Plant breeding

Identifying novel alleles of rice blast resistance

genes Pikh and Pita through allele mining
G. Ramkumar, A.K. Biswal, K. Madhan Mohan, K. Sakthivel, A.K.P. Sivaranjani, C.N. Neeraja, T.
Ram, S.M. Balachandran, R.M. Sundaram, M.S. Prasad, B.C. Viraktamath, and M.S. Madhav,
Directorate of Rice Research, Rajendranagar, Hyderabad 500030, India
E-mail: sheshu_24@yahoo.com

Rice is the staple food for more than half of the world’s population. Demand for
rice continues to increase due to the ever-increasing rice consumer base.
However, the present rate of increase in rice production (2000-09) has slowed
down (1.21%) compared with that of previous decades (1970-90, 2.49%; 1990-
2000, 1.70%) due to various biotic and abiotic stresses (Khush and Jena 2009).
Among the biotic stresses, blast, a disease caused by Magnaporthe oryzae, is the
most devastating, leading to a 90% yield loss in rice. Although many resistant
varieties have already been bred, the breakdown of blast resistance causes yield
instability in several rice-growing areas in the world. Of the various strategies
tried, enhancement of host-plant resistance is considered one of the important
approaches to tackle this disease. Pyramiding of two or more effective resistance
genes/alleles into elite cultivars enhances the durability and spectrum of
resistance. Hence, identifying superior alleles of effective resistance genes can be
immensely helpful in increasing the degree of resistance and can be valuable in
breeding durable resistance for blast disease (Ramkumar et al 2010).
Of the various blast resistance genes characterized, Pikh and Pita are the
two major genes found to confer broad-spectrum resistance to blast in India.
With the identification and molecular characterization of Pikh (Madhav et al
2005) and Pita genes (Bryan et al 2000), it is now possible to dissect the naturally
occurring allelic variation in these genes from a wide range of germplasm
through allele mining. In our study, we used a sequence-based allele mining
strategy to isolate novel resistance alleles of Pikh and Pita from landraces and
wild Oryza species and studied their effectiveness against blast.
The plant materials used in this study were 27 landraces collected from
northeastern India, known for its rich diversity, and 127 accessions of different
Oryza species collected from IRRI. The landraces and wild species were screened
for disease resistance using the most virulent pathogen race in southern India,
NLR-1; the spray and punch inoculation methods were employed. Inoculations
were repeated twice with three replications. Linked markers—RM206, TRS26,

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and TRS33 for Pikh (Madhav et al 2005) and YL153, YL154, YL155, and YL87 for
Pita (Jia et al 2002)—were also used to screen materials based on marker profiles
to downsize the number of genotypes for allele mining.
Using available gene sequence information at NCBI, primer pairs were
designed to amplify the entire allelic region of the Pikh gene in such a way that
the forward primer targets the upstream region (~500 bp before the transcription
start site) and the reverse primer targets the 3′ UTR region. These primers were
optimized for PCR and the Pikh and Pita alleles were amplified from the selected
genotypes using high-fidelity Taq DNA polymerase (Fermentas, USA). PCR was
performed in a 20-µL reaction using standard protocol with the thermal profile of
94 °C for 5 min (initial denaturation), followed by 35 cycles of denaturation at 94
°C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 2 min for the
Pikh allele and 5 min for the Pita allele, respectively, and a final extension of 10
min at 72 °C. The amplicons were cloned using the ZeBaTA cloning vector (Chen
et al 2009). Four clones per amplicon were sequenced at BioServe Biotechnologies
Pvt. Ltd., Hyderabad. The high- quality sequences (Phred score >20 per base)
were compared with the reported gene sequence using clustal W
(www.ebi.ac.uk/Tools/clustralw/). The structural features of the gene and
amino acid (aa) sequence variation were analyzed by FGENESH software
(available at www.softberry.com). Conserved domains such as NBS, LRR, and p
loop were analyzed based on available literature information and using the
conserved domain database of the NCBI
(www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).
Based on phenotyping data, highly resistant and susceptible landraces
and wild Oryza accessions were selected. Using the optimized primers, the Pikh
and Pita allelic regions were amplified, cloned, and sequenced from 10 and four
genotypes, respectively. The sequence information has been submitted to
GenBank (Tables 1 and 2). Sequence comparison at the nucleotide level of
various Pikh alleles with the reported Tetep Pikh allele revealed that alleles of the
wild species exhibited more variation than did those from the landraces (Table
1). These sequence polymorphisms altered the gene structure in such a way that
susceptible alleles (LS1 and LS2) did not have any open reading frame (ORF) for
functional protein; all the other remaining alleles have shown ORFs. The
resistant (LR1, LR2) and susceptible alleles (LS1, LS2) of the landraces showed
considerable differences in nucleotide level, indicating common polymorphism
of 45 SNPs and four indels in the coding region and 10 SNPs in the noncoding
region (upstream to transcription start site), whereas alleles derived from wild
species did not show any common variation among resistant and susceptible
alleles.

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Table 1. Genotypes selected for Pikh allele mining with phenotype for blast
resistance and allele sequence details (R = resistant, S = susceptible).

Name Pheno- Pikh InDels Allele InDels Protein Substitution

type acc. no. SNPs in the length SNPs in length in protein InDels in
with in the entire of in ORF (AA) level (aa) protein
code entire allele Pikh ORF of Pikh level
allele (bp) (bp)
(bp)
Tetep R AY914077 – – 991 – – 330 – –
Ammana Bavo LR1 GU258506 13 1 1,206 2 1 401 3 2
insertion insertion insertions
Boha Thulasi LR2 GU258507 7 1 1,206 – 1 401 – 2
Joha insertion insertion insertions
Podumoni Ahu LS1 GU258508 7 – 0 – – 0 – –
Bizor II LS2 GU258499 19 1 0 – – 0 – –
insertion
O. rufipogon WR1 GU258500 45 2 1,023 37 2 340 20 2
(acc. no. insertions insertions/ insertions/
106507) 1 deletion 1 deletion
O. barthii WR2 GU258501 48 1 1,029 29 1 343 15 1
(acc. no. insertion insertion/ insertion/
100931) 1 deletion 1 deletion
O. latifolia WR3 GU258505 54 6 537 42 2 178 19 3
(acc. insertions deletions deletions
o.101207)
O. rufipogon WS1 GU258502 19 1 1,206 3 2 401 2 2
(acc. no. insertion/ insertions insertions
106279) 1
deletion
O. rufipogon WS2 GU258503 56 1 1,068 34 1 356 16 1
(acc. no. insertion insertion insertion
106512)
O. rufipogon WS3 GU258504 47 2 1,146 35 2 381 18 2
(acc. no. insertions insertions insertions
81978)

Although the selected Pikh alleles code for the NBS-LRR class of
proteins, sequence variations were observed in the protein sequences and also in
the conserved domains of proteins when these were compared with Pikh of
Tetep. The wild rice WR1 protein has a substitution of 20 aa residues, a deletion
of 28 aa, and an insertion of 13 and 25 aa at two regions, leading to an overall
increased protein length of 10 aa in Tetep’s Pikh. These variations included one
aa substitution (phenylalanine replaced tyrosine) in the NBS domain and another
aa substitution (alanine replaced threonine) in the LRR domain. WR2 protein has

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a substitution of 15 aa, an insertion of 46 aa, and a deletion of 33 aa, leading to an

increase in the final protein length of 13 aa. This protein has the same LRR
domain as that of Pikh (Tetep) but does not have the homology in the NBS
domain. WR3 protein had substitutions of 19 aa and deletions at three places,
which led to a loss of 152 aa in Pikh (Tetep). This protein also does not share a
significant homology in the NBS and LRR domains of Tetep Pikh. The proteins
from landraces (LR1, LR2) have shown the presence of two insertions of 46 and
25 aa (Fig. 1) and substitution of 4 aa and 1 aa, respectively, leading to the
addition of 71 aa in protein length in both cases. In the LR1 protein, the NBS
domain has one aa substitution (glycine replaced glutamic acid), whereas the
LRR domain did not have any change. The LR2 protein did not have any change
in NBS and LRR domains. When the effect of resistance alleles of Pikh on disease
incidence was tested with a set of 25 isolates, the resistance allele derived from
wild species WR-1 (O. rufipogon accession no. 106507) showed higher resistance
than all other alleles (including Tetep), which has substitutions in both NBS and
LRR domains compared with the Pikh allele derived from Tetep. WR-1 (O.
rufipogon accession no. 106507) was further checked with inked markers of
different blast genes (20 tested) and was shown to have only one allele of Pikh.

Fig. 1. Comparison of amino acid sequence of the reported Pikh gene with Pikh alleles of LR1 and LR2.

The sequence comparison of Pita alleles with the reported Tadukan Pita
allele revealed the presence of more variations in the noncoding region than in
the coding region (Table 2). The resistance (LR1, LR2) and susceptible alleles
(LS1, LS2) of landraces differed considerably at the nucleotide level with the
presence of two SNPs and five SNPs, respectively. Both susceptible alleles (LS1
and LS2) also showed a variation similar to that of the Tadukan allele
(resistance)—presence of eight common SNPs at the nucleotide level, leading to
one aa substitution (serine instead of phenylalanine) at 641 residue at the protein
level. Interestingly, Bryan et al (2000) also reported one aa substitution (serine
instead of alanine) among resistant (Tadukan, Yashiro-mochi) and susceptible
(C101A51) sources. Individual susceptible alleles have one more substitution in
addition to the common variation when compared with Tadukan Pita. However,
there was no significant change in the amino acid length (902 aa) of the deduced

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protein of the various Pita alleles, except for LR1 (Table 2). Upon comparative
analysis of the protein sequences with the reported Pita, the Pita protein of LR1
showed a substitution of 3 aa in the LRR domain and a deletion of 19 aa. This
protein has the pat super family (patatin and phospholipases) domain from 705
to 747 aa, which was not present in the reported Pita of Tadukan. However, there
was no change in the NBS domain (which has three other kinds of domain). The
Pita protein of LR2 and LS1 showed 2 aa substitutions in the LRR domain, while
LS2 showed a single aa substitution in the LRR domain of the protein. However,
we observed much conservation in the NBS of these proteins.

Table 2. Genotypes selected for Pita allele mining with phenotype for blast
resistance and allele sequence details (R = resistant, S = susceptible).

Name Phenotype Pita accession SNPs/ SNPs Protein Substitutions/

with code no. InDels in /InDels in length deletions in the
the entire ORF (AA) of protein level
allele Pita

Tadukan R AF207842 – – – –
Ammana Bavo LR1 GU269201 9 SNPs / SNPs/ 888 3 substitutions/
1 deletion 1 deletion 1 deletion
Boha Thulasi LR2 GU269202 21 5 902 4
Joha
Podumoni Ahu LS1 GU269203 7 3 902 2
Bizor II LS2 GU269204 19 4 902 2

In conclusion, many functional Pikh and Pita alleles were successfully

cloned from a range of landraces and wild species. Among the selected
genotypes, Pikh alleles showed more variations than Pita alleles. Since the Pikh
alleles of wild species have much more diverse sequences than those of the
landraces, it is presumed that the Pikh allele might have migrated to different
wild species long before and it could have transferred to cultivated rice. Allelic
sequence analysis revealed the presence of several variations in nucleotide and
protein sequences of the alleles and changes in the conserved domains, which
may explain the resistance/susceptibility of the gene. As these alleles have
different functional polymorphisms at the protein level, the interaction with
corresponding Avr proteins may also change. The efficacy of the identified
resistance alleles of both genes (in LR1, LR2, WR1, WR2, and WR3) can be further
tested using all the isolates collected from India. This can become a resource for

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use in conjunction with known alleles to develop durable resistance in upcoming

blast resistance breeding programs.

References
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Acknowledgment
We thank the Department of Biotechnology, Government of India, New Delhi, for funding
assistance.

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