U. D. Singh

MicroRNA regulated defense responses in Triticum aestivum L. during Puccinia graminis f.sp. tritici infection

Plants have evolved diverse mechanism to recognize pathogen attack and triggers defense responses. These defense responses alter host cellular function regulated by endogenous, small, non-coding miRNAs. To understand the mechanism of miRNAs regulated cellular functions during stem rust infection in wheat, we investigated eight different miRNAs viz. miR159, miR164, miR167, miR171, miR444, miR408, miR1129 and miR1138, involved in three different independent cellular defense response to infection. The investigation reveals that at the initiation of disease, accumulation of miRNAs might be playing a key role in hypersensitive response (HR) from host, which diminishes at the maturation stage. This suggests a possible host-fungal synergistic relation leading to susceptibility. Differential expression of these miRNAs in presence and absence of R gene provides a probable explanation of miRNA regulated R gene mediated independent pathways.

Molecular breeding for the development of multiple disease resistance in Basmati rice

Marker assisted backcross breeding for combining three resistance genes (xa13 and Xa21 for Bacterial Blight, Pi54 for blast) and a major QTL (qSBR11-1 for resistance to Sheath blight) in Basmati rice.

Molecular diversity in rice blast resistance gene Pi-ta makes it highly effective against dynamic population of Magnaporthe oryzae

Rice blast is one of the important diseases of rice which can be effectively managed by the deployment of resistance genes. Pi-ta is one of the major blast resistant genes effective against pathogen populations in different parts of India. We analysed allelic variants of Pi-ta from 48 rice lines selected after phenotyping of 529 rice landraces across three eco-geographical blast hot spot regions. Besides, Pi-ta orthologue sequences of 220 rice accessions belonging to wild and cultivated species of rice were also included in the study for a better evo–devo perspective of the diversity present in the gene and the selection pressures acting on this locus. We obtained high nucleotide variations (SNPs and insertion–deletions) in the intronic region. We also identified 64 haplotypes based on nucleotide polymorphism in these alleles. Pi-ta orthologues of Indian landraces were scattered in eight major haplotypes indicating its heterogenous nature. We identified a total of 47 different Pi-ta protein variants on the basis of deduced amino acid residues amongst the orthologues. Five unique and novel Pi-ta variants were identified for the first time in rice landraces exhibiting different reaction types against the Magnaporthe oryzae population. A high value of Pinon/syn was observed only in the leucine-rich domain of the alleles cloned from Indian landraces, indicating strong selective forces acting on this region. The detailed molecular analysis of the Pi-ta orthologues provides insights to a high degree of inter- and intraspecific relationships amongst the Oryza species. We identified rice landraces possessing the effective alleles of this resistance gene which can be used in future blast resistance breeding programmes.

Extensive sequence variation in rice blast resistance gene Pi54 makes it broad spectrum in nature.

Rice blast resistant gene, Pi54 cloned from rice line, Tetep, is effective against diverse isolates of Magnaporthe oryzae. In this study, we prospected the allelic variants of the dominant blast resistance gene from a set of 92 rice lines to determine the nucleotide diversity, pattern of its molecular evolution, phylogenetic relationships and evolutionary dynamics, and to develop allele specific markers. High quality sequences were generated for homologs of Pi54 gene. Using comparative sequence analysis, InDels of variable sizes in all the alleles were observed. Profiling of the selected sites of SNP (Single Nucleotide Polymorphism) and amino acids (N sites ≥ 10) exhibited constant frequency distribution of mutational and substitutional sites between the resistance and susceptible rice lines, respectively. A total of 50 new haplotypes based on the nucleotide polymorphism was also identified. A unique haplotype (H_3) was found to be linked to all the resistant alleles isolated from indica rice lines. Unique leucine zipper and tyrosine sulfation sites were identified in the predicted Pi54 proteins. Selection signals were observed in entire coding sequence of resistance alleles, as compared to LRR domains for susceptible alleles. This is a maiden report of extensive variability of Pi54 alleles in different landraces and cultivated varieties, possibly, attributing broad-spectrum resistance to Magnaporthe oryzae. The sequence variation in two consensus region: 163 and 144 bp were used for the development of allele specific DNA markers. Validated markers can be used for the selection and identification of better allele(s) and their introgression in commercial rice cultivars employing marker assisted selection.

Transposon-based high sequence diversity in Avr-Pita alleles increases the potential for pathogenicity of Magnaporthe oryzae populations

Magnaporthe oryzae causes rice blast that is one of the most devastating diseases of rice worldwide. Highly variable nature of this fungus has evolved itself against major resistance genes in newly released rice varieties. Understanding the population structure of this fungus is essential for proper utilization of the rice blast resistance genes in rice crop plants. In the present study, we analyzed 133 isolates of M. oryzae from ten countries to find the allelic variation of Avr-Pita gene that is triggering Pita-mediated resistance in rice plant. The diversity analysis of these alleles showed higher level of nucleotide variation in the coding regions than the noncoding regions. Evolutionary analysis of these alleles indicates that Avr-Pita gene is under purifying selection to favor its major alleles in 133 isolates analyzed in this study. We hypothesize that the selection of favorable Avr-Pita allele in these isolates may occur through a genetic mechanism known as recurrent selective sweeps. A total of 22 functional Avr-Pita protein variants were identified in this study. Insertion of Pot3 transposable element into the promoter of Avr-Pita gene was identified in virulent isolates and was suggested that mobility of repeat elements in avirulence genes of M. oryzae seems to help in emergence of new virulent types of the pathogen. Allele-specific markers developed in this study will be helpful to identify a particular type of Avr-Pita allele from M. oryzae population which can form the basis for the deployment of Pita gene in different epidemiological regions.

Positive selection pressure on rice blast resistance allele Piz-t makes it divergent in Indian land races

Abstract Allelic variants of the broad-spectrum blast resistance gene, Piz-t, have been analyzed in 48 rice lines selected after phenotyping across three blast hot-spot regions of India. Single Nucleotide Polymorhisms in the form of transitions were more frequent than the transversions in the alleles. On the basis of nucleotide polymorphism, 46 haplotypes have been identified, with major haplotypes forming three main haplogroups. The Piz-t alleles formed mostly region-specific clusters. Resistant and susceptible Piz-t alleles were grouped into separate sub-clusters. The value of Tajimas D was negative indicating positive selection on Piz-t locus. Sequence variations were more abundant in the leucine rich repeats (LRR) region than in the NB-ARC (nucleotide-binding adaptor shared by APAF-1,R proteins, and CED-4) region, indicating that the LRR region has played a more important role in the evolution of this allele. The detailed molecular analysis of the Piz-t locus provide insights to high degree of inter-and intra-specific relationship among the Indian land races of rice which will help in the selection of better alleles for future rice breeding programs.

Introgression of multiple disease resistance into a maintainer of Basmati rice CMS line by marker assisted backcross breeding

Globally, blast incited by Magnaporthe oryzae and sheath blight (ShB) by Rhizoctonia solani Kuhn forms two major fungal diseases that cause significant yield loss in rice. Pusa 6B, the Basmati quality maintainer line of the popular superfine grain aromatic rice hybrid Pusa RH10, is highly susceptible to both the diseases. The rice cultivar Tetep was used as the donor for transferring a major blast resistant gene, Pi54 and three ShB resistant quantitative trait loci (QTLs) namely, qSBR11-1, qSBR11-2 and qSBR7-1 into Pusa 6B using a marker assisted backcross breeding scheme with restricted number of backcrosses. Plants heterozygous for the alleles of interest and phenotypic similarity to the recurrent parent were used for generating BC1F2 population by selfing selected BC1F1 plants. Selected BC1F2 plants homozygous for Pi54 were selfed to generate BC1F3 families that were subjected to a step-wise reductive screening for the three ShB resistant QTLs. Final selections were advanced to BC1F5 generation through selfing while subjecting to stringent phenotypic selection. The advanced selections carrying blast and ShB resistant genes (Pi54, qSBR11-1, qSBR11-2, qSBR7-1) in the background of Pusa 6B were resistant to highly virulent strains of rice blast as well as ShB isolates without compromising the grain and cooking quality of Pusa 6B. Marker assisted transfer of blast and ShB resistance into Pusa 6B will aid in developing CMS lines with inbuilt resistance to these diseases. When combined with restorer lines possessing resistant genes/QTLs for these diseases, the improved Pusa6A lines will aid in development of improved Pusa RH10 and other novel aromatic hybrids with resistance to blast and ShB diseases. The present study demonstrates successful use of a restricted backcrossing strategy for introgression of multiple loci conferring resistance to two important fungal diseases in rice.

Marker assisted pyramiding of major blast resistance genes Pi9 and Pita in the genetic background of an elite Basmati rice variety, Pusa Basmati 1

Basmati is a premium quality rice of India which is highly priced in the international market. Pusa Basmati 1, an elite Basmati rice variety is highly susceptible to rice blast caused by Magnaporthe oryzae. Therefore, pyramiding blast resistance genes is essential to effectively combat the blast disease and increase the durability of resistance genes. The blast resistance genes Pi9 and Pita have been earlier demonstrated to be effective in Basmati growing regions of the country. Therefore, in the present study, monogenicnear isogenic lines Pusa 1637-18-7-6-20 and Pusa 1633-3-88-16-1 carrying Pi9 and Pita, respectively, were intercrossed to generate pyramided lines through marker assisted foreground, background and phenotypic selection for recurrent parent phenotype. The pyramided lines carrying Pi9+Pita were found to be either at par or superior to the recurrent parent Pusa Basmati 1 for agro-morphological, grain and cooking quality traits. Further, these pyramided lines were also found to show resistance against three virulent pathotypes of M. oryzae namely, Mo-nwi-kash 1, Mo-nwi-lon2 and Mo-ei-ran1, when evaluated under artificial inoculation conditions as well as in the natural epiphytotic conditions of uniform blast nursery at two locations. The developed pyramided lines are the potential sources of blast resistance genes in the Basmati improvement program and can also be released for commercial cultivation after required testing.

Phenotyping of improved rice lines and landraces for blast and sheath blight resistance

Globally, two major fungal diseases of rice,viz., blast caused by Magnaporthe oryzae and sheath blight incited by Rhizoctonia solani Kuhn. occur in all agro-ecosystems resulting in significant yield losses. In the present study, a set of 100 rice lines were evaluated for blast and sheath blight resistance. Twenty seven genotypes were identified to be resistant against M. oryzae isolate (Mo-ni-0066). However, evaluation of these genotypes with highly virulent M. oryzae Karnataka isolate (Mo-si-mnd) revealed that only two genotypes viz., Bhusan San and P1490-03 were resistant. Same set of genotypes were screened for sheath blight resistance. Four entries, viz., BPL7-12, BML27-1, BML 21-1 and Kajarahwa possess high degree of tolerance to sheath blight but none of the rice entries was completely resistant.

Identification and validation of rice blast resistance genes in Indian rice germplasm

Blast disease caused by Magnaporthe oryzae is a major constraint in rice production. Identification of new donors for blast resistance is a pre-requisite for effective utilization of diverse germplasm for marker assisted incorporation of blast resistance into improved varieties. Therefore, in the present study, a set of 100 diverse rice germplasm accessions were evaluated for 11 blast resistance genes namely Pikm, Pik, Pikh, Pi1, Pi5, Pi54, Pib, Piz5, Piz, Pi9 and Pish, both at genotypic and phenotypic level. Genotyping with gene based/gene linked markers could identify six genotypes from the germplasm possessing as many as six resistance specific alleles. A total of 34 and 67 germplasm lines were found to possess resistance alleles for two genes, Pikm and Pik, respectively. Phenotypic validation using artificial inoculation in the germplasm was carried out with 4 diverse isolates under controlled conditions. The congruence between marker genotype and disease phenotype on a set of monogenic lines for blast resistance in the LTH background was used to compute Disease Resistance Index (DRI) in the germplasm. Cumulative DRI for each genotype was computed over all the marker loci. The genotypes Heibao, Kalinga-I, Vijetha, Anjali, Bhaubhog, Sada Kaijam, Kala Jeera had high cumulative resistance score. Allelic Cumulative Disease Resistance Index (ACDRI), a measure for comparing the effectiveness of markers was calculated and markers linked to Pikm, Pik, Piz5, Pi1 were found to possess higher accuracy and better correlation with expected patterns of resistance under artificial inoculation. Based on disease resistance index, 25 germplasm accessions were found carrying blast resistance specific alleles at different loci and were fully validated for disease phenotype, which are valuable in breeding for resistance, allele mining and functional genomics studies.