Gouri Sankar Laha

Marker assisted introgression of bacterial blight resistance in Samba Mahsuri, an elite indica rice variety

Samba Mahsuri (BPT5204) is a medium slender grain indica rice variety that is very popular with farmers and consumers across India because of its high yield and excellent cooking quality. However, the variety is susceptible to several diseases and pests, including bacterial blight (BB). We have used PCR based molecular markers in a backcross-breeding program to introgress three major BB resistance genes (Xa21, xa13 and xa5) into Samba Mahsuri from a donor line (SS1113) in which all the three genes are present in a homozygous condition. At each backcross generation, markers closely linked to the three genes were used to select plants possessing these resistance genes (foreground selection) and microsatellite markers polymorphic between donor and recurrent parent were used to select plants that have maximum contribution from the recurrent parent genome (background selection). A selected BC4F1 plant was selfed to generate homozygous BC4F2 plants with different combinations of BB resistance genes. The three-gene pyramid and two-gene pyramid lines exhibited high levels of resistance against the BB pathogen. Under conditions of BB infection, the three-gene pyramid lines exhibited a significant yield advantage over Samba Mahsuri. Most importantly, these lines retain the excellent grain and cooking qualities of Samba Mahsuri without compromising the yield as determined in multi-location trials. This work demonstrates the successful application of marker-assisted selection for targeted introgression of multiple resistance genes into a premium quality rice variety.

Introduction of bacterial blight resistance into Triguna, a high yielding, mid-early duration rice variety.

Bacterial blight (BB) is a serious disease of rice in India. We have used molecular marker-assisted selection in a backcross breeding program to introgress three genes (Xa21, xa13, and xa5) for BB resistance into Triguna, a mid-early duration, high yielding rice variety that is susceptible to BB. At each generation in the backcross program, molecular markers were used to select plants possessing these resistance genes and to select plants that have maximum contribution from the Triguna genome. A selected BC3F1 plant was selfed to generate homozygous BC(3)F(2) plants with different combinations of BB resistance genes. Plants containing the two-gene combination, Xa21 and xa13, were found to exhibit excellent resistance against BB. Single plant selections for superior agronomic characteristics were performed on the progeny of these plants, from BC(3)F(3) generation onwards. The selected plants were subjected to yield trials at the BC(3)F(8) generation and were found to have a significant yield advantage over Triguna. The newly developed lines are being entered into national multi-location field trials. This work represents a successful example of the application of molecular marker-assisted selection for BB resistance breeding in rice.

Update on Bacterial Blight of Rice: Fourth International Conference on Bacterial Blight

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Inheritance of bacterial blight resistance in the rice cultivar Ajaya and high-resolution mapping of a major QTL associated with resistance.

The cultivar Ajaya (IET 8585) exhibits durable broad-spectrum resistance to bacterial blight (BB) disease of rice and is widely used as a resistance donor. The present study was carried out to decipher the genetics of BB resistance in Ajaya and map the gene(s) conferring resistance. Genetic analysis in the F2 indicated a quantitative/additive nature of resistance governed by two loci with equal effects. Linked marker analysis and allelic tests revealed that one of the resistance genes is xa5. Sequence analysis of a 244 bp region of the second exon of the gene-encoding Transcription factor IIAγ (the candidate gene for xa5) confirmed the presence of xa5. Bulked-segregant analysis (BSA) revealed the putative location of the two quantitative trait loci (QTLs)/genes associated with resistance on chromosomes 5 and 8. Composite interval mapping located the first locus on Chr. 5S exactly in the genomic region spanned by xa5 and the second locus (qtl BBR 8.1) on Chr. 8L. Owing to its differential disease reaction with a set of seven hyper-virulent isolates of Xanthomonas oryzae, a map location on Chr. 8L, which was distinct from xa13 and data from allelism tests, the second resistance locus in Ajaya was determined to be novel and was designated as xaAj. A contig map spanning xaAj was constructed in silico and the genomic region was delimited to a 13.5 kb physical interval. In silico analysis of the genomic region spanning xaAj identified four putatively expressed candidate genes, one of which could be involved in imparting BB resistance in Ajaya along with xa5.

Nucleotide diversity analysis of three major bacterial blight resistance genes in rice.

Nucleotide sequence polymorphisms among R gene alleles influence the process of co-evolutionary interaction between host and pathogen by shaping the response of host plants towards invading pathogens. Here, we present the DNA sequence polymorphisms and diversities present among natural alleles of three rice bacterial blight resistance genes, Xa21, Xa26 and xa5. The diversity was examined across different wild relatives and cultivars of Oryza species. Functional significance of selected alleles was evaluated through semi-quantitative reverse transcription polymerase chain reaction and real time PCR. The greatest nucleotide diversity and singleton variable sites (SVS) were present in Xa26 (π = 0.01958; SVS = 182) followed by xa5 and Xa21 alleles. The highest frequency of single nucleotide polymorphisms were observed in Xa21 alleles and least in xa5. Transition bias was observed in all the genes and ‘G’ to ‘A’ transitions were more favored than other form of transitions. Neutrality tests failed to show the presence of selection at these loci, though negative Tajima’s D values indicate the presence of a rare form of polymorphisms. At the interspecies level, O. nivara exhibited more diversity than O. sativa. We have also identified two nearly identical resistant alleles of xa5 and two sequentially identical alleles of Xa21. The alleles of xa5 showed basal levels of expression while Xa21 alleles were functionally not expressed.

Population genomic insights into variation and evolution of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae ( Xoo) is a serious pathogen of rice causing bacterial leaf blight disease. Resistant varieties and breeding programs are being hampered by the emergence of highly virulent strains. Herein we report population based whole genome sequencing and analysis of 100 Xoo strains from India. Phylogenomic analysis revealed the clustering of Xoo strains from India along with other Asian strains, distinct from African and US Xo strains. The Indian Xoo population consists of a major clonal lineage and four minor but highly diverse lineages. Interestingly, the variant alleles, gene clusters and highly pathogenic strains are primarily restricted to minor lineages L-II to L-V and in particularly to lineage L-III. We could also find the association of an expanded CRISPR cassette and a highly variant LPS gene cluster with the dominant lineage. Molecular dating revealed that the major lineage, L-I is youngest and of recent origin compared to remaining minor lineages that seems to have originated much earlier in the past. Further, we were also able to identify core effector genes that may be helpful in efforts towards building durable resistance against this pathogen.