Genetic Enhancement for Biotic Stress Resistance in Basmati Rice through Marker-Assisted Backcross Breeding.

Pusa Basmati 1509 (PB1509) is one of the major foreign-exchange-earning varieties of Basmati rice; it is semi-dwarf and early maturing with exceptional cooking quality and strong aroma. However, it is highly susceptible to various biotic stresses including bacterial blight and blast. Therefore, bacterial blight resistance genes, namely, xa13 + Xa21 and Xa38, and fungal blast resistance genes Pi9 + Pib and Pita were incorporated into the genetic background of recurrent parent (RP) PB1509 using donor parents, namely, Pusa Basmati 1718 (PB1718), Pusa 1927 (P1927), Pusa 1929 (P1929) and Tetep, respectively. Foreground selection was carried out with respective gene-linked markers, stringent phenotypic selection for recurrent parent phenotype, early generation background selection with Simple sequence repeat (SSR) markers, and background analysis at advanced generations with Rice Pan Genome Array comprising 80K SNPs. This has led to the development of Near isogenic lines (NILs), namely, Pusa 3037, Pusa 3054, Pusa 3060 and Pusa 3066 carrying genes xa13 + Xa21, Xa38, Pi9 + Pib and Pita with genomic similarity of 98.25%, 98.92%, 97.38% and 97.69%, respectively, as compared to the RP. Based on GGE-biplot analysis, Pusa 3037-1-44-3-164-20-249-2 carrying xa13 + Xa21, Pusa 3054-2-47-7-166-24-261-3 carrying Xa38, Pusa 3060-3-55-17-157-4-124-1 carrying Pi9 + Pib, and Pusa 3066-4-56-20-159-8-174-1 carrying Pita were identified to be relatively stable and better-performing individuals in the tested environments. Intercrossing between the best BC3F1s has led to the generation of Pusa 3122 (xa13 + Xa21 + Xa38), Pusa 3124 (Xa38 + Pi9 + Pib) and Pusa 3123 (Pi9 + Pib + Pita) with agronomy, grain and cooking quality parameters at par with PB1509. Cultivation of such improved varieties will help farmers reduce the cost of cultivation with decreased pesticide use and improve productivity with ensured safety to consumers.

From farm to plate: Spatio-temporal characterization revealed compositional changes and reduced retention of γ-oryzanol upon processing in rice.

Background: Antioxidants detain the development and proliferation of various non-communicable diseases (NCDs). γ-oryzanol, a group of steryl ferulates and caffeates, is a major antioxidant present in rice grain with proven health benefits. The present study evaluated the distribution and dynamics of γ-oryzanol and its components in spatial and temporal scales and also delineated the effect of processing and cooking on its retention. Methods: Six rice varieties (four Basmati and two non-Basmati) belonging to indica group were analyzed at spatial scale in four different tissues (leaf blades, leaf sheaths, peduncle and spikelets) and temporal scale at three developmental stages (booting, milky and dough). Additionally, the matured grains were fractioned into husk, embryo, bran, and endosperm to assess differential accumulation in these tissues. Further, milling and cooking of the samples was done to assess the retention upon processing. After extraction of γ-oryzanol by solvent extraction method, individual components were identified by UPLC-QToF-ESI-MS and quantified by RP-HPLC. Results: The non-seed tissues were significantly different from the seed tissues for composition and quantitative variation of γ-oryzanol. Cycloartenyl caffeate was predominant in all the non-seed tissues during the three developmental stages while it showed significant reduction during the growth progression toward maturity and was totally absent in the matured grains. In contrary, the 24-methylenecycloartanyl ferulate, campesteryl ferulate and ß-sitosteryl ferulate showed significant increment toward the growth progression to maturity. Milling caused significant reduction, retaining only an average of 58.77% γ-oryzanol. Cooking of brown rice in excess water showed relatively lower average retention (43.31%) to samples cooked in minimal water (54.42%). Cooked milled rice showed least mean retention of 21.66%. Conclusion: The results demonstrate prominent compositional variation of γ-oryzanol during different growth stages. For the first time, the study demonstrated that ferulate esters of γ-oryzanol were predominant in the seed tissues while caffeate esters were dominant in non-seed tissues. Basmati cultivars show differential expression of γ-oryzanol and its components compared to non-Basmati cultivars. Cooking in excess water causes maximum degradation of γ-oryzanol. Post-harvest losses due to milling and cooking indicate the necessity of biofortification for γ-oryzanol content in rice grain.

Genome-wide association study reveals novel genomic regions governing agronomic and grain quality traits and superior allelic combinations for Basmati rice improvement.

Background: Basmati is a speciality segment in the rice genepool characterised by explicit grain quality. For the want of suitable populations, genome-wide association study (GWAS) in Basmati rice has not been attempted. Materials: To address this gap, we have performed a GWAS on a panel of 172 elite Basmati multiparent population comprising of potential restorers and maintainers. Phenotypic data was generated for various agronomic and grain quality traits across seven different environments during two consecutive crop seasons. Based on the observed phenotypic variation, three agronomic traits namely, days to fifty per cent flowering, plant height and panicle length, and three grain quality traits namely, kernel length before cooking, length breadth ratio and kernel length after cooking were subjected to GWAS. Genotyped with 80K SNP array, the population was subjected to principal component analysis to stratify the underlying substructure and subjected to the association analysis using Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway (BLINK) model. Results: We identified 32 unique MTAs including 11 robust MTAs for the agronomic traits and 25 unique MTAs including two robust MTAs for the grain quality traits. Six out of 13 robust MTAs were novel. By genome annotation, six candidate genes associated with the robust MTAs were identified. Further analysis of the allelic combinations of the robust MTAs enabled the identification of superior allelic combinations in the population. This information was utilized in selecting 77 elite Basmati rice genotypes from the panel. Conclusion: This is the first ever GWAS study in Basmati rice which could generate valuable information usable for further breeding through marker assisted selection, including enhancing of heterosis.

Population Structure of a Worldwide Collection of Tropical Japonica Rice Indicates Limited Geographic Differentiation and Shows Promising Genetic Variability Associated with New Plant Type.

Abating the approaching yield plateau in rice requires taking advantage of potential technologies that requires knowledge on genetic diversity. Hybrid breeding, particularly in indica rice, requires the recruitment of large genetic variability from outside because the available genetic diversity of the cultivated pool has already been utilized to a great extent. In this study, we examined an assembly of 200 tropical japonica lines collected worldwide for population genetic structure and variability in yield-associated traits. Tested along with 30 indica and six wild rice lines belonging to India, the tropical japonica lines indicated great phenotypic variability, particularly related to new plant type (NPT) phenology, and formed six clusters. Furthermore, a marker-based characterization using a universal diversity marker panel classified the genotype assembly into four clusters, of which three encompassed tropical japonica lines, while the last cluster included mostly indica lines. The population structure of the panel also revealed a similar pattern, with tropical japonica lines forming three subpopulations. Remarkable variation in the allelic distribution was observed between the subpopulations. Superimposing the geographical sources of the genotypes over the population structure did not reveal any pattern. The genotypes sourced closer to the center of origin of rice showed relatively little diversity compared with the ones obtained from other parts of the world, suggesting migration from a common region of origin. The tropical japonica lines can be a great source of parental diversification for hybrid development after confirming the presence of widely compatible genes.

Population Dynamics of Wide Compatibility System and Evaluation of Intersubspecific Hybrids by indica-japonica Hybridization in Rice.

The exploitation of heterosis through intersubspecific hybridisation between indica and japonica has been a major breeding target in rice, but is marred by the cross incompatibility between the genomes. Wide compatibility (WC) is a triallelic system at the S5 locus on chromosome 6 that ensures the specificity of hybridisation within and between indica and japonica. The S5n allele that favours intercrossing is sparsely distributed in the rice gene pool and therefore warrants identification of diverse WC sources to develop superior intersubspecific hybrids. In this study, we have identified several novel WC sources through the marker-assisted screening of a large set of 950 rice genotypes. Seventeen percent of the genotypes carried S5n, which fell into two subpopulations. The WC genotypes showed wide phenotypic and genotypic variability, including both indica and japonica lines. Based on phenotypic performance, the WC varieties were grouped into three clusters. A subset of 41 WC varieties was used to develop 164 hybrids, of which WC/japonica hybrids showed relative superiority over WC/indica hybrids. The multilocation evaluation of hybrids indicated that hybrids derived from WC varieties, such as IRG137, IRG143, OYR128, and IRGC10658, were higher yielding across all the three different locations. Most of the hybrids showed the stability of performance across locations. The identified diverse set of wide compatible varieties (WCVs) can be used in the development of intersubspecific hybrids and also for parental line development in hybrid rice breeding.

Molecular Breeding for Improving Productivity of Oryza sativa L. cv. Pusa 44 under Reproductive Stage Drought Stress through Introgression of a Major QTL, qDTY12.1.

Increasing rice production is quintessential to the task of sustaining global food security, as a majority of the global population is dependent on rice as its staple dietary cereal. Among the various constraints affecting rice production, reproductive stage drought stress (RSDS) is a major challenge, due to its direct impact on grain yield. Several quantitative trait loci (QTLs) conferring RSDS tolerance have been identified in rice, and qDTY12.1 is one of the major QTLs reported. We report the successful introgression of qDTY12.1 into Pusa 44, a drought sensitive mega rice variety of the northwestern Indian plains. Marker-assisted backcross breeding (MABB) was adopted to transfer qDTY12.1 into Pusa 44 in three backcrosses followed by four generations of pedigree selection, leading to development of improved near isogenic lines (NILs). Having a recurrent parent genome (RPG) recovery ranging from 94.7-98.7%, the improved NILs performed 6.5 times better than Pusa 44 under RSDS, coupled with high yield under normal irrigated conditions. The MABB program has been modified so as to defer background selection until BC3F4 to accelerate generational advancements. Deploying phenotypic selection alone in the early backcross generations could help in the successful recovery of RPG. In addition, the grain quality could be recovered in the improved NILs, leading to superior selections. Owing to their improved adaptation to drought, the release of improved NILs for regions prone to intermittent drought can help enhance rice productivity and production.

Genome-Wide Association Study Reveals Marker-Trait Associations for Early Vegetative Stage Salinity Tolerance in Rice.

Rice germplasm is a rich resource for discovering genes associated with salt tolerance. In the current study, a set of 96 accessions were evaluated for seedling stage salinity tolerance and its component traits. Significant phenotypic variation was observed among the genotypes for all the measured traits and eleven accessions with high level of salt tolerance at seedling stage were identified. The germplasm set comprised of three sub-populations and genome-wide association study (GWAS) identified a total of 23 marker-trait associations (MTAs) for traits studied. These MTAs were located on rice chromosomes 1, 2, 5, 6, 7, 9, and 12 and explained the trait phenotypic variances ranging from 13.98 to 29.88 %. Twenty-one MTAs identified in this study were located either in or near the previously reported quantitative trait loci (QTLs), while two MTAs namely, qSDW2.1 and qSNC5 were novel. A total of 18 and 13 putative annotated candidate genes were identified in a genomic region spanning ~200 kb around the MTAs qSDW2.1 and qSNC5, respectively. Some of the important genes underlying the novel MTAs were OsFBA1,OsFBL7, and mTERF which are known to be associated with salinity tolerance in crops. These MTAs pave way for combining salinity tolerance with high yield in rice genotypes through molecular breeding.

Drought Tolerant Near Isogenic Lines of Pusa 44 Pyramided With qDTY2.1 and qDTY3.1, Show Accelerated Recovery Response in a High Throughput Phenomics Based Phenotyping.

Reproductive stage drought stress (RSDS) is a major challenge in rice production worldwide. Cultivar development with drought tolerance has been slow due to the lack of precise high throughput phenotyping tools to quantify drought stress-induced effects. Most of the available techniques are based on destructive sampling and do not assess the progress of the plant's response to drought. In this study, we have used state-of-the-art image-based phenotyping in a phenomics platform that offers a controlled environment, non-invasive phenotyping, high accuracy, speed, and continuity. In rice, several quantitative trait loci (QTLs) which govern grain yield under drought determine RSDS tolerance. Among these, qDTY2.1 and qDTY3.1 were used for marker-assisted breeding. A set of 35 near-isogenic lines (NILs), introgressed with these QTLs in the popular variety, Pusa 44 were used to assess the efficiency of image-based phenotyping for RSDS tolerance. NILs offered the most reliable contrast since they differed from Pusa 44 only for the QTLs. Four traits, namely, the projected shoot area (PSA), water use (WU), transpiration rate (TR), and red-green-blue (RGB) and near-infrared (NIR) values were used. Differential temporal responses could be seen under drought, but not under unstressed conditions. NILs showed significant level of RSDS tolerance as compared to Pusa 44. Among the traits, PSA showed strong association with yield (80%) as well as with two drought tolerances indices, stress susceptibility index (SSI) and tolerance index (TOL), establishing its ability in identifying the best drought tolerant NILs. The results revealed that the introgression of QTLs helped minimize the mean WU per unit of biomass per day, suggesting the potential role of these QTLs in improving WU-efficiency (WUE). We identified 11 NILs based on phenomics traits as well as performance under imposed drought in the field. The study emphasizes the use of phenomics traits as selection criteria for RSDS tolerance at an early stage, and is the first report of using phenomics parameters in RSDS selection in rice.

Genetic Analysis and Molecular Mapping of the Quantitative Trait Loci Governing Low Phytic Acid Content in a Novel LPA Rice Mutant, PLM11.

Breeding rice varieties with a low phytic acid (LPA) content is an effective strategy to overcome micronutrient deficiency in a population which consume rice as a staple food. An LPA mutant, Pusa LPA Mutant 11 (PLM11), was identified from an ethyl methane sulfonate (EMS)-induced population of Nagina 22. The present study was carried out to map the loci governing the LPA trait in PLM11 using an F2:3 population derived from a cross between a high phytic acid rice variety, Pusa Basmati 6, with PLM11. The genotyping of the F2 population with 78 polymorphic SSR markers followed by the estimation of phytic acid content in the seeds harvested from 176 F2 plants helped in mapping a major QTL, qLPA8.1, explaining a 22.2% phenotypic variation on Chromosome 8. The QTL was delimited to a 1.96 cM region flanked by the markers RM25 and RM22832. Since there are no previous reports of a QTL/gene governing the LPA content in rice in this region, the QTL qLPA8.1 is a novel QTL. In silico analysis based on the annotated physical map of rice suggested the possible involvement of a locus, Os08g0274775, encoding for a protein similar to a phosphatidylinositol 3- and 4-kinase family member. This needs further validation and fine mapping. Since this QTL is currently specific to PLM11, the linked markers can be utilized for the development of rice varieties with reduced phytic acid (PA) content using PLM11 as the donor, thus enhancing the bioavailability of mineral micronutrients in humans.

Discovery of a Novel Induced Polymorphism in SD1 Gene Governing Semi-Dwarfism in Rice and Development of a Functional Marker for Marker-Assisted Selection.

The semi-dwarfing allele, sd1-d, has been widely utilized in developing high-yielding rice cultivars across the world. Originally identified from the rice cultivar Dee-Geo-Woo-Gen (DGWG), sd1-d, derived from a spontaneous mutation, has a 383-bp deletion in the SD1 gene. To date, as many as seven alleles of the SD1 gene have been identified and used in rice improvement, either with a functional single-nucleotide polymorphism (SNP), with insertion-deletions (InDels), or both. Here, we report discovery of a novel SNP in the SD1 gene from the rice genotype, Pusa 1652. Genetic analysis revealed that the inheritance of the semi-dwarfism in Pusa 1652 is monogenic and recessive, but it did not carry the sd1-d allele. However, response to exogenous gibberellic acid (GA3) application and the subsequent bulked segregant and linkage analyses confirmed that the SD1 gene is involved in the plant height reduction in Pusa 1652. Sequencing of the SD1 gene from Pusa 1652 revealed a novel transition in exon 3 (T/A) causing a nonsense mutation at the 300th codon. The stop codon leads to premature termination, resulting in a truncated protein of OsGA20ox2 obstructing the GA3 biosynthesis pathway. This novel recessive allele, named sd1-bm, is derived from Bindli Mutant 34 (BM34), a γ-ray induced mutant of a short-grain aromatic landrace, Bindli. BM34 is the parent of an aromatic semi-dwarf cultivar, Pusa 1176, from which Pusa 1652 is derived. The semi-dwarfing allele, sd1-bm, was further validated by developing a derived cleaved amplified polymorphic sequence (dCAPS) marker, AKS-sd1. This allele provides an alternative to the most widely used sd1-d in rice improvement programs and the functional dCAPS marker will facilitate marker-assisted introgression of the semi-dwarf trait into tall genotypes.

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.