLs does not make this
approach very attractive to the rice breeders. In
contrast to the QTLs, monogenic resistance mostly
offers complete resistance against a set of pathogen
races. Such type of resistance is said to be
governed by gene-for-gene hypothesis (Flor 1971),
which implies that for every resistance gene
present in the host plant, there is a corresponding
Avr gene in the pathogen and the interaction
between R:Avr genes lead to incompatibility.
IDENTIFICATION AND MAPPING OF BLAST
RESISTANCE GENES
Genome wide analysis for the presence of disease
resistance genes and their chromosomal locations
are now known to a greater extent. It was reported
that the distribution of R-genes among twelve
chromosomes of rice is not random and has
followed a clustering pattern (Singh et al 2006).
Our analysis showed that most of the genes have
been found to be clustered on three hot spots of
chromosomes 6, 11 & 12 (Fig.1). Maximum number
(23) of blast resistance genes were reported on
chromosome 11 whereas from chromosome 3, no
blast resistance gene has been identified. Till date,
a total of 73 blast resistance genes have been
mapped in different rice varieties. Of these, 38.7%
have originated from japonica cultivars while
almost equivalent i.e. 36.5 % have been reported to
be mapped in the indica rice lines. Interestingly,
24.7 % of the mapped genes have also been
identified from the wild species of rice (Fig. 2).
Figure 1. Chromosome-wise distribution of mapped blast
resistance genes in rice
Genomics and Crop Improvement: Relevance and Reservation 205

on identification and genetics of different blast

resistance genes in rice, Pi-b was the first R-gene to
be cloned by the group of Takuji Sasaki in Japan in
the year 1999 (Wang et al 1999). However, even
after being the first R-gene to be cloned, not much
progress has been made in the utilization of Pi-b
for developing blast resistant cultivars.

The Pi-ta gene was cloned in the next year

Figure 2. Status of blast resistance genes cloned from
different Oryza sub species
While most of the mapped genes have been
reported from Japanese and Chinese laboratories, a
substantial number of them have also been
reported from other countries like USA, Korea,
France and India. Four rice blast resistance genes
i.e. Pi-kh or Pi-54 (Sharma et al 2005a), Pi-tp(t)
(Barman et al 2004), Pi-38(Gowda et al 2006) and
Pi-42(t) (Kumar et al 2010) have been mapped in
India. Of these, Pi-kh (Pi-54) has already been
cloned from rice line Tetep using positional
cloning approach (Sharma et al 2005b).
CLONING OF BLAST RESISTANCE GENES
in USA, with simultaneous cloning and
characterization of the corresponding Avr-Pi-ta
gene from M. oryzae (Orbach et al 2000; Bryan et al
2000). After a gap of five years, Pi-kh (now,
designated as Pi-54) was cloned and characterized
by our group from indica rice line Tetep (Sharma et
al 2005b Sharma et al 2010). Four genes located in
the region near to centromere on chromosome 6
were cloned in 2006. Of these, Pi-d-2 and Pi-9 were
cloned in China while Pi-2 and Piz-t from USA
(Chen et al 2006, Qu et al 2006, Zhou et al 2006). In
2007, two genes Pi-36 and Pi-37 were cloned, both
by the same group of Qinghua Pan in China (Liu et
al 2007 Lin et al 2007). The second gene to be
cloned from Pi-k locus was Pi-k-m in Japan
(Ashikawa et al 2008). One resistance gene was
cloned in each of the years 1999, 2000, 2005 and
2008 (Fig. 3). Four blast resistance genes have
been cloned in 2009. These were Pi-5 in Korea (Lee
et al 2009), Pi-t and Pi-21 in Japan (Hayashi et al
2006; Fukuoka et al 2009) and Pi-d3 in China
(Shang et al 2009).

Molecular cloning, functional validation and

determination of resistance spectrum of R-genes
are pre-requisites for their utilization in breeding
programmes effectively. The validated genes can
be used in the development of blast resistant
varieties conferring durable and broad spectrum
resistance to the pathogen. Further
characterization of the gene structure and its
domain architecture helps in understanding the
molecular mechanism of resistance response. This
information can further be utilized for engineering
durable resistance in modern rice varieties. Out of
14 genes cloned till date in rice, 5 have been cloned
in China, 4 in Japan, 3 in USA and one each in Figure 3. Cloning of blast resistance genes over the
India and Korea. After more than 75 years of study years
206 Institute of Biotechnology, Acharya NG Ranga Agricultural University, Hyderabad 500 030 India
Four genes have been cloned in 2006 and in 2009
and two were cloned in 2007. The rapid increase in
the rate of cloning of these genes during the last
five years is mainly due to the availability of high
quality rice genome sequence data in the public
domain (IRGSP 2005). Once a skeleton map of a
resistance locus is made, the genome sequence is
used to identify candidate blast resistance gene by
using various comparative genome approaches.
SOURCES OF CLONED BLAST RESISTANCE GENES
Rice varieties which have been used as a source of
resistance genes are very important for their
utilization in rice improvement programmes. Even
O. sativa subspecies indica or subspecies japonica
containing blast resistance genes have to be used
continuously in breeding programmes. Out of 14
genes cloned till date, 6 genes have been cloned
from five indica rice cultivars namely Tadukan,
Tetep, Digu, 5173 and Kasalath where as 5 genes
have been cloned from japonica rice cvs. Tohoku
IL9, Toride, St. No. 1, Tsuyake and Tjaha. The
diversity available in wild rice varieties has also
been tapped and a dominant blast resistance gene
Pi-9 has been cloned from Oryza minuta. The gene
Pi-5 has been cloned from RIL 260, which was
originally derived from a cross of resistant West
African upland rice cv. Moroberekan and
susceptible rice line Co 39. The recently cloned
recessive gene for blast resistance, Pi-21 has been
isolated from upland Japanese rice variety
Owarihatamochi.
STRATEGIES USED FOR CLONING RICE BLAST
RESISTANCE GENES
The most prevalent strategy that has been used to
clone blast resistance genes in rice is the very
tedious and time taking map based or positional
cloning approach. However, in the post-genomic
era, map based approach has sometimes been used
in combination with comparative in silico
approaches to simplify the cloning process. It has
resulted in significant reduction in time taken to
clone a gene. The Pi-b gene, which is located on
chromosome 6 at 154.1cM (Table 1), was finely
mapped between RFLP markers within a distance
of 0.015cM. Chromosome walking was performed
by constructing a cosmid library spanning the Pi-b
locus and three cosmid clones containing putative
Pi-b gene were used for genomic sequencing
(Wang et al 1999). They shortlisted 2 candidate
genes on the basis of homology searches of the
deduced amino acid sequences showing similarity
to the nucleotide binding site (NBS) found
predominantly in plant disease resistance genes.
Finally, transcriptional analysis of the
candidate genomic region and sequence analysis of
the cDNA clone helped in the identification and
cloning of the Pi-bgene (Wang et al 1999).
Molecular markers associated with the Pi-ta gene
located on chromosome 12 at 50.4 cM were
identified using RAPD markers and bulked
segregant analysis (Wu et al 1995). To clone this
gene BAC library was constructed and after
chromosome walking specific BAC clones were
sequenced. The putative Pi-ta gene was identified
in a 7.5 kb fragment on the basis of similarity to
central NBS region of NBS-LRR (leucine rich
repeats) resistance genes (Bryan et al 2000). The
third gene to be cloned in the series of rice blast
resistance genes was Pi-kh (Pi-54) by our group
(Sharma et al 2005b). The gene was first tagged
with RAPD marker S129700 and mapped to the long
arm of rice chromosome 11 (Sharma et al 2005a)
and isolated by using map based cloning approach
(Sharma et al 2005b). In case of the Pi-d2 gene, fine
mapping was done to identify closely linked CAPS
markers CAPS1 and CAPS8 (Chen et al 2006). Pi-d2
covering, an interval of 180 kb P1- artificial
chromosome (PAC) contig was identified using
rice genome sequence data. The candidate gene
was identified through gene prediction analysis
and bioinformatics analysis of this DNA sequence.
Out of the predicted 33 genes in this region, one
was predicted to encode a receptor like kinase
(RLK) gene (Chen et al 2006). Pi-9 gene is the first
and the only blast resistance gene which has been
derived and cloned from a wild species of rice.
This gene was fine mapped on rice chromosome 6
(Liu et al 2002). A slightly different approach was
used to clone this gene.
Genomics and Crop Improvement: Relevance and Reservation 207

Table 1. List of blast resistance genes cloned from different rice line.

Gene Cloning References

Name Donar cv. Chr. (cM) Associated Markers strategy and
year
Pib Tohoku IL9 (J) 2 154.1 Os02g57310, b213, b28, Map Based, 1999 Shinoda 1971,
b2, b3989, RM208, Hayasaka et al 1995,
S1916, G7031 Wang et al. 1999,
Fjellstorm et al 2004,
Pita Tadukan (I) 12 50.4 Os12g18360, SP4B9, Map Based, 2000 Bryan 2000, Hayashi 2006,
SP9F3, ta642, ta801, Jia 2002
ta3, ta577, Pi-ta 440, Pi-
ta 1042, Pi-ta 403
Pi54 Tetep (I) 11 101.9 TRS26, TRS33, RM206 Map Based, 2005 Sharma et al 2005a, 2005b,
h
( Pi-k ) 2010
Pid-2 Digu (I) 6 65.8 CAPS1, CAPS 8, Map Based, 2006 Chen et al 2006
Os06g29810
Pi9 O. minuta, 75-1- 6 58.7 Os06g17900 Map Based, 2006 Qu et al 2006
127 (I),
Pi-2, 5173 (I) 6 58.7 Z4792, Z5765, Z56592 Map Based, 2006 Zhou et al 2006, Hayashi et
Piz-t Fukunishiki 1, al 2006
Toride1 (J),
Pi36 Kasalath (I) 8 21.6- Os08g05440, CRG3 Map Based, 2007 Liu et al
25.2 2007
Pi37 St. No. 1 (J) 1 136.1 RM543, FPSM1, RM302, In Silico Map Chen et al 2005
RM212 Based, 2008
Pikm Tsuyake (J) 11 115.1- K2167, K4731, K6441, Map Based, 2008 Hayashi et al 2006,
117 85H07554, k3952 Ashikawa et al 2008

Pi5 RIL260, 9 31.3-33 JJ113-T3, JJ817 Map Based, 2008 Jeon et al 2003
Pit Tjaha (J) 1 12.2 t311, t256, t8042 Map Based, 2009 Hayashi et al 2009
Pid3 Digu (I) - - - In Silico Homology Shang et al 2009
Based, 2009
pi21 Owarihatamochi 4 58.6 RM16913, RM1359 Map Based, 2009 Fukuoka et al 2009

Two BAC clones of 58 kb and 40 kb,
forming a contig of 76 kb from the mapped region
were fully sequenced and the computational
analysis of DNA and predicted protein sequences
were performed. Six candidate genes similar to the
NBS-LRR disease R-genes were predicted.
Sequence comparison and phylogenetic analysis
along-with the characterisation of Pi-9 susceptible
mutants helped in the short listing of two
candidate genes. Complementation test and the
analysis of resistance spectrum of these candidate
genes finally lead to the identification of the actual
Pi-9 gene (Qu et al 2006). Two different R- genes
Pi-2 and Piz-t which are allelic to each other were
cloned simultaneously from two different rice
varieties. Pi-2 gene was earlier mapped at a
distance of 2.2 cM with R2123 and RG64 markers
on chromosome 6 at 58.7 cM, on the same locus
where Pi-9 gene was earlier mapped (Liu et al
2002). High resolution genetic mapping and
sequence analyses were applied to identify three
Pi-2 candidate genes. Finally, a single candidate for
Pi-2 was identified by characterization of the
susceptible mutant derived from Pi-2 carrying
plants. Piz-t gene, which is allelic to the Pi-2 was
cloned by allele mining of different candidates
from the same locus (Zhou et al 2006). Another
blast resistance gene Pi-36 was mapped on
chromosome 8 (Liu et al 2002).
A 17 kb interval spanning the gene locus
was sequenced and three candidates were
indentified on the basis of similarity to NBS-LRR
class of R-genes. These 3 genes were analyzed by
complementation test to identify the Pi-36 gene
(Liu et al 2007). Similar strategy of in silico map
based cloning was applied to clone the Pi-37 gene.
In this study, four NBS-LRR candidates were
transferred to susceptible rice line and finally the
Pi37 gene was cloned (Lin et al 2007). One of the
hot spots for blast resistance genes in rice is the Pi-
k locus on chromosome 11 which contains at least
208 Institute of Biotechnology, Acharya NG Ranga Agricultural University, Hyderabad 500 030 India
five genes. Pi-kh which was first cloned from this
locus from rice line Tetep (Sharma et al 2005b),
later on found to be a new gene because of its large
distance from the Pi-k locus. It was subsequently
designated as Pi-54 (Sharma et al 2010). On this
locus another gene Pi-km was found to be closely
linked to k2167 and k4731 markers (Hayashi et al
2006). They found 19 putative genes in 180 kb fine
mapped region of rice line Nipponbare genome
sequence. Of these, 6 candidates were found to
encode NBS-LRR class proteins. By comparing
with the corresponding DNA sequence from the
resistant rice line Tsuyake, two most probable
candidates were identified. Complementation test
of these two candidates performed independently
and in combination with each other uniquely
revealed that the resistance provided by Pi-km is
conferred by a combination of both of these genes,
residing adjacent to each other at the Pi-k locus
(Ashikawa et al 2008).
A very similar approach was applied for
the cloning of Pi-5 gene located on chromosome 9,
which again resulted in the revelation of two
closely placed genes which were responsible for
resistance conferred by Pi-5 gene (Jeon et al 2003).
Another blast resistance gene Pi-t was identified in
the Indonesian rice variety by Kiyosawa in 1972
and later the gene was reported to be tightly linked
to SNP marker t256, on chromosome 1 (Hayashi et
al 2006). A PCR polymorphism based analysis
identified a 34 kb region containing this gene. The
gene was identified by sequencing of this region
and using this sequence for gene prediction and
comparison with susceptible variety Nipponbare
(Hayashi et al 2009).
A new approach was used to identify and
clone Pi-d3 gene. Based on the genome wide
analysis of NBS-LRR genes in rice, the alleles of
NBS-LRR pseudogenes were used as co-
seggregating markers for blast susceptibility in the
F2 population developed from a cross between
blast-resistant indica variety Digu and the
susceptible japonica variety, Taipei 309. PCR-based
molecular markers were developed to amplify the
allelic gene pairs in both the parents. The
candidate pseudo gene was found on chromosome
6, which is an NBS-LRR allele, and was named Pi-
d3, owing to the location of this gene close to
earlier identified Pi-d(t)1 and Pi-d2 (Shang et al
2009). The most recent report for cloning of a blast
resistance gene is that of a recessive resistance
gene pi-21 which confers partial resistance to
different strains of M. oryzae. High resolution
mapping of this gene on chromosome 4 at 58.6 cM,
delimited the Pi-21 locus to a 1705-bp region
containing a single gene (Fukuoka et al 2009). It
clearly shows that with the availability of genome
sequence of rice in public domain, the pace of
cloning of blast resistance genes has increased and
new genes or alleles are being cloned from diverse
sources of resistance.
RESISTANCE GENE AND PROTEIN TYPES
The involvement of a particular protein in a
resistance signaling pathway can be predicted on
the basis of its domain composition. Most of the
rice blast resistance genes that have been
characterized till date encode NBS-LRR class of R-
proteins. However, the exact domain architecture
varies among these predicted proteins. Twelve of
these proteins have one conserved NBS domains.
The number of LRRs in each of them is variable,
and ranges from two repeats in Pi-54(Pi-kh) to 25
such repeats in the Pi-37 (Table 2). In addition to
conserved NBS-LRR domains, Pi-b protein also has
a cysteine rich domain, while Pi-9 and Pi-t also
have one and two CC domains, respectively. In the
Pi-54 (Pi-kh) protein, an additional zinc finger
domain is also present in between the NBS and
LRR domains. The protein encoded by Pid-2, is
found to be different from other cloned blast
resistance proteins of rice as it has a receptor like
kinase (RLK) domain in addition to an
extracellular trans-membrane (TM) domain. The
pi21 gene, which confers a race-non specific
resistance towards blast pathogen, has been shown
to encode a proline rich protein having heavy
metal binding domain as well as a protein-protein
interaction motif. Out of 14 R-proteins, 3 have
been shown to be localized in the cytoplasm, while
only one has been reported to be localized on
plasma membrane.
Genomics and Crop Improvement: Relevance and Reservation
EXPRESSION PATTERN AND MECHANISM OF ACTION
OF BLAST RESISTANCE GENES
Out of 14 genes cloned from rice, 7 genes have
been shown to express constitutively and seem to
have no effect of the fungal inoculation on their
expression (Table 2). In contrast, the expression of
Pi-bgene was induced significantly by the increase
in temperature and darkness, conditions favouring
disease development, whereas no effect of fungal
inoculation could be detected (Wang et al 1999).
The Pi-kh (Pi-54) gene has been reported as
pathogen inducible in nature (Sharma et al 2005b).
In case of Pi-5 gene, which comprises two
adjacently placed genes; one gene was induced by
fungal inoculation while the other one was found
to express constitutively (Lee et al 2008). The
expression of Pi-t gene was also not induced by the
blast pathogen but there was a significant
reduction in the expression level in disease
favourable conditions (Fukuota et al 2009). For the
development of long lasting blast resistant
varieties pathogen inducible gene could be more
effective and durable.
All of these R-genes are expected to
function by following gene-for-gene hypothesis
(Flor 1971). The corresponding Avr genes from
only a few of them have been studied. Avr-Pi-ta
was cloned simultaneously with the Pi-ta gene and
the protein pair was shown to interact directly
(Orbach et al 2000). The genes Pi-b, Pi-kh (Pi-54),
and Pi-37 have also been predicted to have their
corresponding Avr genes (Table 2). The
corresponding Avr gene for Pi-z (t) has also been
cloned and characterized (Li et al 2009). However,
till date except Avr-Pi-ta, protein products of none
of these genes have been shown to interact
physically. It is expected that with the availability
of M. oryzae genome sequence, many more Avr
genes will be cloned in future. This will help in
understanding molecular mechanism of blast
resistance in rice in greater detail.
UNDERSTANDING MOLECULAR BASIS OF BLAST
RESISTANCE IN RICE LINE TETEP
Rice line Tetep has been used in breeding rice
varieties for blast resistance for the past many
209
years. This is an indica type line which possesses
durable resistance to blast pathogen across India.
The first report on studying the genetics of blast
resistance in Tetep came in 1981 which showed
that this line contain a blast resistance gene which
is allelic to Pi-kh indentified from rice line HR22
(Kioysawa 1981). Since the Tetep line is tall, late
maturing and agronomically very inferior, most of
the time rice breeders avoided to use this line in
their breeding programmes. Tetep is also one of
the differential lines used for the characterization
of blast pathogen M oryzae.
In 1996, we started evaluation of rice line
Tetep for its effectiveness against the race flora of
M. oryzae from North Western Himalayan region
of India. Our major emphasis was on molecular
characterization of genetic variability in M. oryzae
pathogen population prevalent in NorthWestern
Himalayan region, identification of resistance (R)
genes effective against rice blast pathogen,
molecular mapping and positional cloning of rice
blast resistance gene, Pi-kh (now Pi-54). We
obtained very high variability in pathogenicity of
different isolates of M. grisea prevalent in North-
Western Himalayan region using DNA markers
and differential hosts (Sharma et al 2002). Besides,
we found that indica type rice line Tetep that
contain Pi-kh (in addition to other blast resistance
genes) which is highly effective against the
pathogen population of NorthWestern
Himalayan region of India (Sharma et al 2002;
Rathour et al 2004). For the mapping of Pi-kh gene
with DNA markers, rice line Tetep was used as a
resistant donor to develop a mapping population.
An F2 mapping population consisting of 205 plants
was generated by crossing Tetep with HP2216, a
highly susceptible cultivar.Inoculation with
specific isolate (PLP-1) of M grisea at seedling
stage showed that the Pi-kh gene inherited as a
single dominant gene in this population (Sharma
et al 2005a). More than 170 sequence tagged
microsatellite (STMS), sequence tagged sites (STS),
expressed sequence tag (EST) and simple sequence
repeat (SSR) markers used for genotyping of a
population of 208 F2 individuals.
210 Institute of Biotechnology, Acharya NG Ranga Agricultural University, Hyderabad 500 030 India

Table 2. Characteristic features of cloned rice blast resistance genes

Gen Protei Protein Type/Domain Localizati Expressio Promote GFG Promoter / Referenc
e n Size Architecture on n Pattern r Variety es
Nam in AA Transformed
e
Pib 1251 NBS-LRR Cytoplasmi Inducible 3.5 Kb R-Avr Native Wang et
1 NBS+17LRR+Cys Rich c (Pr) by-T, (Pr) Nipponbare al. 1999
Darkness,
No effect of
Mo
Pita 928 NBS-LRR Cytoplasmi Low 2.4kb Avr- Native Orbach et
1NBS+ LRD (10X) c Constitutive Pita Nipponbare al. 2000;
expression Bryan et
al. 2000.
Pi54 330 NBS-ZNF-LRR PM (Pr) Inducible 0.5 kb R-Avr Native Sharma et
(Pi- 1NBS+2LRR+1ZNF by Mo (Pr) Taipei 309 al. 2005;
h
k) Rai et al.
2009,
Sharma et
al.2010.
Pid- 825 RLK+ PM Constitutive NI NI 35S Chen et
2 Extraceluular/TM/Intracell Localized Nipponbare al. 2006.
ular kinase/Intracellular
kinase
Pi9 1032 NBS-LRR NI Constitutive NI NI Native Qu et al.
1NBS+17LRRs+ Taipae 309 2006
CCDomain (non-TIR)
Pi-2, 1030 NBS-LRR NI Constitutive NI AvrPi Native Zhou et al.
Piz-t NBS, Non-TIR, 17 LRRs z-t Nippanbare 2006
Pi36 1056 CC-NBS-LRR Constitutive NI NI Q1063 Liu et al.
2007
Pi37 1290 NBS-LRR Cytoplasm Slightly Native Avr Q1063 Lin et al.
1NBS +25 LRRs induced by Pi37 2007
Magnoport (Pr)
he oryzae
Pikm 1143 NBS-LRR NI Constitutive Native NI Native Ashikawa
1NBS + non-TIR + 16 Nipponbare et al. 2008
LRRs
1021 NBS-LRR
1NBS + 13 LRRs
Pi5 1025 CC-NBS-LRR NI Induced by 0.5kb NI Native Lee et al.
pathogen Dongjin (J) 2009
1063 Constitutive
Pit 989 CC-NBS-LRR NI No Renovat NI Ubiquitin/Nipponba Hayashi et
1NBS + 18 LRRs + 2CC induction or re, Native/ al. 2009
by Nipponbare
M.oryzae
Disease
favourable
condition
reduces the
expression
level.
Pid3 924 NBS-LRR NI Constitutive 3 kb NI Native/TP309 Shang et
1 NBS + 13 LRRs UTR al. 2009
pi21 263 Proline Rich Protein Cytoplasmi Slowly NI NI - Fukuoka
Heavy metal binding c Inducible et al 2009
machine + PP interaction by fungus
motif (3-6 hr)
AA = Numbers of amino acid residue, NI = no information available, GFG = available gene for gene information, Pr = predicted, Mo
= Magnaporthe oryzae, PP = protein-protein, RLK = receptor like kinase, CC = coiled coil, NBS = nucleotide binding site, LRR =
leucine rich repeat, TIR = toll interleukin receptor
Genomics and Crop Improvement: Relevance and Reservation
Genotyping combined with bioinformatics
analysis, high resolution map was constructed and
gene was mapped between two SSR markers,
TRS26 and TRS33 at 0.7 cM and 0.5 cM,
respectively. Later the Pi-kh gene was cloned
(Sharma et al 2005b). High titer genomic library
was also prepared for the identification of a
genomic clone containing Pi-kh gene with its
complete upstream and downstream sequences
from the rice blast resistant line Tetep. Structural
analysis of protein reveled that Pi-kh has central
nucleotide binding site (NBS) domain, zinc-finger
domain besides a unique leucine rich repeats
(LRR) domain (Madhav et al 2008).
With the advances in molecular genetics
and genomics of rice, the Pik locus has now been
mapped more precisely. Since there are two
reports on the mapping of Pi-kh gene from
different rice lines, there is some confusion in the
naming of this gene. The name of Pi-kh gene cloned
from the rice line Tetep has thus been designated
as per the standard guidelines of Committee on
Gene Symbolization, Nomenclature and Linkage
(CGSNL) and its physical location on rice
chromosome 11, which is ~2.5Mb away from the
Pik locus mapped recently. Hence, Pi-kh gene
cloned from Tetep was designated as Pi54 (Sharma
et al 2010). Functional complementation of cloned
Pi54 gene has already been reported Kumar et al
(2006). The complete story of expression analysis
of Pi54 (Pi-kh) gene complemented using transgenic
approach and its effectiveness against different
strains of M. oryzae has also been reported (Rai et
al 2009).
Analysis of structural organization of Pi-kh
(Pi54) locus in both japonica and indica rice lines
was performed (Kumar et al 2007a). For this
analysis, a 50 kb upstream and downstream
sequences flanking to the Pi-kh (Pi54) locus (100 kb
region) was selected to know the structural
organization. In this region, 15 genes in the
sequence of japonica and 16 genes in indica lines
were predicted and annotated. Average percent
GC content of japonica and indica genes in this
211
region was 49.3% and 53.15%, respectively. Both
japonica and indica sequences were polymorphic for
microsettalites having mono-, di-, tri-, tetra-, and
penta-nucleotide repeats. Sequence analysis of
specific blast resistant Pi-kh (Pi54) allele of Tetep
and susceptible allele of Nipponbare showed
differences in number and distributions of motifs
involved in phosphorylation, resulting resistance
phenotype in rice line Tetep (Kumar et al 2007a).
The resistance gene analogues (RGAs) were
amplified from the genomic DNA of 10 rice lines
having varying degree of resistance to M. oryzae by
using degenerate primers and various RGAs were
mapped in silico on different rice chromosomes
(Kumar et al 2007b ). Out of 98 RGAs obtained in
this study, 65 RGA clones showed more than 95%
homology with already cloned RGAs. Using
sequence homology approach, RGAs isolated in
this study were physically mapped on 23 loci on
chromosomes 1, 2, 3, 4, 5, 6, 7,8,10, 11 and 12.
Twenty RGAs were mapped near to the
chromosomal regions containing known
genes/QTLs for rice blast, bacterial leaf blight, and
sheath blight resistance.
Resistance gene dependent accumulation
of expressed sequence tags (ESTs) was studied in a
blast resistant, indica rice cultivar Tetep after
challenge inoculation with an incompatible race of
M. oryzae (Dikshit et al 2009). The nucleotide
sequence of 287 randomly selected cDNA clones
from the rice cDNA library constructed from the
RNA isolated after challenge inoculation of the
host was obtained and submitted in NCBI
Genbank (Accession Nos. DN475717 - DN475431).
Of these 184 (63%) ESTs were highly
representative of the rice transcriptomes. A set of
178 unique transcripts was identified after
assembly of 287 ESTs into unigenes. These
unigenes were categorized into 17 functional
groups (Dikshit et al 2009). The unigenes obtained
in this study were physically located on the
pseudomolecules of rice genome. This information
can be used for determining the arrays of genes
being expressed during Oryza sativa-M. oryzae
interactions which will be helpful in
212 Institute of Biotechnology, Acharya NG Ranga Agricultural University, Hyderabad 500 030 India
understanding molecular basis of disease
resistance.
ALLELE MINING FOR
BLAST RESISTANCE GENES
The availability of complete genome sequences of
rice subspecies indica and japonica in public domain
(http://rgp.dna.affrc.go.jp;
http://www.genomics.org.cn) has been an
important resource for the analysis of the genomes
of different rice cultivars to develop improved rice
varieties. This genome sequence information of
rice will also be useful to facilitate rapid and
inexpensive polymerase chain reaction strategies
such as primer walking to isolate useful alleles of
cloned and functionally validated rice genes from
a wide range of rice cultivars and wild species. The
available sequence data of rice will also assist allele
mining in other cereals like wheat which have
conserved synteny at genome level (Singh et al
2007). Allele mining approach will be important
for giving rice breeders direct access to key alleles
conferring resistance to biotic and abiotic stresses.
Mining is mainly used to find the novel alleles of
that gene and identification of SNPs within the
genes. Different methods can be used for SNP
detection in various crops. These are
oligonucleotide hybridization, nuclease cleavage of
mismatches, oligonucleotide ligation, primer
extension and direct sequencing.
In the recent years, TILLING (target
induced local lesions in genome) has been
developed as one of the most important allele
mining methods. It is a reverse genetic high
throughput approach used to identify SNPs and/or
InDels in the gene/genes of interest in a
population developed by mutation. It was
developed in late 1990s by Clairc McCullum for
characterizing the function of two chromo
methylase genes in Arabidopsis at the university of
Washington and Fred Hutchinson Cancer Research
Centre USA (Henikoff et al 2004).
The first step in tilling is creation of
mutagenized population by using chemical
mutagens such as ethyl methane sulphonate
(EMS). In this approach, seeds are first treated with
EMS and grown to produce M1. Then M2
population is developed by selfing of M1. From
M2 plants, leaf samples are collected for DNA
extraction and used for screening of mutants. Only
one M2 individual for each M1 is randomly
selected for DNA extraction and to avoid sampling
of same mutation. The M2 progeny can be self-
fertilized and the resulting M3 seeds can be
preserved in long term storage. Once the mutant
population is prepared, the genomic DNA targets
need to be selected. To evaluates the probable
effect of induced or natural polymorphisms on
gene function (Gilchrist et al 2005), a software,
codon optimised to detect deleterious lesions
(CODDLE, http://www.proweb.org/input) is used.
This software tool helps in designing of suitable
oligos for the amplification of specific target. The
next step is to isolate genomic DNA from the
population and normalization of DNA
concentration. Uniform quantity of DNA is crucial
to ensure accurate results. After normalization, the
samples can be pooled together. In general, for
diploid organisms a pool of DNA containing about
eight individual plants DNA can be successfully
used in mutation detection. The sensitivity of
mutation detection is decreased in the large pool
size since the proportion of heteroduplexes in the
reaction is reduced as compared to homoduplexes
(Henikoff et al 2003). The pooled DNA is arrayed
into 2D 96 well microtiter plates and amplified
using gene specific primers. The forward and
reverse primers are differentially labeled at 5` end
with IRD700 and IRD800 dye for fluorescent
detection at ~700 nm and ~800 nm, respectively.
The PCR products consist of heteroduplexes and
homoduplexes obtained from pooled DNA. These
are formed by heating (denaturing) and cooling
(annealing) of pooled samples which contain
mutants and the wild type. The amplified samples
are treated with endonuclease enzyme CEL I for a
short duration. The enzyme CEL I, isolated from
celery, which specifically recognizes mismatches in
the heteroduplex and also cleaves DNA on the 3`
side of the mismatch (McCallum et al 2000). After
digesting PCR product with CEL 1 enzyme, the
labeled DNA fragments are detected on a
denaturing polyacrylamide gel using a LI-COR
Genomics and Crop Improvement: Relevance and Reservation
4300 DNA analysis system. On separation 3 types
of fragments are obtained i.e. a full length product
and two cleaved fragments (one IRD700 labeled,
one IRD800 labeled). The sum of the cleaved
fragments should be equal to size of the full length
PCR product. The size of the cleaved fragments
can be calculated by comprising with the
standards. The approximate location of the
mutation could be identified and further
confirmed by sequencing. Once mutations are
identified, the software project aligned related
sequences and evaluate SNPs (PARSESNP,
http://www.proweb.org/ parsesnp/) can then be
used to display the locations of the polymorphisms
in a gene/ genes in different formats (Barkley et al
2008).
Rice has been the focus of a few TILLING
studies because of availability of extensive
molecular and genetic information. In 2005, a
report was published on the generation of a large
mutation population (60,000) using multiple
chemical mutagens on IR64, a widely grown indica
rice. This study demonstrated that TILLING was
suitable for reverse genetic studies with mutations
detected in two genes; inspite of the fact that the
mutational density in the population was fairly
low. In addition, extensive phenotypic variation
was assessed for the various chemical mutagens
used to develop the mutant population and
albinism was a predominant phenotype
irrespective of the mutagen used (Wu et al 2005).
In another study, EMS and Az-MNU were used to
induce an elevated mutational density in rice, with
57 polymorphisms identified from 10 target genes
by TILLING (Till et al 2007). Raghavan et al (2007)
demonstrated the efficacy of TILLING in rice to
detect mutations by separation of products on
agarose gels. These results were analogous to
pooling DNA and detecting mutations on a LI-
COR DNA analyzer.
To uncover the natural genetic variation as
opposed to induced mutation, another approach
named as ecotilling is used. This method is
applicable to species not amenable to chemical
213
mutagenesis. This approach can be rapidly used to
screen through many samples with a gene of
interest. It can identify naturally occurring SNPs
and / or small INDELS and their putative function
(Gilchrist et al 2005). The method was also found
to be useful to detect polymorphism in
microsatellite repeats. The method slightly differs
from TILLING in the sense that except using pools
from mutagenised lines, DNA from different
ecotypes are mixed with the reference DNA in a
ratio of 1:1. Rest of the steps are similar to that of
TILLING.
HIGH THROUGHPUT GENOTYPING
Illuminas GoldenGate assay, based on their
BeadArray/BeadChip technology, is one of the
most popular platforms providing a cost effective
assay for genotyping a fairly large collection of
samples for a customized pool of SNP markers in
parallel. The size of pool may range from 96 to
1536 SNPs. In this protocol high multiplexing can
be performed in a single reaction through highly
specific extension and amplification steps. The
most important feature of this assay is that
genomic DNA is directly used for genotyping and
does not require prior PCR amplification of the
target locus.
The DNA sample used in this assay is
activated for binding in paramagnetic particles.
This step is highly robust and requires minimum
input of DNA (250 ng at 50 ng/l). The
hybridization buffer, assay oligonucleotides and
paramagnetic particles are combined with
activated DNA for hybridization of DNA on
magnetic particles. Three oligonucleotides i.e. One
LSO (Locus Specific Oligo) and two ASO (Allele
Specific Oligo) are designed for each SNP locus in
this assay. All three oligonucleotide sequences
contain the regions of genomic complementarities
and universal PCR primer sites. The LSO also
contains a unique address sequence
complementary to a particular bead type. During
the hybridization process, oligonucleotides are
hybridized to the genomic DNA samples and
bound to paramagnetic particles. Following
214 Institute of Biotechnology, Acharya NG Ranga Agricultural University, Hyderabad 500 030 India
hybridization, several wash steps are performed to
retain hybridized oligonucleotides. The
mishybridized and excess assay oligonucleotides
are washed away during this step. After oligo
hybridization, a polymerase with high specificity
for 3` match is added which extends the ASO(s)
that perfectly match the target sequence at the SNP
sites. The polymerase used in this assay has no
strand displacement or exonuclease activity. Hence
this polymerase fills the gap between the ASO and
LSO. When reaches the LSO, the polymerase
simply drops off the genomic DNA. High locus
specificity is achieved in this assay since both the
ASO and LSO oligos hybridize to the same target
site. A DNA ligase seals the nick between the
extended sequence of the ASO and the LSO to
form PCR templates that can be amplified with
universal PCR primers, which carries allele specific
fluorescent label. After thermal cycling and
downstream-processing the single-stranded, dye-
labeled DNA products (the GoldenGate assay
products) are hybridized to their complement bead
type through their unique address sequences.
Hybridization of the GoldenGate assay products
onto the Sentrix Arrays allows for the readout of
the highly multiplexed SNP genotyping assay.
After the hybridization, Bead Array Reader is used
to analyze fluorescence signal on the Array Matrix
or BeadChip (Shen et al 2005).
SNP genotyping based on golden gate
assay has been found very successful in
constructing genetic maps, undertaking trait
mapping and association mapping. Golden gate
assay have been developed for several crop species
such as barley, wheat, soybean and maize. In 2008,
a report was published on using golden gate assay
in complex genome of soyabean for high
throughput genotyping. They used 384 SNPs
through the sequencing of 5 diverse accessions that
are parents of 3 Recombinant Inbred line (RIL)
mapping population. Allelic data was successfully
generated for 89% of SNP loci (342 out of 384) and
when this data was used in 3 Recombinant Inbred
line (RIL), it indicated that complex nature of
soyabean genome had little impact on conversion
of discovered SNP into usable assay (Hyten et al
2008). Custom golden gate assay containing 1536
SNPs developed from SNP database of maize and
used to genotype two recombinant inbreed lines
population (Zang 3 x 87-1 and B73 x By804) and
154 diverse inbreed lines (Yan et al 2009). Total 975
SNP markers were found to be polymorphic in at
least one of the 2 mapping population with the
polymorphic rate of 38.5% in Zang 3x87-1 and
52.6% in B73 x By804. The polymorphic rate for
individual SNP marker in pair wise comparison of
genotype tested, ranged from 03-63.8% with an
average of 36.3%. They stated that golden gate
assay appeared to be equally useful for diversity
analysis, marker trait association studies and
marker assisted breeding. Akhunov et al (2009)
had employed 53 homozygous tetraploid and 38
hexaploid wheat for SNP genotyping using
Illumina golden gate assay. After removing the
low quality data, genotyping error rate was 0%
and 1% for tetraploid and hexaploid wheat,
respectively. They demonstrated that this assay is
very efficient for high throughput genotyping of
polyploidy wheat and opening new possibilities
for the analysis of genetic variation in wheat and
dissection of genetic basis of complex trait using
association mapping approach. It is believed that
such golden gate assay should be available for
majority of crop species including rice.
ALLELE MINING FOR RICE BLAST RESISTANCE GENES
The success of any allele mining programme, for
the identification of novel alleles of genes is
dependent on the proper choice of genetic
material. The genetic material used for allele
mining should be representative of diverse genetic
origins, from which it can potentially offer novel
alleles. For this purpose, wild ancestors and local
land races are regarded as a potential reservoir of
useful alleles hidden in unattractive phenotypes
(Tanksley et al 1996). In rice, the greatest allelic
diversity has been commonly observed in the
accessions of Oryza spp. (Londo et al 2006;
Vaughan et al 2005). Using PCR-based approach,
Sharma et al (2009) analyzed 19 lines of O. sativa
for mining alleles of three blast resistant genes (Pi-
ta, Pi54[Pi-kh] and Pi-z(t)). Pi54(Pi-kh) and Pi-z(t)
alleles were more variable than Pi-ta allele in
Indian land races of rice.
Genomics and Crop Improvement: Relevance and Reservation 215

similar with the Pi-ta protein in all accessions of six

studied Oryza spp. It suggests that the homology
of Pi-ta was conserved before the divergence of
these Oryza spp. Yoshida and Miyashita (2009)
analyzed nucleotide diversity of Pi-ta gene in 26
accessions of wild rice (Oryza rufipogon), cultivated
rice (O. sativa) and two other related wild rice
species (O. meridionalis and O. officinalis). They
Figure 4. PCR Amplification of different fragments of Pi- detected dimorphic pattern of nucleotide
ta allele using 13 sets of primers in Beesginsali. bp- Base polymorphism and low nucleotide diversity at
pairs, M- Molecular weight marker and 1-13 set of replacement sites in LRD region of Pi-ta gene in
primers used for primer walking of Pi-ta gene both resistant and susceptible accessions of O.
rufipogon.
Coding region of Pi-ta allele was highly conserved
in all the land races whereas coding region of
Pi54(Pi-kh) and Pi-z(t) alleles were more variable.
The alleles of Pi-ta and Pi-z(t) gene have also been
explored (Gupta et al 2009; Thakur et al 2009) from
land races of rice collected from north-western
Himalayan region and Western Ghats of India by
utilizing the available sequence information of
these genes. The Pita allele was amplified (Fig. 4)
and sequenced from the land races and sequence
data were analyzed for the detection of SNPs and
InDels using bioinformatics tools. Subsequently,
the data was correlated with resistance
phenotypes. The Pi-ta region of the Indian land
races was conserved but some variations were
found in the coding region. Thakur et al (2009)
obtained 405 segregating sites and 150 InDel sites
in exonic region of Pi-z(t) amplified from 10 land
races (Fig. 5). NB-ARS domain was predicted in all Figure 5. Nucleotide diversity in exonic region of Pi-z(t)
the Pi-z(t) alleles cloned from the Indian land races allele amplified from 10 land races.
of rice except from land race Kijun. They also
observed that variation among amino acid The allele diversity of Pi-km genes, Pi-km1-
sequences in NB-ARS domain was very high. The TS and Pi-km2-TS in 16 elite rice cultivars and 35
Pita allele has also been mined by Wang et al land races have also been studied by PCR based
(2008) using molecular approaches like RT-PCR. In approach (Ashikawa et al 2009). By examining the
this study, a panel of 51 rice accessions consisting allele diversity of these two genes, they identified a
of cultivated rice, invasive weedy species of rice large number of polymorphic sites among these
and wild relatives, including AA and CC type alleles. However, the sequences were grouped into
genomes were sequenced to investigate the four haplotypes. Rice being one of the most
existence of variations in the exonic and intronic studied crop plants, we can expect many more
regions of the gene. reports on allele mining for blast resistance in
future. It will help in identification of novel
All Pi-ta variants were found to fall into variants of blast resistance genes which can be
two major clades and these variations were highly used in rice improvement breeding programmes.
216 Institute of Biotechnology, Acharya NG Ranga Agricultural University, Hyderabad 500 030 India
ACKNOWLEDGEMENTS
TR Sharma is thankful to the National Agricultural
Technology Project (ICAR) and Department of
Biotechnology, Govt. of India for financial
assistance.
LITERATURE CITED
Akhunov E, Nicolet C and Dvorak J. 2009. Single
nucleotide polymorphism genotyping in
polyploid wheat with the Illumina GoldenGate
assay. Theor Appl Genet 119:507-517.
Ashikawa I, Hayashi N, Yamane H, Kanamori H,
Wu J, Matsumoto T, Ono K and Yano M.
2008. Two adjacent nucleotide-binding site-
leucine-rich repeat class genes are required to
confer Pikm-specific rice blast resistance.
Genetics 180: 22672276.
Ashikawa I, Wu J, Matsumoto T and Ishikawa R.
2010. Haplotype diversity and molecular
evolution of the rice Pikm locus for blast
resistance. J Gen Plant Pathol 76:37-42.
Ballini E, Morel JB, Droc G, Price A, Courtois B,
Notteghem JL and Tharreau DA. 2008.
Genome-wide meta-analysis of rice blast
resistance genes and quantitative trait loci
provides new insights into partial and
complete resistance. MPMI 21: 859-868.
Barkley NA and Wang ML. 2008. Application of
TILLING and EcoTILLING as Reverse Genetic
Approaches to Elucidate the Function of Genes
in Plants and Animals. Curr Genomics 9:212-
226.
Barman SR, Gawda M, Venu RC and Chattoo BB.
2004. Identification of a major blast resistance
gene in the rice cultivar 'Tetep'. Plant Breed 123:
300-302.
Bryan GT, Wu KS, Farrall L, Jia Y, Hershey HP,
McAdams SA, Faulk KN, Donaldson GK,
Tarchini R and Valent B. 2000. A single amino
acid difference distinguishes resistant and
susceptible alleles of the rice blast resistance
gene Pi-ta. Plant Cell 12:20332045.
Chen X, Shang J, Chen D, Lei C, Zou Y, Zhai W,
Liu G, Xu J, Ling Z, Cao G, Ma B, Wang Y,
Zhao X, Li S and Zhu L. 2006. A B-lectin
receptor kinase gene conferring rice blast
resistance. Plant J 46:794804.
Dikshit R, Bhargava A, Dalal V, Plaha P, Singh
NK and Sharma TR. 2009. Accumulation of
defence response-related and unique
expressed sequence tags during the
incompatible interaction in the Oryza sativa
Magnaporthe oryzae pathosystem. J Phytopathol
157:483-489.
Fjellstrom R, Conaway-Bormans CA, McClung
AM, Marchetti MA, Shank AR, Park WD.
2004. Development of DNA markers suitable
for marker assisted selection of three Pi genes
conferring resistance to multiple Pyricularia
grisea pathotypes. Crop Sci 44:17901798.
Flor HH. 1971. Current status of the gene-for-gene
concept. Annu Rev Phytopathol 9:275-296.
Fukuoka S, Saka N, Koga H, Ono K, Shimizu T,
Ebana K, Hayashi N, Takahashi A, Hirochika
H, Okuno K and Yano M. 2009. Loss of
function of a proline-containing protein
confers durable disease resistance in rice.
Science 325: 998-1001.
Gilchrist EJ and Haughn GW. 2005. TILLING
without a plough: A new method with
applications for reverse genetics. Curr Opin
Plant Biol 8:211-215.
Gowda M, Barman SR and Chattoo BB. 2006.
Molecular mapping of a novel blast resistance
gene Pi38 in rice using SSLP and AFLP
markers. Plant Breed 125: 596-599.
Gupta YK, Singh PK, Thakur S, Sonah H, Upreti
HC, Rathour R, Variar M, Prashanthi SK,
Singh AK, Singh UD, Singh NK and Sharma
TR 2009. Exploring the allelic variants of blast
resistance gene Pi-ta in Indian land races of
rice. 5th Int Conf, on Plant Pathology, IARI,
Indian Agric Res Inst, New Delhi, 151p.
Hayasaka H, Miyao A, Yano M, Matsunaga K and
Sasaki T. (1996) RFLP mapping of a rice blast
resistance gene Pi-k. Breed Sci 46 (Suppl 2): 68 .
Hayashi K, Yoshida H and Ashikawa I. 2006.
Development of PCR-based allele specific and
InDel marker sets for nine rice blast resistance
genes. Theor Appl Genet 113: 251-260.
Hayashi K. and Yoshida H. 2009.
Refunctionalization of the ancient rice blast
disease resistance gene Pit by the recruitment
Genomics and Crop Improvement: Relevance and Reservation
of a retrotrasnposon as a promoter. Plant J 57:
413-425.
Henikoff S and Comai L. 2003. Single-nucleotide
mutations for plant functional genomics.
Annu. Rev. Plant Biol. 54:375-401.
Henikoff S, Till BJ and Comai L. 2004. TILLING:
Traditional mutagenesis meets functional
genomics. Plant Physiol 135:630-636.
Hyten DL, Song Q, Choi IY, Yoon MS, Specht
JE, Matukumalli LK, Nelson RL, Shoemaker
RC, Young ND and Cregan PB. 2008. High-
throughput genotyping with the GoldenGate
assay in the complex genome of soybean. Theor
Appl Genet 116:945-952.
IRGSP, International Rice Genome Sequencing
Project. 2005. - The map based sequence of the
rice genome. Nature 436:793-800.
Jeon JS, Chen D, Yi GH, Wang GL and Ronald
PC. 2003 Genetics and physical mapping of
Pi5(t), a locus associated with broad- spectrum
resistance to the rice blast. Mol Gen Genomics
269: 280-289.
Kiyosawa S and Chao CI. 1980. Identification of
blast resistance genes in Korean rice variety,
Tonigil. Japan J Breed 30: 73-82.
Kiyosawa S and Ling ZZ. 1984. Identification of
genes for rice blast resistance in Chinese
differential varieties with Japanese fungus
strains. Scientia Sincia 27: 273-283.
Kiyosawa S. 1967a. The inheritance of resistance of
the Zenith type varieties of rice to blast fungus.
Japan J Breed 17: 99-107.
Kiyosawa S. 1967b Inheritance of resistance in rice
variety Pi No. 4 to blast. Japan J Breed 17: 165-
172.
Kiyosawa S. 1981. Genetic analysis of blast
resistance. Oryza 18: 196203.
Koide Y, Kobayashi N, Xu D and Fukuta Y. 2009.
Resistance genes and selection DNA markers
for blast disease in rice (Oryza sativa L.). JARQ
43: 255-280
Kumar P, Pathania S, Katoch P, Sharma TR,
Plaha P and Rathour R. 2010. Genetics and
physical mapping of blast resistance gene Pi-
42(t) on the short arm of rice chromosome 12.
Mol Breed 25: 217-228.
217
Kumar SP, Dalal V, Singh NK and Sharma TR.
2007a. Comparative analysis of the 100 kb
region containing the Pi-kh locus between
indica and japonica rice lines. Genom, Proteom
Bioinform 5:35-44.
Kumar SP, Gautam N, Rai AK, Upreti HC, Singh
NK and Sharma TR. 2006. Genetic
transformation of rice line Taipei 309 with
cloned blast resistance gene Pi-kh. In: 2nd Int
Rice Congr, Oct 9-13, 2006, New Delhi, India.
Kumar SP, Dalal V, Singh NK and Sharma TR.
2007b. Cloning and in silico mapping of
resistance gene analogues isolated from rice
lines containing known genes for blast
resistance. J Phytopathol 155:273-280.
Lee SK, Song MY, Seo YS, Kim HK, Ko S, Cao PJ,
Suh JP, Yi G, Roh JH, Lee S, An G, Hahn TR,
Wang GL, Ronald P and Jeon JS. 2009. Rice
Pi5-mediated resistance to Magnaporthe oryzae
requires the presence of two coiled-coil-
nucleotide-binding-leucine-rich repeat genes.
Genetics 181:1627-1638.
Li W, Wang B, Wu J, Lu G, Hu Y, Zhang X, Zhang
Z, Zhao Q, Feng Qi, Zhang H, Wang Z, Wang
G, Han B, Wang Z and Zhou B. 2009. The
Magnaporthe oryzae avirulence gene AvrPiz-t
encodes a predicted secreted protein that
triggers the immunity in rice mediated by the
blast resistance gene Piz-t. MPMI 22: 411-420.
Lin F, Chen S, Que Z, Wang L, Liu X and Pan Q.
2007. The blast resistance gene Pi37 encodes a
nucleotide binding site-leucine-rich repeat
protein and is a member of a resistance gene
cluster on rice chromosome 1. Genetics
177:18711880.
Liu G, Lu G, Zeng L and Wang GL. 2002. Two
broad-spectrum blast resistance genes, Pi9(t)
and Pi2(t), are physically linked on rice
chromosome 6. Mol Genet Genomics 267: 472
480.
Liu X, Lin F, Wang L and Pan Q. 2007. The in
silico map-based cloning of Pi36, a rice coiled-
coil-nucleotide-binding site-leucine-rich repeat
gene that confers race-specific resistance to the
blast fungus. Genetics 176:25412549.
218 Institute of Biotechnology, Acharya NG Ranga Agricultural University, Hyderabad 500 030 India
Londo JP, Chiang YC, Hung KH, Chiang TT and
Schaal BA. 2006. Phylogeography of Asian
wild rice, Oryza rufipogon, reveals multiple
independent domestications of cultivated rice,
Oryza sativa. Proc Natl Acad Sci 103:9578-9583.
Madhav MS, Plaha P, Singh NK and Sharma TR.
2008. Molecular characterization of a genomic
fragment containing Pi-kh gene from the
genomic library of Indica rice line Tetep. J
Phytopathol 157: 5.
McCallum CM, Comai L, Greene EA and
Henikoff S. 2000. Targeting induced local
lesions in genomes (TILLING) for plant
functional genomics. Plant Physiol 123: 439-442.
Orbach MJ, Farrall L, Sweigard JA, Chumley FG
and Valent B. 2000. A telomeric avirulence
gene determines efficacy for the rice blast
resistance gene Pi-ta. Plant Cell 12, 20192032.
Purba D, Kiyosawa S, Ando I and Furutani T.
1994. Estimation of functional values of field
resistance genes to blast disease in some rice
varities. Breed Sci 44: 285-293.
Qu S, Liu G, Zhou B, Bellizzi M, Zeng L, Dai L,
Han B and Wang GL. 2006. The broad-
spectrum blast resistance gene Pi9 encodes a
nucleotide-binding site-leucine-rich repeat
protein and is a member of a multigene family
in rice. Genetics 172:19011914.
Raghavan C, Naredo MEB, Wang HH, Atienza G,
Liu B, Qiu FL, McNally KL and Leung H.
2007. Rapid method for detecting SNPs on
agarose gels and its application in candidate
gene mapping. Mol Breed 19:87-101.
Rai AK, Kumar SP, Gautam N, Gupta SK, Chand
D, Singh NK and Sharma TR. 2009 Cloned
rice blast resistance gene Pi-kh confers broad
spectrum resistance to Magnaporthe oryzae. XIV
Int Cong Molecular Plant-Microbe
Interactions, Quebec City, Canada 2009 10687.
Rathour R, Singh BM, Sharma TR and Chauhan
RS. 2004. Population structure of Magnaporthe
grisea from north-western Himalayas and its
implications for blast resistance breeding of
rice. J. Phytopathol 152:304-312.
Sasaki R. 1922. Existence of strains in rice blast
fungus. Int J Plant Prot, Tokyo 9: 631-644.
Shang J, Tao Y, Chen X, Zou Y, Lei C, Wang J, Li
X, Zhao X, Zhang M, Lu Z, Xu J, Cheng Z,
Wan J and Zhu L. 2009. Identification of a new
rice blast resistance gene, Pid3, by genome
wide comparison of paired nucleotide-binding
site-Leucine rich repeat genes and their
pseudogene alleles between the two sequenced
Rice Genomes. Genetics 182: 1303-1311.
Sharma TR, Chauhan RS, Singh BM, Paul R,
Sagar V and Rathore R. 2002. RAPD and
pathotype analysis of Magnaporthe grisea
population from north-western Himalayan
region of India. J Phytopathol 150: 649-656.
Sharma TR, Gupta YK, Thakur S, Singh PK,
Upreti HC, Singh NK, Singh AK, Singh UD,
Rathour R, Kapoor AS, Kaushik RP, Variar
M, Maiti D, Mandal NP, Prashanthi SK and
Hanamaratti NG. 2009 Allele mining for
important blast resistance genes from land
races of rice. In: 6th Int Rice Genet Symp, Manila
Hotel, Manila, Philippines, P 3-59.
Sharma TR, Madhav MS, Singh BK, Shanker P,
Jana TK, Dalal V, Pandit A, Singh A,
Gaikwad K, Upreti HC and Singh NK.
2005b. High-resolution mapping, cloning and
molecular characterization of the gene of rice,
which confers resistance to rice blast. Mol
Genet Genomics 274:569-578.
Sharma TR, Rai AK, Gupta SK and Singh NK.
2010. Broadspectrum blast resistance gen Pi-kh
cloned from rice line Tetep designated as Pi54.
J Plant Biochem Biotech 19: 87-89.
Sharma TR, Shanker P, Singh BK, Jana TK,
Madhav MS, Gaikwad K, Singh NK, Plaha P
and Rathour R. 2005a. Molecular mapping of
rice blast resistance gene Pi-kh in the rice
variety Tetep. J Plant Biochem Biotech 14:127-
133.
Shinoda H. 1971. Studies on the varietal resistance
of rice to blast. 6. Linkage relationship of blast
resistance genes. Bull. Chugoku Agric Exp Stn
Ser A 20 : 125 [In Japanese].
Singh NK, Dalal V, Batra K, Singh BK, Chitra G,
Singh A, Ghazi IA, Yadav M, Pandit A, Dixit
R, Singh PK, Singh H, Koundal KR, Gaikwad
K, Mohapatra T and Sharma TR. 2007. Single-
copy genes define a conserved order between
Genomics and Crop Improvement: Relevance and Reservation
rice and wheat for understanding differences
caused by duplication, deletion, and
transposition of genes Funct Integ Genomics 7:
17-35.
Singh S, Dalal V, Singh NK, Chand S, Singh NK
and Sharma TR. 2006. Genome wide analysis
of disease resistance and defense response
genes in rice. In: 2nd Int Rice Cong, Oct 9-13,
2006, Indian Agric Res Inst, New Delhi, India.
Takahashi Y. 1965. Genetics of resistance to rice
blast disease. In: The rice blast disease. Johns
Hopkins Press, Baltimore, Maryland pp 303-
329.
Tanksley SD, Granadillo S, Fulton TM, Zamir D,
Eshed T, Petiard T, Lopez V and Beckbunn T.
1996. Advanced backcross QTL analysis in a
cross between an elite processing tomato and
its wild relative, L. pimpinelliforum. Theor Appl
Genet 92:213-224.
Thakur S, Gupta YK, Singh PK, Sonah H, Upreti
HC, Rathour R, Variar M, Prashanthi SK,
Singh AK, Singh UD, Singh NK and Sharma
TR. 2009 Cloning and characterization of Pi-
z(t) allele from indian land races of rice. In: 5th
Int Conf, Plant Pathology, Indian Agric Res
Inst, New Delhi.
Till BJ, Cooper J, Tai TH, Colowit P, Greene EA,
Henikoff S and Comai L. 2007. Discovery of
chemically induced mutations in rice by
TILLING. BMC Plant Biol 7:19.
Vaughan DA, Kadowaki K, Kaga A and Tomoka
N. 2005. On the phylogeny and biogeography
of the genus Oryza. Breed Sci 55:113-122.
Wang GL, Mackill DJ, Bonman JM, McCouch SR,
Champoux MC and Nelson RJ. 1994. RFLP
mapping of genes conferring complete and
partial resistance to blast in a durably resistant
rice cultivar. Genetics 136:1421-1434.
Wang X, Jia Y, Shu QY and Wu D. 2008.
Haplotype diversity at the Pi-ta locus in
cultivated rice and its wild relatives.
Phytopathology 98:1305-1311.
Wang ZX, Yano M, Yamanouchi U, Iwamoto M,
Monna L, Hayasaka H, Katayose Y and
219
Sasaki T. 1999. The Pib gene for rice blast
resistance belongs to the nucleotide binding
and leucine-rich repeat class of plant disease
resistance genes. Plant J 19:5564.
Wu JL, Wu C, Lei C, Baraoidan M, Bordeos A,
Madamba MR, Ramos-Pamplona M,
Mauleon R, Portugal A, Ulat VJ, Bruskiewich
R, Wang G, Leach J, Khush G and Leung H.
2005. Chemical- and irradiation-induced
mutants of indica rice IR64 for forward and
reverse genetics. Plant Mol Biol 59:85-97.
Wu KS, Martinez C, Lentini Z, Tohme J,
Chumley FG, Scolnik PA and Valent B. 1995.
Cloning a blast resistance gene by
chromosome walking. In: Rice Genetics III. Proc
Third Int Rice Genetics Symp, 16-20 Oct1995 GS
Khush, ed International Rice Research
Institute, Manila, Philippines, pp 669-674.
Yamada M, Kiyosawa S, Yamaguchi T, Hirano T,
Kobayashi T, Kushibuchi K and Watanabe, S.
1976. Proposal of new method for
differentiating races of Pyricularia orzae Cavara
in Japan. Ann Phytopathol Soc Japan 42: 57-58.
Yamada M, Kiyosawa S, Yamaguchi T, Hirano T,
Kobayashi T, Kushibuchi K and Watanabe, S.
1976. Proposal of new method for
differentiating races of Pyricularia orzae Cavara
in Japan. Ann Phytopathol Soc Japan 42: 57-58.
Yan J, Yang X, Shah T, Villeda H, Li J,
Warburton M, Zhou Y, Crouch HJ and Xu Y.
2009. High-throughput SNP genotyping with
the GoldenGate assay in maize. Mole Breed
10.1007/s11032-009-9343-2.
Yoshida K and Miyashita NT. 2009. DNA
polymorphism in the blast disease resistance
gene Pi-ta of the wild rice Oryza rufipogon and
its related species. Genes Genet Syst 84:121-136.
Zhou B, Qu S, Li G, Dolan M, Sakai H, Lu G,
Bellizzi M and Wang GL. 2006. The eight
amino-acid differences within three leucine-
rich repeats between Pi2 and Piz-t resistance
proteins determine the resistance specificity to
Magnaporthe grisea. Mol Plant Microbe Interact
19: 12161228.