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

Rice, a crucial staple food crop globally, is cultivated in over 117 countries, providing over one-fifth of the world's calories for more than 3 billion people. India, with the world's largest rice-cultivating area (43.39 million hectares), produces one-fourth (22%, or 104.3 million tonnes) of the world's rice, with an average productivity of 2.40 tonnes ha^-1. However, rice production is significantly impacted by numerous biotic and abiotic stresses, including drought, salinity, and nutrient deficiencies. Phosphorus (P) deficiency is a major yield-limiting factor, as P is a vital macronutrient for rice growth and development. The global demand for P fertilizers is steadily increasing, while global phosphate reserves are projected to be depleted within decades, with no readily available substitutes. In India, a significant portion of soils (49.3%) are low in available P, leading to high dependency on imported phosphorus-based fertilizers (90%). This necessitates exploring alternative solutions like improved crop residue management, integrated nutrient management, and the development of low soil P tolerant rice varieties. Genetic improvement of rice's tolerance to P-limiting soils is a key research focus to reduce P fertilizer application, lowering costs for farmers and ensuring sustainable rice production. Significant variability exists in P use efficiency and low soil P tolerance among rice genotypes. The identification of the *Pup1* QTL, which confers tolerance to low soil P, and the successful performance of *Pup1*-containing rice lines in diverse genetic backgrounds, both upland and irrigated, provides an opportunity to improve rice cultivars for low soil P conditions. The *Pup1* QTL has been fine-mapped, and closely linked markers have been developed, enabling its introgression into several rice varieties through marker-assisted backcross breeding (MABB). The cloning of *Pup1* and identification of the underlying gene, *OsPSTOL*, further facilitates genetic improvement. Given the known effectiveness of *Pup1* in improving low soil P tolerance in both irrigated and upland rice and the availability of closely linked and functional markers, this research aimed to genetically enhance the low P tolerance of the elite Indian rice variety Improved Samba Mahsuri (ISM) using MABB.

Previous research has highlighted the significant variability in phosphorus use efficiency among rice genotypes. Studies have identified the *Pup1* QTL as a major contributor to low soil phosphorus tolerance, demonstrating the improved performance of rice lines possessing this QTL in various environments. The *Pup1* QTL has been fine-mapped, leading to the development of closely linked markers that facilitate marker-assisted backcross breeding (MABB) for introgression of *Pup1* into other rice varieties. Furthermore, the cloning of *Pup1* and the identification of the underlying candidate gene, *OsPSTOL1*, have provided valuable insights into the genetic basis of low soil phosphorus tolerance. This knowledge has enabled the development of effective molecular markers for selecting lines with enhanced tolerance to phosphorus deficiency. Past MABB studies have successfully introgressed *Pup1* into several rice varieties, demonstrating the feasibility and effectiveness of this approach.

This study utilized marker-assisted backcross breeding (MABB) to introgress the *Pup1* QTL, responsible for low soil phosphorus tolerance, into the Improved Samba Mahsuri (ISM) rice variety. A new co-dominant marker, K20-1-1, was developed by identifying a 3-bp indel polymorphism between low P-tolerant and sensitive genotypes. This marker was highly specific for *Pup1* and showed perfect co-segregation with the trait phenotype in an F2 population. The MABB process involved several key steps: 1. **Hybridization:** Improved Samba Mahsuri (ISM, recipient parent) was crossed with Swarna (donor parent, possessing *Pup1*) in Rabi 2012-13. 2. **F1 Confirmation:** The resulting F1 progeny were genotyped using the co-dominant marker K20-1-1 to confirm hybridity. 3. **Backcrossing:** True F1 plants were backcrossed with ISM to produce BC1F1 generation. 4. **Foreground Selection:** BC1F1 plants were screened for *Pup1* using K20-1-1 and the functional marker K46-1. Plants positive for *Pup1* were also screened for the bacterial blight resistance genes (Xa21, xa13, xa5) using respective functional markers (pTA248, xa13prom, xa5FM). 5. **Recombinant Selection:** SSR markers (RM28011 and RM1261) flanking *Pup1* were used to select plants with recombination events, minimizing linkage drag. 6. **Background Selection:** 66 polymorphic SSR markers were employed to select plants with maximum recurrent parent (ISM) genome recovery. 7. **BC2F1 Generation:** Selected BC1F1 plants were backcrossed with ISM to produce BC2F1. 8. **Repeating Selection Process:** Steps 4-6 were repeated in the BC2F1 generation. 9. **BC2F2 Generation:** Selected BC2F1 plants were selfed to create BC2F2 and screened using K20-1-1 for *Pup1* homozygosity. 10. **BC2F3 Evaluation:** The best 12 BC2F2 plants, homozygous for *Pup1* and the bacterial blight resistance genes, were selfed to produce BC2F3 lines, which were evaluated for low P tolerance, bacterial blight resistance, and key agronomic traits under both low and normal soil phosphorus conditions. 11. **Multi-location Trials:** A superior line (A-13-144-139, IET 28061) was further evaluated in multi-location trials under different phosphorus application levels. DNA extraction utilized the Miniprep protocol, and PCR protocols were adapted from existing literature for the various markers used in the study. Data were analyzed using SAS 9.2 software.

A new co-dominant marker, K20-1-1, specific for the *Pup1* locus, was successfully developed and utilized in MABB. The MABB strategy effectively introgressed the *Pup1* QTL into the ISM rice variety while retaining bacterial blight resistance. Under low soil phosphorus conditions, *Pup1*-introgressed ISM lines exhibited significantly improved performance compared to the original ISM variety. These lines showed enhanced root system development, increased grain yield, and maintained similar or superior agronomic traits. Under normal soil P conditions, the introgressed lines performed comparably to or better than ISM. The superior line, A-13-144-139 (IET 28061), demonstrated significant yield advantages (16% and 30% under 100% and 50% recommended P application, respectively) compared to ISM in multi-location trials. All *Pup1* introgressed lines maintained bacterial blight resistance. The introgressed lines also retained desirable grain and cooking quality parameters, similar to ISM. Specific findings from the low-P plot evaluation (Table 3) show statistically significant differences for days to 50% flowering, number of productive tillers, flag leaf length, panicle length, root volume, dry root weight, dry shoot weight, thousand grain weight, and grain yield per plant, with *Pup1* introgressed lines exhibiting improved performance. Notably, no significant differences were found between *Pup1* introgressed lines and ISM for plant height, flag leaf width, root length, and shoot length. Under normal P conditions, the *Pup1* introgressed lines performed comparably to or better than ISM across various traits (Supplementary Table 1). Bacterial blight resistance was maintained in the *Pup1* introgressed lines, with similar levels of resistance to ISM, while Samba Mahsuri showed susceptibility (Supplementary Table 2). Grain and cooking quality analysis indicated that *Pup1* introgression did not negatively affect these traits (Table 4). Multi-location trial results (Table 5) demonstrated that line IET 28061 consistently outperformed ISM, showing significant yield improvements.

The successful introgression of *Pup1* into ISM using MABB demonstrates the feasibility of enhancing low soil P tolerance in elite rice varieties while maintaining other desirable traits. The development of a new, highly specific co-dominant marker (K20-1-1) significantly improved the efficiency of the MABB process, overcoming limitations of previously used markers. The substantial yield improvements observed under both low and normal phosphorus conditions highlight the potential of *Pup1* to enhance rice productivity. The retention of bacterial blight resistance underscores the value of combining multiple stress tolerances in a single variety. The consistent performance of line IET 28061 across multiple locations and phosphorus application levels validates its potential for widespread adoption. This research supports the development of resource-efficient rice varieties for sustainable agriculture, reducing reliance on phosphorus fertilizers and minimizing environmental impact.

This study successfully utilized marker-assisted backcross breeding to improve the low soil phosphorus tolerance of the elite Indian rice variety ISM while preserving its yield, grain quality, and bacterial blight resistance. The superior line IET 28061 demonstrates significant yield improvements across multiple locations and phosphorus application levels, showcasing the potential to reduce P fertilizer application in diverse environments. Future research could focus on exploring the broader genetic architecture of low soil P tolerance, dissecting the complex interactions between *Pup1* and other QTLs, and further optimizing breeding strategies for developing superior rice varieties adapted to various environmental conditions.

The study's sample size for some stages of the MABB process might have limited the detection of rare recombination events. Further research with larger populations could provide more detailed insights into the genetic architecture of low phosphorus tolerance. While multi-location trials provided strong evidence of the superior line's performance, long-term evaluation is crucial for validating its stability across various seasons and locations.