Precision mapping and expression analysis of recessive bacterial blight resistance gene xa-45(t) from Oryza glaberrima.

BACKGROUND: Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases of rice leading to huge yield losses in Southeast Asia. The recessive resistance gene xa-45(t) from Oryza glaberrima IRGC102600B, mapped on rice chromosome 8, spans 80 Kb with 9 candidate genes on Nipponbare reference genome IRGSP-1.0. The xa-45(t) gene provides durable resistance against all the ten Xanthomonas pathotypes of Northern India, thus aiding in the expansion of recessive bacterial blight resistance gene pool. Punjab Rice PR127, carrying xa-45(t), was released for wider use in breeding programs. This study aims to precisely locate the target gene among the 9 candidates conferring resistance to bacterial blight disease. METHODS AND RESULTS: Sanger sequencing of all nine candidate genes revealed seven SNPs and an Indel between the susceptible parent Pusa 44 and the resistant introgression line IL274. The genotyping with polymorphic markers identified three recombinant breakpoints for LOC_Os08g42370, and LOC_Os08g42400, 15 recombinants for LOC_Os08g423420 and 26 for LOC_Os08g42440 out of 190 individuals. Relative expression analysis across six time intervals (0, 8, 24, 48, 72, and 96 h) after bacterial blight infection showed over expression of LOC_Os08g42410-specific transcripts in IL274 compared to Pusa 44, with a significant 4.46-fold increase observed at 72 h post-inoculation. CONCLUSIONS: The Indel marker at the locus LOC_Os08g42410 was found co-segregating with the phenotype, suggesting its candidacy towards xa-45(t). The transcript abundance assay provides strong evidence for the involvement of LOC_Os08g42410 in the resistance conferred by the bacterial blight gene xa-45(t).

Conversion of sheath blight susceptible indica and japonica rice cultivars into moderately resistant through expression of antifungal ß-1,3-glucanase transgene from Trichoderma spp.

Rice is an important food crop for three billion people worldwide. The crop is vulnerable to several diseases. Sheath blight caused by fungal pathogen Rhizoctonia solani is a significant threat to rice cultivation accounting for up to 50% yield losses. The pathogen penetrates leaf blades and sheaths, leading to plant necrosis; and major disease resistance gene against the pathogen is not available. This study describes development of sheath blight resistant transgenic indica and japonica rice cultivars through introduction of antifungal ß-1,3-glucanase transgene cloned from Trichoderma. The transgene integration and expression in transformed T0 rice plants was examined by PCR, RT-PCR, qRT-PCR demonstrating up to 5-fold higher expression as compared to non-transgenic plants. The bioassay of T0, T1 and homozygous T2 progeny plants with virulent R. solani isolate revealed that plants carrying high level of ß-1,3-glucanase expression displayed moderately resistant reaction to the pathogen. The optical micrographs of leaf sheath cells from moderately resistant plant after pathogen inoculation displayed presence of a few hyphae with sparse branching; on the contrary, pathogen hyphae in susceptible non-transgenic plant cells were present in abundance with profuse hyphal branching and forming prominent infection cushions. The disease severity in T2 progeny plants was significantly less as compared to non-transgenic plants confirming role of ß-1,3-glucanase in imparting resistance.

Development and validation of a novel core set of KASP markers for the traits improving grain yield and adaptability of rice under direct-seeded cultivation conditions.

The development and utilization of molecular-markers play an important role in genomics-assisted breeding during pyramiding of valuable genes. The aim of present study was to develop and validate a novel core-set of KASP (Kompetitive Allele-Specific PCR) markers associated with traits improving rice grain yield and adaptability under direct-seeded cultivation conditions. The 110 phenotypically validated KASP assays out of 171 designed KASP, include assays for biotic-resistance genes, anaerobic germination, root-traits, grain yield, lodging resistance and early-uniform emergence. The KASP assays were validated for their robustness and reliability at five different levels using diverse germplasm, segregating and advanced population, comparison with SSR markers and on F1s. The present research work will provide (i) breeding material in form of anticipated pre-direct-seeded adapted rice varieties (ii) single improved breeding line with many useful genes and (iii) KASP assay information for the useful QTL/genes providing grain yield and adaptability to rice under direct-seeded cultivation conditions.

High-resolution mapping of the quantitative trait locus (QTLs) conferring resistance to false smut disease in rice.

Rice false smut (RFS), an emerging major fungal disease worldwide caused by Ustilaginoidea virens, affects rice grain quality and yield. RFS cause 2.8-49% global yield loss depending upon disease severity and cultivars. In India, the yield loss due to RFS ranged from 2 to 75%. Identification of the genes or quantitative trait loci (QTLs) governing disease resistance would be of utmost importance towards mitigating the economic losses incurred due to RFS. Here, we report mapping of RFS resistance QTLs from a resistant breeding line RYT2668. The mapping population was evaluated for RFS resistance under the field condition in three cropping seasons 2013, 2015, and 2016. A positive correlation among infected panicle/plant, total smut ball/panicle, and disease score was observed in the years 2013, 2015, and the mean data. A total of seven QTLs were mapped on rice chromosomes 2, 4, 5, 7, and 9 using 2326 single nucleotide polymorphism markers. Of these, two QTLs, qRFSr5.3 and qRFSr7.1a, were associated with the infected panicle per plant, one QTL qRFsr9.1 with total smut ball per panicle, and four QTLs qRFSr2.2, qRFSr4.3, qRFSr5.4, and qRFSr7.1b with disease score. Among them, a novel QTL qRFSr9.1 on chromosome 9 exhibits the largest phenotypic effect. The prediction of putative candidate genes within the qRFSr9.1 revealed four nucleotide-binding sites-leucine-rich repeat (NBS-LRR) domain-containing disease resistance proteins. In summary, our findings mark the hotspot region of rice chromosomes carrying genes/QTLs for resistance to the RFS disease.

Characterization of evolutionarily distinct rice BAHD-Acyltransferases provides insight into their plausible role in rice susceptibility to Rhizoctonia solani.

Plants produce diverse secondary metabolites in response to different environmental cues including pathogens. The modification of secondary metabolites, including acylation, modulates their biological activity, stability, transport, and localization. A plant-specific BAHD-acyltransferase (BAHD-AT) gene family members catalyze the acylation of secondary metabolites. Here we characterized the rice (Oryza sativa L.) BAHD-ATs at the genome-wide level and endeavor to define their plausible role in the tolerance against Rhizoctonia solani AG1-IA. We identified a total of 85 rice OsBAHD-AT genes and classified them into five canonical clades based on their phylogenetic relationship with characterized BAHD-ATs from other plant species. The time-course RNA sequencing (RNA-seq) analysis of OsBAHD-AT genes and qualitative real-time polymerase chain reaction (qRT-PCR) validation showed higher expression in sheath blight susceptible rice genotype. Furthermore, the DNA methylation analysis revealed higher hypomethylation of OsBAHD-AT genes that corresponds to their higher expression in susceptible rice genotype, indicating epigenetic regulation of OsBAHD-AT genes in response to R. solani AG1-IA inoculation. The results shown here indicate that BAHD-ATs may have a negative role in rice tolerance against R. solani AG1-IA possibly mediated through the brassinosteroid (BR) signaling pathway. Altogether, the present analysis suggests the putative functions of several OsBAHD-AT genes, which will provide a blueprint for their functional characterization and to understand the rice-R. solani AG1-IA interaction.

High-resolution genetic mapping of a novel bacterial blight resistance gene xa-45(t) identified from Oryza glaberrima and transferred to Oryza sativa.

KEY MESSAGE: A novel recessive bacterial blight resistance locus designated as a xa-45(t) was identified from Oryza glaberrima accession IRGC 102600B, transferred to O. sativa and mapped to the long arm of chromosome 8 using ddRAD sequencing approach. The identified QTL spans 80 kb region on Nipponbare reference genome IRGSP-1.0 and contains 9 candidate genes. An STS marker developed from the locus LOC_Os08g42410 was found co-segregating with the trait and will be useful for marker-assisted transfer of this recessive resistance gene in breeding programs. Bacterial blight, caused by Xanthomonas oryzae pv. oryzae, is one of the major constraints of rice productivity in Southeast Asia. In spite of having 44 bacterial blight resistance genes from cultivated rice and wild species, the durability of resistance is always at stake due to the continually evolving nature of the pathogen and lack of suitable chemical control. Here, we report high-resolution genetic mapping of a novel bacterial blight resistance gene tentatively designated as a xa-45(t) from an introgression line derived from Oryza glaberrima accession IRGC 102600B. This introgression line was crossed with the susceptible rice indica cultivar cv. Pusa 44 to generate F2 and F2:3 populations for inheritance and mapping studies. The inheritance studies revealed the presence of single recessive locus controlling resistance to the Xanthomonas pathotype seven. A high-density linkage map was constructed using double-digest restriction-associated DNA sequencing of 96 F2 populations along with the parents. The QTL mapping identified a major locus on the long arm of rice chromosome 8 with a LOD score of 33.22 between the SNP markers C8.26737175 and C8.26818765. The peak marker, C8.26810477, explains 49.8% of the total phenotypic variance and was positioned at 202.90 cM on the linkage map. This major locus spans 80 kb region on Nipponbare reference genome IRGSP-1.0 and contains 9 candidate genes. A co-segregating STS marker was developed from the LOC_Os08g42410 for efficient transfer of this novel gene to elite cultivars.