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Identification of Novel Alleles of the Rice Blast Resistance Gene Pi54


Identification of Novel Alleles of the Rice Blast Resistance Gene Pi54

Kumar Vasudevan et al. Sci Rep.

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Rice blast is one of the most devastating rice diseases and continuous resistance breeding is required to control the disease. The rice blast resistance gene Pi54 initially identified in an Indian cultivar confers broad-spectrum resistance in India. We explored the allelic diversity of the Pi54 gene among 885 Indian rice genotypes that were found resistant in our screening against field mixture of naturally existing M. oryzae strains as well as against five unique strains. These genotypes are also annotated as rice blast resistant in the International Rice Genebank database. Sequence-based allele mining was used to amplify and clone the Pi54 allelic variants. Nine new alleles of Pi54 were identified based on the nucleotide sequence comparison to the Pi54 reference sequence as well as to already known Pi54 alleles. DNA sequence analysis of the newly identified Pi54 alleles revealed several single polymorphic sites, three double deletions and an eight base pair deletion. A SNP-rich region was found between a tyrosine kinase phosphorylation site and the nucleotide binding site (NBS) domain. Together, the newly identified Pi54 alleles expand the allelic series and are candidates for rice blast resistance breeding programs.


Figure 1
Figure 1. Selection of rice accessions for Pi54 allele mining.
The 885 rice accessions that were resistant with a phenotypic score between 0 and 3 in the uniform blast nursery (UBN) as well as against any of the five pure blast isolates tested were screened using the Pi54MAS marker. The 329 rice accessions identified as molecular positives were selected as candidates for Pi54 allele mining. The data on number of accessions resistant (score 0–3) against each of the five blast isolates is also presented.
Figure 2
Figure 2. Schematic representation of sequence alignment of newly identified Pi54 alleles.
The DNA sequence alignment of 11 allelic forms of Pi54 isolated from the studied Indian accessions together with Pi54_Tetep as reference is shown. The alleles Pi54_3708 and Pi54_40636 were found identical to recently reported alleles of Pi54 but the remaining nine alleles are unique. The domain regions of the Pi54 alleles are illustrated at the bottom as grey boxes (CC, NBS and LRR) and the brown box within LRR1 represents a zinc finger motif. The unit scale indicates the nucleotide position. The black lines on the bars as well as the numbers on the right indicate nucleotide polymorphisms compared to Pi54_Tetep. Numbers within brackets indicate the non-synonymous SNPs. The gap in the bars for certain alleles indicates 2 bp (Pi54_22419, Pi54_10202 and Pi54_13758) and 8 bp (Pi54_42439 and Pi54_40996) deletions. * indicates the alleles for which non-synonymous SNPs were not calculated due to the presence of deletions causing premature stop codons or a change in the ORF.
Figure 3
Figure 3. SNP rich region in the new Pi54 alleles.
(a) Selection window of the SNP-rich region (between nucleotide position 232 and 429) from the sequence alignment of the eleven studied Pi54 alleles. Dots represent the nucleotides that are identical to the Pi54-Tetep reference and the polymorphisms are represented by single letter code for nucleotides in different colours A (green), T (red), G (black) and C (blue) respectively. The domains and PTM sites are illustrated on top of the reference sequence. The unit scale on top indicates the nucleotide position. (b) Sliding-window analysis of nucleotide diversity (π) observed in the identified Pi54 alleles. The domains are illustrated below the unit scale that represents nucleotide position.
Figure 4
Figure 4. Schematic representation of secondary structure elements, protein-binding sites, exposed/buried and disordered regions within Pi54 proteins.
(a) Protein sequences of alleles with complete ORFs as that of reference sequence Pi54_Tetep were analyzed using ‘PredictProtein’ server. (b) Surface accessibility of AA residues of Pi54 proteins predicted using NetSurfP server. ‘X’ represents polymorphic AA residues identified at least in one of the analysed Pi54 proteins. The conserved AA residues are represented by their respective single letter code. The polymorphic residues predicted as solvent exposed are highlighted in green and the ones predicted as buried are in red. The posttranslational modification sites and domain regions are illustrated.
Figure 5
Figure 5. Phylogenetic relationship among Pi54 alleles.
Analysis was performed using Pi54 sequences identified in our study material together with previously reported Pi54 sequences from wild rice species and cultivars. The ORF for all the sequences (including the previously reported sequences) were extracted from NCBI database based on the reported ORF for Pi54_Tetep reference sequence. Bootstrap values (1000 replications) are mentioned at the branch nodes. The alleles identified in our study material are labelled in yellow and the reference allele in green. The leaf nodes are coloured purple for wild rice, pink for landraces, green for cultivars, black for unknown status or unknown + breeding lines and blue for landraces + unknown/breeding line/cultivar, respectively.

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