## 4 Materials and Methods
### QTL Analysis  
We extracted DNA from leaf samples using the CTAB method and assessed its quality and quantity using a UV spectrophotometer at both 260 nm/280 nm and 260 nm/230 nm. Genomic DNA was isolated from 188 fresh leaf samples using the CTAB method, and the F\({}_{2}\) population was genotyped. We used a working DNA concentration of 40 ng/\(\upmu\)L for genotyping. A set of 1280 markers, comprising 1220 SSRs and 60 InDels, evenly distributed across the rice genome were used to study parental polymorphism. The SSR and InDel markers which have been previously reported in the universal core genetic map for rice [75], the genome-wide InDel marker set for allele discrimination between rice (_Oryza sativa_), and the other seven AA-genome _Oryzaa_ species [76], respectively, were used for the polymorphism study between parents. Some SSR markers for the rice genome were retrieved from the Gramene Marker Database ([[https://gramene.org/archive](https://gramene.org/archive)]([https://gramene.org/archive](https://gramene.org/archive)) (accessed on 27 December 2022)). Markers were selected from all 12 chromosomes of rice (_Oryza sativa_ L.) to ensure comprehensive genome-wide coverage.  
Among the 1280 markers, 103 were polymorphic. We screened the F\({}_{2}\) population using these 103 polymorphic markers. PCR was carried out in a Bio-Rad T100 TM Thermal Cycler (Bio-Rad, Hercules, CA, USA) with a reaction volume of 10 \(\upmu\)L, containing 15 ng of genomic DNA, primers at concentrations ranging from 5 to 10 pM, and Emerald Amp GT PCRMaster Mix (Takara Bio India, New Delhi, India). The details of PCR cycles are as follows: initial denaturation at 94 \({}^{\circ}\)C for 5 min, followed by 35 cycles of denaturation at 94 \({}^{\circ}\)C for 30 s, annealing at 55 \({}^{\circ}\)C for 30 s, extension at 72 \({}^{\circ}\)C for 1 min, and a final extension step at 72 \({}^{\circ}\)C for 10 min. Amplified products were separated using 3% agarose gel electrophoresis in 1 \(\times\) TAE buffer at 120 V for 2 h. Ethidium bromide staining was used to visualize bands and a gel documentation system was employed to document the results.  
SSRs and InDels were scored as co-dominant markers, with \"A\" representing the presence of the JAK-686 allele, \"B\" representing the presence of the BPT 5204 allele, and \"AB\" indicating heterozygous bands. A Chi-square test was conducted to assess the segregation ratio in the F\({}_{2}\) mapping population. We then constructed a linkage map using genotypic data from 188 F\({}_{2}\) plants and 103 polymorphic markers spanning all the rice chromosomes. We developed the genetic linkage map using ICIM and utilized Kosambi's mapping function to convert recombination frequencies into centiMorgan (cM). QTL mapping was carried out through inclusive composite interval mapping of additive QTLs (ICIM-ADD) and inclusive composite interval mapping of epistatic QTLs (ICIM-EPI), implemented in QTL IciMapping v4.1. We considered a QTL significant if it exceeded the logarithm of odds (LOD) threshold of 2.5, determined through a 1000-permutation test. Stepwise regression analysis was utilized to estimate the percentage of phenotypic variance explained (PVE or R\({}^{2}\)) by individual QTLs and to determine their additive effects at the LOD peak positions.