Understanding the nature of blast resistance in combined bacterial leaf blight and blast gene pyramided lines of rice variety tellahamsa.

BACKGROUND: Rice blast and bacterial leaf blight (BLB) are the most limiting factors for rice production in the world which cause yield losses typically ranging from 20 to 30% and can be as high as 50% in some areas of Asia especially India under severe infection conditions. METHODS AND RESULTS: An improved line of Tellahamsa, TH-625-491 having two BLB resistance genes (xa13 and Xa21) and two blast resistance genes (Pi54 and Pi1) with 95% Tellahamsa genome was used in the present study. TH-625-491 was validated for all four target genes and was used for backcrossing with Tellahamsa. Seventeen IBC1F1 plants heterozygous for all four target genes, 19 IBC1F2 plants homozygous for four, three and two gene combinations and 19 IBC1F2:3 plants also homozygous for four, three and two gene combinations were observed. Among seventeen IBC1F1 plants, IBC1F1-62 plant recorded highest recurrent parent genome (97.5%) covering 75 polymorphic markers. Out of the total of 920 IBC1F2 plants screened, 19 homozygous plants were homozygous for four, three and two target genes along with bacterial blight resistance. Background analysis was done in all 19 homozygous IBC1F2 plants possessing BLB resistance (possessing xa13, Xa21, Pi54 and Pi1 in different combinations) with five parental polymorphic SSR markers. IBC1F2-62-515 recovered 98.5% recurrent parent genome. The four, three and two gene pyramided lines of Tellahamsa exhibited varying resistance to blast. CONCLUSIONS: Results show that there might be presence of antagonistic effect between bacterial blight and blast resistance genes since the lines with Pi54 and Pi1 combination are showing better resistance than the combinations with both bacterial blight and blast resistance genes.

Improvement of bacterial blight resistance of the high yielding, fine-grain, rice variety, Gangavati sona through marker-assisted backcross breeding.

Gangavati sona (GS) is a high-yielding, fine-grain rice variety widely grown in the Tungabhadra command area in Karnataka, India; however, it is susceptible to bacterial blight (BB). Therefore, the present study was conducted to improve the GS variety for BB resistance. Three BB-resistant genes (xa5, xa13, and Xa21) were introgressed into the genetic background of susceptible cultivar GS through marker-assisted backcrossing (MABB) by using Improved samba Mahsuri (ISM), a popular, high-yielding, bacterial blight resistant rice variety as a donor parent. Foreground selection was carried out using gene-specific markers, viz., xa5FM (xa5), xa13prom (xa13), and pTA248 (Xa21), while background selection was carried out using well-distributed 64 polymorphic microsatellite markers. The true heterozygote F1 was used as the male parent for backcrossing with GS to obtain BC1F1. The process was repeated in BC1F1 generation, and a BC2F1 plant (IGS-5-11-5) possessing all three target genes along with maximum recurrent parent genome (RPG) recovery (86.7%) was selfed to obtain BC2F2s. At BC2F2, a single triple gene homozygote plant (IGS-5-11-5-33) with 92.6% RPG recovery was identified and advanced to BC2F5 by a pedigree method. At BC2F5, the seven best entries were selected, possessing all three resistance genes with high resistance levels against bacterial blight, yield level, and grain quality features equivalent to better than GS. The improved versions of GS will immensely benefit the farmers whose fields are endemic to BB.

Evolution of pathogenicity-associated genes in Rhizoctonia solani AG1-IA by genome duplication and transposon-mediated gene function alterations.

BACKGROUND: Rhizoctonia solani is a polyphagous fungal pathogen that causes diseases in crops. The fungal strains are classified into anastomosis groups (AGs); however, genomic complexity, diversification into the AGs and the evolution of pathogenicity-associated genes remain poorly understood. RESULTS: We report a recent whole-genome duplication and sequential segmental duplications in AG1-IA strains of R. solani. Transposable element (TE) clusters have caused loss of synteny in the duplicated blocks and introduced differential structural alterations in the functional domains of several pathogenicity-associated paralogous gene pairs. We demonstrate that the TE-mediated structural variations in a glycosyl hydrolase domain and a GMC oxidoreductase domain in two paralogous pairs affect the pathogenicity of R. solani. Furthermore, to investigate the association of TEs with the natural selection and evolution of pathogenicity, we sequenced the genomes of forty-two rice field isolates of R. solani AG1-IA. The genomic regions with high population mutation rates and with the lowest nucleotide diversity are enriched with TEs. Genetic diversity analysis predicted the genes that are most likely under diversifying and purifying selections. We present evidence that a smaller variant of a glucosamine phosphate N-acetyltransferase (GNAT) protein, predicted to be under purifying selection, and an LPMP_AA9 domain-containing protein, predicted to be under diversifying selection, are important for the successful pathogenesis of R. solani in rice as well as tomato. CONCLUSIONS: Our study has unravelled whole-genome duplication, TE-mediated neofunctionalization of genes and evolution of pathogenicity traits in R. solani AG1-IA. The pathogenicity-associated genes identified during the study can serve as novel targets for disease control.

DRR Dhan 58, a Seedling Stage Salinity Tolerant NIL of Improved Samba Mahsuri Shows Superior Performance in Multi-location Trials.

BACKGROUND: Improved Samba Mahsuri (ISM) is an elite, high-yielding, bacterial blight resistant, fine-grained rice variety with low glycaemic index. It is highly sensitive to salt stress, particularly at seedling stage, which significantly reduces its yield potential in coastal areas. A salinity tolerant QTL, Saltol, associated with seedling stage tolerance was previously mapped on chromosome 1 (10.6-11.5 Mb) from the Indian landrace, Pokkali and is effective in different genetic backgrounds. The objective of this study was to enhance salinity tolerance of ISM by incorporating the Saltol QTL through marker-assisted backcross breeding using the breeding line, FL478 (Pokkali/IR29). RESULTS: Foreground selection was carried out at each generation using five Saltol-specific markers and three bacterial blight resistance genes, Xa21, xa13 and xa5. Background selection was conducted using 66 well distributed polymorphic SSR markers and at the BC3F2 generation, a single plant with maximum recurrent parent genome recovery (95.3%) was identified and advanced to the BC3F4 generation. Based on bacterial blight resistance, seedling stage salinity tolerance and resemblance to ISM, four advanced breeding lines were selected for testing in replicated experiments near Hyderabad, India. A promising near-isogenic line, DRR Dhan 58, was evaluated in multi-location trials-coastal salinity and it showed significant salinity tolerance, resistance to bacterial blight disease, high yield and excellent grain quality during the 2019 and 2020 trials. DRR Dhan 58 was 95.1% similar to ISM based on genotyping with the 90 K SNP chip. Whole genome resequencing analysis of Pokkali and FL478 which were salinity tolerant checks, ISM and DRR Dhan 58 showed a high degree of relatedness with respect to the candidate gene loci for Saltol and OsSKC1 (Shoot K+ Concentration 1). CONCLUSION: DRR Dhan 58, possessing Saltol and three bacterial blight resistance genes (Xa21, xa13 and xa5) in the genetic background of the Indian mega-variety of rice, Samba Mahsuri, was developed for potential cultivation in areas prone to seedling stage salinity, as well as areas with endemic bacterial blight disease. This entry had a 24% yield advantage over the recurrent parent ISM under coastal saline conditions in multi-location trials and was recently released for commercial cultivation in India.

Intra-regional diversity of rice bacterial blight pathogen, Xanthomonas oryzae pv. oryzae, in the Andaman Islands, India: revelation by pathotyping and multilocus sequence typing.

AIM: To investigate the genetic and pathogenic variability of Xanthomonas oryzae pv. oryzae causing bacterial blight in rice on the remote Andaman Islands, India. METHODS AND RESULTS: A total of 27 yellow-pigmented bacterial isolates representing rice fields of Andaman Islands incited blight on the susceptible-rice cultivar, C14-8. Phenotypic, pathogenic traits and 16S rRNA gene sequences revealed their identity as X. oryzae pv. oryzae. Virulence profiling indicated the prevalence of seven pathotypes of X. oryzae pv. oryzae on the Islands. Pathotypes-VI and -VII were highly virulent, whereas the pathotype-I was less virulent. Multilocus sequence typing based on nucleotide sequence polymorphism in nine housekeeping genes dnaK; fyuA; gyrB (two loci): rpoD; fusA; gapA; gltA and lepA clustered 27 isolates into 17 sequence types (STs) segregated into two clonal-complexes (CC). While CC-I comprised of isolates from Andaman Island, the CC-II is a mixture of isolates representing mainland India and Andaman Island. The data revealed trans-boundary pathogen introduction and a consequent intra-regional diversification on these islands due to the deployment of different rice cultivars in different regions. CONCLUSIONS: Genotyping and pathotyping of sland isolates revealed seven pathotypes distributed in two clonal complexes with strong indications for trans-boundary movement and consequent diversification of the bacterial pathogen. Highly virulent pathotypes of X. oryzae pv. oryzae that could overcome combinations of R-genes, xa13+Xa21 as well as xa5+xa13 were found prevalent in the Andaman Islands SIGNIFICANCE AND IMPACT OF THE STUDY: Genetic and virulence analysis of X. oryzae pv. oryzae in the Andaman Islands revealed introduction and host-mediated regional diversification and local adaptation of X oryzae pv. oryzae. The study calls for the need of multi-gene pyramiding for durable disease resistance and establishing stringent quarantine measures for safeguarding island agricultural practices in the future.

Marker assisted improvement of low soil phosphorus tolerance in the bacterial blight resistant, fine-grain type rice variety, Improved Samba Mahsuri.

Improved-Samba-Mahsuri (ISM), a high-yielding, popular bacterial blight resistant (possessing Xa21, xa13, and xa5), fine-grain type, low glycemic index rice variety is highly sensitive to low soil phosphorus (P). We have deployed marker-assisted backcross breeding (MABB) approach for targeted transfer of Pup1, a major QTL associated with low soil P tolerance, using Swarna as a donor. A new co-dominant marker, K20-1-1, which is specific for Pup1 was designed and used for foreground selection along with functional markers specific for the bacterial blight resistance genes, Xa21, xa13, and xa5. A set of 66 polymorphic SSR marker were used for the background selection along with a pair of flanking markers for the recombination selection in backcross derived progenies and in BC2F2 generation, 12 plants, which are homozygous for Pup1, all the three bacterial blight resistance genes and possessing agro-morphological traits equivalent to or better than ISM were selected and selfed to produce BC2F3s. They were evaluated in plots with low soil P and normal soil P at ICAR-IIRR, Hyderabad for their low soil P tolerance, and bacterial blight resistance and superior lines were advanced to BC2F6. One of the lines, when tested at multiple locations in India was found promising under both normal as well as low soil P conditions.

Marker Assisted Gene Pyramiding (MAGP) for bacterial blight and blast resistance into mega rice variety "Tellahamsa".

Bacterial blight (BB) and fungal blast diseases are the major biotic constraints that limit rice productivity. To sustain yield improvement in rice, it is necessary to developed yield potential of the rice varieties by incorporation of biotic stress resistance genes. Tellahamsa is a well-adapted popular high yielding rice variety in Telangana state, India. However, the variety is highly susceptible to BB and blast. In this study, simultaneous stepwise transfer of genes through marker-assisted backcross breeding (MABB) strategy was used to introgress two major BB (Xa21 and xa13) and two major blast resistance genes (Pi54 and Pi1) into Tellahamsa. In each generation (from F1 to ICF3) foreground selection was done using gene-specific markers viz., pTA248 (Xa21), xa13prom (xa13), Pi54MAS (Pi54) and RM224 (Pi1). Two independent BC2F1 lines of Tellahamsa/ISM (Cross-I) and Tellahamsa/NLR145 (Cross-II) possessing 92% and 94% recurrent parent genome (RPG) respectively were intercrossed to develop ICF1-ICF3 generations. These gene pyramided lines were evaluated for key agro-morphological traits, quality, and resistance against blast at three different hotspot locations as well as BB at two locations. Two ICF3 gene pyramided lines viz., TH-625-159 and TH-625-491 possessing four genes exhibited a high level of resistance to BB and blast. In the future, these improved Tellahamsa lines could be developed as mega varieties for different agro-climatic zones and also as potential donors for different pre-breeding rice research.

Marker Assisted Forward Breeding to Combine Multiple Biotic-Abiotic Stress Resistance/Tolerance in Rice.

BACKGROUND: Unfavorable climatic changes have led to an increased threat of several biotic and abiotic stresses over the past few years. Looking at the massive damage caused by these stresses, we undertook a study to develop high yielding climate-resilient rice, using genes conferring resistance against blast (Pi9), bacterial leaf blight (BLB) (Xa4, xa5, xa13, Xa21), brown planthopper (BPH) (Bph3, Bph17), gall midge (GM) (Gm4, Gm8) and QTLs for drought tolerance (qDTY1.1 and qDTY3.1) through marker-assisted forward breeding (MAFB) approach. RESULT: Seven introgression lines (ILs) possessing a combination of seven to ten genes/QTLs for different biotic and abiotic stresses have been developed using marker-assisted selection (MAS) breeding method in the background of Swarna with drought QTLs. These ILs were superior to the respective recurrent parent in agronomic performance and also possess preferred grain quality with intermediate to high amylose content (AC) (23-26%). Out of these, three ILs viz., IL1 (Pi9+ Xa4+ xa5+ Xa21+ Bph17+ Gm8+ qDTY1.1+ qDTY3.1), IL6 (Pi9+ Xa4+ xa5+ Xa21+ Bph3+ Bph17+ Gm4+ Gm8+ qDTY1.1+ qDTY3.1) and IL7 (Pi9+ Xa4+ xa5+ Bph3+ Gm4+ qDTY1.1+ qDTY3.1) had shown resistance\tolerance for multiple biotic and abiotic stresses both in the field and glasshouse conditions. Overall, the ILs were high yielding under various stresses and importantly they also performed well in non-stress conditions without any yield penalty. CONCLUSION: The current study clearly illustrated the success of MAS in combining tolerance to multiple biotic and abiotic stresses while maintaining higher yield potential and preferred grain quality. Developed ILs with seven to ten genes in the current study showed superiority to recurrent parent Swarna+drought for multiple-biotic stresses (blast, BLB, BPH and GM) together with yield advantages of 1.0 t ha- 1 under drought condition, without adverse effect on grain quality traits under non-stress.

Pectin induced transcriptome of a Rhizoctonia solani strain causing sheath blight disease in rice reveals insights on key genes and RNAi machinery for development of pathogen derived resistance.

KEY MESSAGE: RNAi mediated silencing of pectin degrading enzyme of R. solani gives a high level of resistance against sheath blight disease of rice. Rice sheath blight disease caused by Rhizoctonia solani Kuhn (telemorph; Thanatephorus cucumeris) is one of the most devastating fungal diseases which cause severe loss to rice grain production. In the absence of resistant cultivars, the disease is currently managed through fungicides which add to environmental pollution. To explore the potential of utilizing RNA interference (RNAi)-mediated resistance against sheath blight disease, we identified genes encoding proteins and enzymes involved in the RNAi pathway in this fungal pathogen. The RNAi target genes were deciphered by RNAseq analysis of a highly virulent strain of the R. solani grown in pectin medium. Additionally, pectin metabolism associated genes of R. solani were analyzed through transcriptome sequencing of infected rice tissues obtained from six diverse rice cultivars. One of the key candidate gene AG1IA_04727 encoding polygalacturonase (PG), which was observed to be significantly upregulated during infection, was targeted through RNAi to develop disease resistance. Stable expression of PG-RNAi construct in rice showed efficient silencing of AG1IA_04727 and suppression of sheath blight disease. This study highlights important information about the existence of RNAi machinery and key genes of R. solani which can be targeted through RNAi to develop pathogen-derived resistance, thus opening an alternative strategy for developing sheath blight-resistant rice cultivars.

Proteomic and transcriptomic approaches to identify resistance and susceptibility related proteins in contrasting rice genotypes infected with fungal pathogen Rhizoctonia solani.

The devastating sheath blight disease caused by Rhizoctonia solani Kuhn (teleomorph: Thanatephorus cucumeris) causes major yield loss in most rice growing regions of the world. In this study, two moderately tolerant and four susceptible genotypes of rice were selected for R. solani induced proteome analysis using two-dimensional polyacrylamide gel electrophoresis. Forty five differentially expressed proteins (DEPs) were identified and analyzed by Mass Spectrometry. Based on their functions, these proteins were classified into different groups, viz., photosynthesis, resistance and pathogenesis, stress, cell wall metabolism and cytoskeleton development associated proteins, and hypothetical or uncharacterized proteins. Expression of 14 genes encoding DEPs was analyzed by quantitative PCR which showed consistency in transcripts and genes expression pattern. Furthermore, the expression of 16 other genes involved in diverse biological functions was analyzed. Up-regulation of these genes in the tolerant genotype Pankaj during sheath blight disease suggested efficient genetic regulation of this cultivar under stress. Also, expression analysis of conserved microRNAs (miRNAs) and their target genes revealed important role of miRNAs in post-transcriptional gene regulation during development of rice sheath blight disease. Genome-wide discovery of miRNAs and further characterization of DEPs and genes will help in better understanding of the molecular events during sheath blight disease development in rice.

Marker-assisted improvement of the elite restorer line of rice, RPHR-1005 for resistance against bacterial blight and blast diseases.

This study was carried out to improve the RPHR-1005, a stable restorer line of the popular medium slender grain type rice hybrid, DRRH-3 for bacterial blight (BB) and blast resistance through marker-assisted backcross breeding (MABB). Two major BB resistance genes, Xa21 and Xa33 and a major blast resistance gene, Pi2 were transferred to RPHR-1005 as two individual crosses. Foreground selection for Xa21, Xa33, Pi2, Rf3 and Rf4 was done by using gene-specific functional markers, while 59 simple sequence repeat (SSR) markers polymorphic between the donors and recipient parents were used to select the best plant possessing target resistance genes at each backcross generation. Backcrossing was continued till BC2F2 and a promising homozygous backcross derived line possessing Xa21+ Pi2 and another possessing Xa33 were intercrossed to stack the target resistance genes into the genetic background of RPHR-1005. At ICF4, 10 promising lines possessing three resistance genes in homozygous condition along with fine-grain type, complete fertility restoration, better panicle exertion and taller plant type (compared to RPHR-1005) were identified.

Development of Gene-Pyramid Lines of the Elite Restorer Line, RPHR-1005 Possessing Durable Bacterial Blight and Blast Resistance.

RPHR-1005, the stable restorer line of the popular medium slender (MS) grain type rice hybrid, DRRH-3 was improved in this study for resistance against bacterial blight (BB) and blast diseases through marker-assisted backcross breeding (MABB). In this study, four major resistance genes (i.e., Xa21 and Xa33 for BB resistance and Pi2 and Pi54 for blast resistance) have been transferred to RPHR-1005 using RPBio Patho-1 (possessing Xa21 + Pi2), RPBio Patho-2 (possessing Xa21 + Pi54) and FBR1-15EM (possessing Xa33) as the donors. Foreground selection was carried out using PCR-based molecular markers specific for the target resistance genes and the major fertility restorer genes, Rf3 and Rf4, while background selection was carried out using a set of parental polymorphic rice SSR markers and backcrossing was continued uptoBC2 generation. At BC2F2, plants possessing the gene combination- Xa21 + Pi2, Xa21 + Pi54 and Xa33 in homozygous condition and with >92% recovery of the recurrent parent genome (RPG) were identified and intercrossed to combine all the four resistance genes. Twenty-two homozygous, pyramid lines of RPHR-1005 comprising of three single-gene containing lines, six 2-gene containing lines, eight 3-gene containing lines, and five 4-gene containing lines were identified among the double intercross lines at F3 generation (DICF3). They were then evaluated for their resistance against BB and blast, fertility restoration ability and for key agro-morphological traits. While single gene containing lines were resistant to either BB or blast, the 2-gene, 3-gene, and 4-gene pyramid lines showed good level of resistance against both and/or either of the two diseases. Most of the 2-gene, 3-gene, and 4-gene containing pyramid lines showed yield levels and other key agro-morphological and grain quality traits comparable to the original recurrent parent and showed complete fertility restoration ability, with a few showing higher yield as compared to RPHR-1005. Further, the experimental hybrids derived by crossing the gene-pyramid lines of RPHR-1005 with APMS6A (the female parent of DRRH-3), showed heterosis levels equivalent to or higher than DRRH-3. The results of present study exemplify the utility of MABB for targeted improvement of multiple traits in hybrid rice.

Identification and fine-mapping of Xa33, a novel gene for resistance to Xanthomonas oryzae pv. oryzae.

Broadening of the genetic base for identification and transfer of genes for resistance to insect pests and diseases from wild relatives of rice is an important strategy in resistance breeding programs across the world. An accession of Oryza nivara, International Rice Germplasm Collection (IRGC) accession number 105710, was identified to exhibit high level and broad-spectrum resistance to Xanthomonas oryzae pv. oryzae. In order to study the genetics of resistance and to tag and map the resistance gene or genes present in IRGC 105710, it was crossed with the bacterial blight (BB)-susceptible varieties 'TN1' and 'Samba Mahsuri' (SM) and then backcrossed to generate backcross mapping populations. Analysis of these populations and their progeny testing revealed that a single dominant gene controls resistance in IRGC 105710. The BC(1)F(2) population derived from the cross IRGC 105710/TN1//TN1 was screened with a set of 72 polymorphic simple-sequence repeat (SSR) markers distributed across the rice genome and the resistance gene was coarse mapped on chromosome 7 between the SSR markers RM5711 and RM6728 at a genetic distance of 17.0 and 19.3 centimorgans (cM), respectively. After analysis involving 49 SSR markers located between the genomic interval spanned by RM5711 and RM6728, and BC(2)F(2) population consisting of 2,011 individuals derived from the cross IRGC 105710/TN1//TN1, the gene was fine mapped between two SSR markers (RMWR7.1 and RMWR7.6) located at a genetic distance of 0.9 and 1.2 cM, respectively, from the gene and flanking it. The linkage distances were validated in a BC(1)F(2) mapping population derived from the cross IRGC 105710/SM//2 × SM. The BB resistance gene present in the O. nivara accession was identified to be novel based on its unique map location on chromosome 7 and wider spectrum of BB resistance; this gene has been named Xa33. The genomic region between the two closely flanking SSR markers was in silico analyzed for putatively expressed candidate genes. In total, eight genes were identified in the region and a putative gene encoding serinethreonine kinase appears to be a candidate for the Xa33 gene.

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.