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

Rice is a global staple cultivated in over 117 countries and provides more than one-fifth of human caloric intake. India accounts for about one-fourth of global rice production but faces significant yield losses due to biotic and abiotic stresses. Phosphorus (P) deficiency is a critical macronutrient limitation in rice soils; global P fertilizer demand is rising while reserves are finite, and India depends heavily on P fertilizer imports. Nearly all Indian soils are low to medium in available P, necessitating alternatives such as improved nutrient management and development of low soil P tolerant rice varieties. Considerable genetic variability for P-use efficiency exists in rice, and the major QTL Pup1 confers tolerance to low soil P across different genetic backgrounds. With Pup1 fine-mapped, cloned, and functional/linked markers available, the authors aimed to improve the elite Indian variety Improved Samba Mahsuri (ISM)—a high-yielding, fine-grain, low glycemic index, bacterial blight (BB) resistant variety that is highly sensitive to low soil P—by introgressing Pup1 through marker-assisted backcross breeding (MABB).

Previous studies have identified significant variation among rice genotypes for phosphorus uptake and use efficiency. The major QTL Pup1, which enhances P uptake and confers tolerance to P-deficient soils, has been mapped and fine-mapped, with closely linked and functional markers developed. Pup1 has been introgressed into several rice varieties (IR-64, IR-72, Dodokan, Batur, Situ) using marker-assisted backcrossing, improving performance under both upland and irrigated conditions. Pup1’s candidate gene OsPSTOL1, a protein kinase from the tolerant variety Kasalath, increases root growth and P acquisition, and is absent from most modern low-P-sensitive irrigated varieties and from Nipponbare. Earlier markers for Pup1 (e.g., K46-1, K46-2, and CAPS-based K20-1) were dominant or required restriction digestion, limiting routine deployment; nevertheless, they facilitated Pup1 introgression and validation. ISM is a near-isogenic derivative of Samba Mahsuri with pyramided BB resistance genes Xa21, xa13, xa5 via MABB, retaining high yield and quality but exhibiting strong sensitivity to low soil P. These findings motivated targeted Pup1 introgression into ISM using improved marker strategies and standard MABB components: foreground selection for the target locus, recombinant selection with flanking markers to reduce linkage drag, and background selection to accelerate recurrent parent genome recovery.

Plant materials: ISM (recurrent parent), a BB-resistant, fine-grain, low glycemic variety, and Swarna (MTU 7029) (donor parent carrying Pup1 and tolerant to low soil P) were used. Marker development: PCR products from CAPS marker K20-1 were sequenced from Vandana (Pup1+) and ISM (Pup1−). Multiple sequence alignment with Nipponbare and Kasalath reference sequences (chromosome 12) identified a 3-bp indel near the K20-1 reverse primer. Primers were designed (Primer3) to develop a new co-dominant marker K20-1-1 targeting this indel. K20-1-1 yields 89-bp amplicon in tolerant genotypes (Kasalath, Vandana) and 92-bp in sensitive genotypes (ISM), resolved on 8% native PAGE. Co-segregation was validated in an F2 population (n=135) from ISM × Swarna. MABB scheme: Cross ISM × Swarna; confirm true F1s using K20-1-1. Backcross true F1s to ISM to generate BC1F1. Foreground selection for Pup1 used K20-1-1 and Pup1 functional marker K46-1; recombinant selection used flanking SSRs RM28011 and RM1261 (~1.5 Mb flanking Pup1); BB resistance genes were monitored using pTA248 (Xa21), xa13prom (xa13), xa5FM (xa5). Background selection used 66 polymorphic SSRs across the genome to maximize recurrent parent genome recovery (RPG). The MABB cycle (foreground, recombinant, background selection) was repeated to BC2F1; a plant with recombination on both sides of Pup1, fixed BB resistance genes, and maximum RPG was selfed to BC2F2. BC2F2 plants were genotyped with K20-1-1 to identify Pup1 homozygotes, selfed to BC2F3. Phenotyping and evaluations: BC2F3 lines were evaluated at ICAR-IIRR, Hyderabad in low-P plots (Available P <2 kg ha−1, no P fertilizer) and normal-P plots (Available P >20–25 kg ha−1, recommended P). Traits recorded included days to 50% flowering, plant height, productive tillers, flag leaf length/width, panicle length, shoot length, root length, root volume (water displacement), dry shoot and root weights, 1000-grain weight, grain yield per plant, and grain type. BB resistance was assessed in normal-P plots using clip inoculation with Xanthomonas oryzae pv. oryzae (Xoo) isolate DX-020 (10^8–10^9 cfu/ml); lesion length and IRRI-SES scores were recorded 14 days post-inoculation. Grain and cooking quality were measured for four selected lines (milling %, head rice recovery, kernel dimensions, L/B ratio, VER, water uptake, cooked kernel length, elongation ratio, alkali spreading value, amylose content, gel consistency, grain type, chalkiness). A superior Pup1 ISM line (A-13-144-139) was nominated as IET 28061 and tested in AICRIP multi-location trials (Cuttack, Maruteru, Hyderabad) under 100% and 50% recommended P application. Standard PCR conditions and marker resolution protocols were followed (details provided, including PCR cycling for K20-1-1 and gel systems for markers). Data were analyzed using SAS 9.2.

- Marker development and validation: The new co-dominant marker K20-1-1 targets a 3-bp indel near Pup1; it amplifies 89 bp in tolerant (Kasalath, Vandana) and 92 bp in sensitive (ISM) genotypes and perfectly co-segregated with tolerance in an F2 population (n=135): all 103 tolerant plants carried the tolerant allele (homozygous/heterozygous), and all 32 sensitive plants carried the sensitive allele. - MABB progress and genome recovery: 35 F1 seeds obtained; 29 germinated; 20 true F1s identified by K20-1-1. In BC1F1 (n=130), 79 Pup1+ plants were identified by foreground selection; 11 recombinants detected with flanking markers (RM28011/RM1261). Two BC1F1 plants (#A-13, #A-54) were homozygous for all three BB genes; #A-13 had the highest recurrent parent genome recovery (RPG) of 78.03%. In BC2F1 (n=124), 63 Pup1+ plants were identified; 4 recombinants at RM28011 were selected; RPG among selected plants ranged from 84.85% to 89.23%; #A-13-144 (RPG 89.23%) was advanced. In BC2F2 (n=231), 76 plants were homozygous for Pup1; 12 best plants (fixed for Pup1 and BB genes, near-ISM phenotype) were advanced to BC2F3. - Performance under low P (Available P <2 kg ha−1): Pup1-introgressed BC2F3 lines were equivalent or superior to ISM for agro-morphological traits. Significant improvements over ISM were observed in productive tillers, flag leaf length, panicle length, root volume, dry root weight, dry shoot weight, 1000-grain weight, and grain yield per plant. No significant differences among lines were observed for plant height, flag leaf width, root length, and shoot length within the introgressed set. The entry A-13-144-139 performed best for grain yield under low P, with several other lines (A-13-144-192, -170, -53, -12, -55, -135) performing similarly. - Performance under normal P (>20–25 kg P ha−1): Pup1 lines were equivalent to ISM for days to 50% flowering, plant height, panicle length, shoot length, and 1000-grain weight, and showed equal or better root volume, dry root/shoot weights, productive tillers, and grain yield. - Bacterial blight resistance: All Pup1-introgressed lines and ISM showed resistant reactions to Xoo DX-020 (lesion length <1 cm; IRRI-SES score 1), while Samba Mahsuri (BPT 5204) was susceptible (lesion length 18.5 cm; SES score 9), confirming retention of Xa21, xa13, and xa5. - Grain and cooking quality: Four selected lines (A-13-144-53, -170, -139, -192) had medium-slender grains like ISM and comparable quality traits. Representative means across lines: milling ~66%, head rice recovery up to 55.8%, kernel length ~4.9–5.1 mm, L/B ~2.7–2.9, cooked kernel length ~8.4–8.8 mm, elongation ratio ~1.7–1.8, amylose ~22–24.5%, gel consistency ~22 mm, chalkiness absent. - Multi-location yield advantage (AICRIP, IET 28061): A-13-144-139 showed significant yield gains over ISM. At 100% recommended P: 28.05% (Cuttack), 18.39% (Maruteru), 1.80% (Hyderabad), mean +16.63%. At 50% P: 19.75% (Cuttack), 43.72% (Maruteru), 10.09% (Hyderabad), mean +30.19%. Performance was equivalent to Swarna under both P regimes.

The study demonstrates that targeted introgression of Pup1 into ISM via MABB can effectively overcome the variety’s sensitivity to low soil phosphorus while retaining its key attributes, including bacterial blight resistance conferred by Xa21, xa13, and xa5, and desirable grain quality. The new co-dominant marker K20-1-1 simplified and improved foreground selection for Pup1 by enabling direct discrimination of homozygous and heterozygous states without CAPS digestion, and showed perfect co-segregation with phenotype. The three-tiered MABB strategy—foreground selection for Pup1, recombinant selection using flanking SSRs (RM28011, RM1261) to minimize linkage drag, and background selection using genome-wide SSRs—accelerated recovery of the ISM genome to nearly 90% by BC2F1 and produced lines with minimal donor segments. Physiologically, Pup1 (via OsPSTOL1) enhances root system architecture (root length, surface area, and volume), enabling greater acquisition and utilization of P under deficiency and contributing to higher biomass and yield. Consistent with prior reports, Pup1 effects were also partially expressed under sufficient P, reflected in improved root traits and biomass even in normal-P plots, without compromising flowering time, plant height, or grain quality. Multi-location evaluation confirmed significant yield advantages under both full and reduced P fertilization, indicating potential for reducing P fertilizer inputs while maintaining or improving yields. The retention of strong BB resistance and grain quality ensures agronomic and market acceptability of the improved ISM lines.

Using marker-assisted backcross breeding, the authors successfully improved low soil phosphorus tolerance of the elite Indian rice variety Improved Samba Mahsuri by introgressing the major QTL Pup1 while retaining bacterial blight resistance, yield, and grain and cooking quality. The newly developed co-dominant marker K20-1-1 facilitates efficient selection for Pup1. Pup1-introgressed ISM lines exhibited superior performance under low P and equivalent or better performance under normal P. A top-performing line (A-13-144-139; IET 28061) showed significant multi-location yield advantages under both 100% and 50% recommended P, supporting the feasibility of reduced P fertilizer use and cultivation in P-deficient soils. These lines serve as valuable genetic stocks for improving other elite varieties for combined low P tolerance and BB resistance. Future work includes final-stage multi-location validation and potential varietal release, and broader deployment in breeding programs targeting nutrient-use efficiency and multi-stress resilience.