Identification and Characterization of Fusarium fujikuroi Pathotypes Responsible for an Emerging Bakanae Disease of Rice in India.

The bakanae disease of rice, or foolish seedling disease, is a well-known pathogen infecting rice hosts. Several studies have characterized Fusarium fujikuroi isolates collected from distant geographical regions and within similar geographical areas for secondary metabolite production, population structure, and diversity analysis, but none have attempted to characterize the isolates for virulence in a differential set of rice genotypes. Based on the disease response, a set of five rice genotypes with differing resistance levels were selected as a differential set for further characterization of the pathogen. Ninety-seven Fusarium fujikuroi isolates collected from different rice-growing areas of the country during the years 2011 to 2020 were characterized and evaluated for bakanae disease. Rice genotypes PB1509 and C101A51 were found to be highly susceptible and highly resistant, respectively. Further, based on the disease response, the isolates were grouped into 15 pathotypes. Pathotype 1, with the maximum isolates (19), was observed to be most prevalent, followed by pathotypes 2 and 3. Pathotype 8 was classified as highly virulent, as all the genotypes were susceptible, except for C101A51. When we compared the pathotype distribution in different states, pathotypes 11 and 15 were found to have originated from the state of Punjab. A positive correlation could be established between six pathotype groups and the gene expression of virulence-related genes such as acetylxylan (FFAC), exopolygalacturanase (FFEX), and pisatin demethylase (FFPD). The present study provides the distribution profiles of different pathotypes in Basmati-growing states of India, which will be further helpful for the deployment of breeding strategies and bakanae disease management.

Rice Pangenome Genotyping Array: an efficient genotyping solution for pangenome-based accelerated genetic improvement in rice.

The advent of the pangenome era has unraveled previously unknown genetic variation existing within diverse crop plants, including rice. This untapped genetic variation is believed to account for a major portion of phenotypic variation existing in crop plants. However, the use of conventional single reference-guided genotyping often fails to capture a large portion of this genetic variation leading to a reference bias. This makes it difficult to identify and utilize novel population/cultivar-specific genes for crop improvement. Thus, we developed a Rice Pangenome Genotyping Array (RPGA) harboring probes assaying 80K single-nucleotide polymorphisms (SNPs) and presence-absence variants spanning the entire 3K rice pangenome. This array provides a simple, user-friendly and cost-effective (60-80 USD per sample) solution for rapid pangenome-based genotyping in rice. The genome-wide association study (GWAS) conducted using RPGA-SNP genotyping data of a rice diversity panel detected a total of 42 loci, including previously known as well as novel genomic loci regulating grain size/weight traits in rice. Eight of these identified trait-associated loci (dispensable loci) could not be detected with conventional single reference genome-based GWAS. A WD repeat-containing PROTEIN 12 gene underlying one of such dispensable locus on chromosome 7 (qLWR7) along with other non-dispensable loci were subsequently detected using high-resolution quantitative trait loci mapping confirming authenticity of RPGA-led GWAS. This demonstrates the potential of RPGA-based genotyping to overcome reference bias. The application of RPGA-based genotyping for population structure analysis, hybridity testing, ultra-high-density genetic map construction and chromosome-level genome assembly, and marker-assisted selection was also demonstrated. A web application (http://www.rpgaweb.com) was further developed to provide an easy to use platform for the imputation of RPGA-based genotyping data using 3K rice reference panel and subsequent GWAS.

Molecular profiling of BADH2 locus reveals distinct functional allelic polymorphism associated with fragrance variation in Indian aromatic rice germplasm.

Allelic variability of the aroma gene, betaine aldehyde dehydrogenase 2 (BADH2) was studied in a random subset of indigenous aromatic rice germplasm along with a few exotic aromatic accessions. Use of functional markers of four badh2 alleles identified that the test panel possessed only two alleles, badh2-E7 and badh2-p-5'UTR. Two other alleles, badh2.2 and badh2-E4-5.2 were absent. Based on the alleles present, four functional polymorphisms (FP) were detected, namely FP1 to FP4. 188 genotypes possessed FP1 having both the badh2-p-5'UTR and badh2-E7 (71.8%) alleles. The badh2 allele with FP1 is named badh2-E7-p. 39 genotypes (14.9%) possessed only the badh2-p-5'UTR allele (FP3), while three genotypes were found to carry only the badh2-E7 allele (FP2). We also found that 32 genotypes (12.2%) did not have any of the target aroma alleles tested in this study (FP4). Interestingly, for badh2-p-5'UTR marker, the expected 198 bp amplicon for the non-aromatic allele could not be detected among any of the genotypes tested. Instead, an amplicon of 456 bp length appeared with 100% presence in the non-aromatic checks. Notwithstanding, the 456 bp allele also showed a 16% presence among the aromatic lines. This article forms the first report of this allele, named badh2-p1, among aromatic rice. Quantification of 2-Acetyl-1-Pyrroline (2AP) content and sensory evaluation among the test genotypes showed that those with FP1 are highly aromatic than the genotypes carrying other types of FPs. But, a few strongly aromatic lines showed lower 2AP content. The BADH2 characterization carried out in this study is suggestive of identifying the additional gene(s)/ allele(s) governing aroma among the Indian fragrant rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01181-9.

Marker Aided Incorporation of Saltol, a Major QTL Associated with Seedling Stage Salt Tolerance, into Oryza sativa 'Pusa Basmati 1121'.

Pusa Basmati 1121 (PB1121), an elite Basmati rice cultivar is vulnerable to salinity at seedling stage. A study was undertaken to impart seedling-stage salt tolerance into PB1121 by transferring a quantitative trait locus (QTL), Saltol, using FL478 as donor, through marker assisted backcrossing. Sequence tagged microsatellite site (STMS) marker RM 3412, tightly linked to Saltol was used for foreground selection. Background recovery was estimated using 90 genome-wide STMS markers. Systematic phenotypic selection helped in accelerated recovery of recurrent parent phenome (RPP). A set of 51 BC3F2 lines homozygous for Saltol were advanced to develop four improved near isogenic lines (NILs) of PB1121 with seedling stage salt tolerance. The background genome recovery in the NILs ranged from 93.3 to 99.4%. The improved NILs were either similar or better than the recurrent parent PB1121 for yield, grain and cooking quality and duration. Biochemical analyses revealed significant variation in shoot and root Na+ and K+ concentrations. Correlation between shoot and root Na+ concentration was stronger than that between root and shoot K+ concentration. The effect of QTL integration into the NILs was studied through expression profiling of OsHKT1;5, one of the genes present in the Saltol region. The NILs had significantly higher OsHKT1;5 expression than the recurrent parent PB1121, but lower than FL478 on salt exposure validating the successful introgression of Saltol in the NILs. This was also confirmed under agronomic evaluation, wherein the NILs showed greater salt tolerance at seedling stage. One of the NILs, Pusa1734-8-3-3 (NIL3) showed comparable yield and cooking quality to the recurrent parent PB1121, with high field level seedling stage salinity tolerance and shorter duration. This is the first report of successful introgression of Saltol into a Basmati rice cultivar.

Mapping quantitative trait loci responsible for resistance to Bakanae disease in rice.

BACKGROUND: Bakanae or foot rot disease caused by Fusarium fujikuroi [teleomorph: Gibberella fujikuroi (Sawada) Ito] is emerging as a serious disease in rice. The disease causes both quantitative and qualitative losses to the grains under the field conditions. Breeding for resistance to Bakanae disease is a promising strategy to manage this emerging disease. In this study, we used a population of 168 F14 recombinant inbred lines (RILs) derived from two indica rice parents Pusa 1342, a highly resistant variety and Pusa Basmati 1121, a highly susceptible variety to map quantitative trait loci (QTLs) governing resistance against Bakanae disease. RESULTS: The disease reaction of 168 F14 RILs were measured on the seedlings inoculated using Fusarium fujikuroi culture using high-throughput screening protocol under glasshouse conditions. Utilizing inclusive composite interval mapping, three QTLs governing resistance to Bakanae were identified, namely qBK1.1, qBK1.2 and qBK1.3 which accounted 4.76, 24.74 and 6.49 % of phenotypic variation, respectively. The major effect QTL designated qBK1.2 was mapped in 0.26 Mb region between RM5336 and RM10153. A total of 55 annotated genes were identified within the identified QTL region qBK1.2. CONCLUSIONS: The novel QTLs identified in this study are useful resource for efficiently breeding rice cultivars resistant to Bakanae disease. This is the first report on identification of QTLs governing resistance against Bakanae in rice using inclusive composite interval mapping strategy in a RIL population.

Marker-aided Incorporation of Xa38, a Novel Bacterial Blight Resistance Gene, in PB1121 and Comparison of its Resistance Spectrum with xa13 + Xa21.

Basmati rice is preferred internationally because of its appealing taste, mouth feel and aroma. Pusa Basmati 1121 (PB1121) is a widely grown variety known for its excellent grain and cooking quality in the international and domestic market. It contributes approximately USD 3 billion to India's forex earning annually by being the most traded variety. However, PB1121 is highly susceptible to bacterial blight (BB) disease. A novel BB resistance gene Xa38 was incorporated in PB1121 from donor parent PR114-Xa38 using a modified marker-assisted backcross breeding (MABB) scheme. Phenotypic selection prior to background selection was instrumental in identifying the novel recombinants with maximum recovery of recurrent parent phenome. The strategy was effective in delimiting the linkage drag to <0.5 mb upstream and <1.9 mb downstream of Xa38 with recurrent parent genome recovery upto 96.9% in the developed NILs. The NILs of PB1121 carrying Xa38 were compared with PB1121 NILs carrying xa13 + Xa21 (developed earlier in our lab) for their resistance to BB. Both NILs showed resistance against the Xoo races 1, 2, 3 and 6. Additionally, Xa38 also resisted Xoo race 5 to which xa13 + Xa21 was susceptible. The PB1121 NILs carrying Xa38 gene will provide effective control of BB in the Basmati growing region.

Improvement of Basmati rice varieties for resistance to blast and bacterial blight diseases using marker assisted backcross breeding.

Marker assisted backcross breeding was employed to incorporate the blast resistance genes, Pi2 and Pi54 and bacterial blight (BB) resistance genes xa13 and Xa21 into the genetic background of Pusa Basmati 1121 (PB1121) and Pusa Basmati 6. Foreground selection for target gene(s) was followed by arduous phenotypic and background selection which fast-tracked the recovery of recurrent parent genome (RPG) to an extent of 95.8% in one of the near-isogenic lines (NILs) namely, Pusa 1728-23-33-31-56, which also showed high degree of resemblance to recurrent parent, PB6 in phenotype. The phenotypic selection prior to background selection provided an additional opportunity for identifying the novel recombinants viz., Pusa 1884-9-12-14 and Pusa 1884-3-9-175, superior to parental lines in terms of early maturity, higher yield and improved quality parameters. There was no significant difference between the RPG recovery estimated based on SSR or SNP markers, however, the panel of SNPs markers was considered as the better choice for background selection as it provided better genome coverage and included SNPs in the genic regions. Multi-location evaluation of NILs depicted their stable and high mean performance in comparison to the respective recurrent parents. The Pi2+Pi54 carrying NILs were effective in combating a pan-India panel of Magnaporthe oryzae isolates with high level of field resistance in northern, eastern and southern parts of India. Alongside, the PB1121-NILs and PB6-NILs carrying BB resistance genes xa13+Xa21 were resistant against Xanthomonas oryzae pv. oryzae races of north-western, southern and eastern parts of the country. Three of NILs developed in this study, have been promoted to final stage of testing during the ​Kharif 2015 in the Indian National Basmati Trial.

Development and evaluation of near-isogenic lines for major blast resistance gene(s) in Basmati rice.

KEY MESSAGE: A set of NILs carrying major blast resistance genes in a Basmati rice variety has been developed. Also, the efficacy of pyramids over monogenic NILs against rice blast pathogen Magnaporthe oryzae has been demonstrated. Productivity and quality of Basmati rice is severely affected by rice blast disease. Major genes and QTLs conferring resistance to blast have been reported only in non-Basmati rice germplasm. Here, we report incorporation of seven blast resistance genes from the donor lines DHMASQ164-2a (Pi54, Pi1, Pita), IRBLz5-CA (Pi2), IRBLb-B (Pib), IRBL5-M (Pi5) and IRBL9-W (Pi9) into the genetic background of an elite Basmati rice variety Pusa Basmati 1 (PB1). A total of 36 near-isogenic lines (NILs) comprising of 14 monogenic, 16 two-gene pyramids and six three-gene pyramids were developed through marker-assisted backcross breeding (MABB). Foreground, recombinant and background selection was used to identify the plants with target gene(s), minimize the linkage drag and increase the recurrent parent genome (RPG) recovery (93.5-98.6 %), respectively, in the NILs. Comparative analysis performed using 50,051 SNPs and 500 SSR markers revealed that the SNPs provided better insight into the RPG recovery. Most of the monogenic NILs showed comparable performance in yield and quality, concomitantly, Pusa1637-18-7-6-20 (Pi9), was significantly superior in yield and stable across four different environments as compared to recurrent parent (RP) PB1. Further, among the pyramids, Pusa1930-12-6 (Pi2+Pi5) showed significantly higher yield and Pusa1633-7-8-53-6-8 (Pi54+Pi1+Pita) was superior in cooking quality as compared to RP PB1. The NILs carrying gene Pi9 were found to be the most effective against the concoction of virulent races predominant in the hotspot locations for blast disease. Conversely, when analyzed under artificial inoculation, three-gene pyramids expressed enhanced resistance as compared to the two-gene and monogenic NILs.

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

BACKGROUND AND AIMS: Basmati rice grown in the Indian subcontinent is highly valued for its unique culinary qualities. Production is, however, often constrained by diseases such as bacterial blight (BB), blast and sheath blight (ShB). The present study developed Basmati rice with inbuilt resistance to BB, blast and ShB using molecular marker-assisted selection. METHODOLOGY: The rice cultivar 'Improved Pusa Basmati 1' (carrying the BB resistance genes xa13 and Xa21) was used as the recurrent parent and cultivar 'Tetep' (carrying the blast resistance gene Pi54 and ShB resistance quality trait loci (QTL), qSBR11-1) was the donor. Marker-assisted foreground selection was employed to identify plants possessing resistance alleles in the segregating generations along with stringent phenotypic selection for faster recovery of the recurrent parent genome (RPG) and phenome (RPP). Background analysis with molecular markers was used to estimate the recovery of RPG in improved lines. PRINCIPAL RESULTS: Foreground selection coupled with stringent phenotypic selection identified plants homozygous for xa13, Xa21 and Pi54, which were advanced to BC(2)F(5) through pedigree selection. Marker-assisted selection for qSBR11-1 in BC(2)F(5) using flanking markers identified seven homozygous families. Background analysis revealed that RPG recovery was up to 89.5%. Screening with highly virulent isolates of BB, blast and ShB showed that the improved lines were resistant to all three diseases and were on a par with 'Improved Pusa Basmati 1' for yield, duration and Basmati grain quality. CONCLUSIONS: This is the first report of marker-assisted transfer of genes conferring resistance to three different diseases in rice wherein genes xa13 and Xa21 for BB resistance, Pi54 for blast resistance, and a major QTL qSBR11-1 have been combined through marker-assisted backcross breeding. In addition to offering the potential for release as cultivars, the pyramided lines will serve as useful donors of gene(s) for BB, blast and ShB in future Basmati rice breeding programmes.