Identification and Characterization of a Large Effect QTL from Oryza glumaepatula Revealed Pi68(t) as Putative Candidate Gene for Rice Blast Resistance.

BACKGROUND: Field resistance is often effective and durable as compared to vertical resistance. The introgression line (INGR15002) derived from O. glumaepatula has proven broad spectrum field resistance for both leaf and neck blast. RESULTS: Quantitative Trait Loci (QTL) analysis of INGR15002, led to the identification of two major QTL - qBL3 contributing about 34% and 32% phenotypic variance towards leaf and neck blast resistance, respectively and qBL7 contributing about 25% of phenotypic variance for leaf blast. Further, qBL3 was fine mapped, narrowed down to 300 kb region and a linked SNP maker was identified. By combining mapping with microarray analysis, a candidate gene, Os03g0281466 (malectin-serine threonine kinase), was identified in the fine mapped region and named as Pi68(t). The nucleotide variations in the coding as well as upstream region of the gene was identified through cloning and sequence analysis of Pi68(t) alleles. These significant variations led to the non-synonymous changes in the protein as well as variations (presence/absence) in four important motifs (W-box element; MYC element; TCP element; BIHD1OS) at promoter region those are associated with resistance and susceptible reactions. The effect of qBL3 was validated by its introgression into BPT5204 (susceptible variety) through marker-assisted selection and progeny exhibiting resistance to both leaf and neck blast was identified. Further, the utility of linked markers of Pi68(t) in the blast breeding programs was demonstrated in elite germplasm lines. CONCLUSIONS: This is the first report on the identification and characterization of major effect QTL from O. glumaepatula, which led to the identification of a putative candidate gene, Pi68(t), which confers field resistance to leaf as well as neck blast in rice.

Field level evaluation of rice introgression lines for heat tolerance and validation of markers linked to spikelet fertility.

Rice lines derived from wild species and mutants can serve as a good resource for favorable alleles for heat tolerance. In all, 48 stable lines including 17 KMR3/O. rufipogon introgression lines (KMR3 ILs), 15 Swarna/O. nivara ILs (Swarna ILs) along with their parents, Nagina 22 (N22) and its 4 EMS induced mutants and 7 varieties were evaluated for heat tolerance under irrigated conditions under field in two seasons, wet season 2012 using poly cover house method and dry season 2013 using late sown method. Spikelet fertility (SF), yield per plant (YP) and heat susceptibility index (HSI) for these two traits were considered as criteria to assess heat tolerance compared to control. Four KMR3 ILs and eight Swarna ILs were identified as heat tolerant based on SF and YP and their HSIs in both wet and dry seasons. S-65 and S-70 showed low SF and high YP consistently in response to heat in both seasons. We provide evidence that SF alone may not be the best criterion to assess heat tolerance and including YP is important as lines with low SF but high YP and vice versa were identified under heat stress. Out of 49 SSR markers linked to spikelet fertility, 18 were validated for five traits. RM229 in wet season and RM430 and RM210 in dry season were significantly associated with both SF and its HSI under heat stress. RM430 was also significantly associated with both YP and its HSI in dry season. Thirty two candidate genes were identified close to nine markers associated with traits under heat stress.

Nucleotide variation and identification of novel blast resistance alleles of Pib by allele mining strategy.

Pib is one of significant rice blast resistant genes, which provides resistance to wide range of isolates of rice blast pathogen, Magnaporthe oryzae. Identification and isolation of novel and beneficial alleles help in crop enhancement. Allele mining is one of the best strategies for dissecting the allelic variations at candidate gene and identification of novel alleles. Hence, in the present study, Pib was analyzed by allele mining strategy, and coding and non-coding (upstream and intron) regions were examined to identify novel Pib alleles. Allelic sequences comparison revealed that nucleotide polymorphisms at coding regions affected the amino acid sequences, while the polymorphism at upstream (non-coding) region affected the motifs arrangements. Pib alleles from resistant landraces, Sercher and Krengosa showed better resistance than Pib donor variety, might be due to acquired mutations, especially at LRR region. The evolutionary distance, Ka/Ks and phylogenetic analyzes also supported these results. Transcription factor binding motif analysis revealed that Pib (Sr) had a unique motif (DPBFCOREDCDC3), while five different motifs differentiated the resistance and susceptible Pib alleles. As the Pib is an inducible gene, the identified differential motifs helps to understand the Pib expression mechanism. The identified novel Pib resistant alleles, which showed high resistance to the rice blast, can be used directly in blast resistance breeding program as alternative Pib resistant sources.

Identification of abiotic stress miRNA transcription factor binding motifs (TFBMs) in rice.

Plant growth and yield are affected by many abiotic stresses like salinity, drought, cold and heavy metal; these stresses trigger up and down-regulate several genes through various transcription factors (TFs). Transcription factor binding motifs (TFBMs), located in the upstream region of the genes, associate with TFs to regulate the gene expression. Many factors, including the activation of miRNAs, which are encoded by genes having independent transcription units, regulate the gene expression. TFBMs in the regulatory region of miRNA sequences influence the miRNA expression, which in turn influences the expression of other genes in the cell. However, the current level of information available on TFBMs of miRNA involved in abiotic stress related defense pathway(s) is limited and in-depth studies in this direction may lead to a better understanding of their role in expression and regulation of defense responses in plants. In this study, various aspects related to genomic positions of pre-miRNA, prediction of TSS and TATA box positions and identification of known, unique motifs at regulatory regions of all the reported miRNAs of rice associated with different abiotic stresses are discussed. Sixteen motifs were identified in this study, of which nine are known cis-regulatory elements associated with various stresses, two strong motifs, (CGCCGCCG, CGGCGGCG) and five unique motifs which might play a vital role in the regulation of abiotic stresses related miRNA genes. Common motifs shared by miRNAs that are involved in more than one abiotic stresses were also identified. The motifs identified in this study will be a resource for further functional validation.