Combining ability and heat-stress response in rice hybrids derived from mutation-derived M₆ lines

Elevated temperatures are the main challenge to rice agriculture in Pakistan and around the world because rice production is highly susceptible to heat stress, demanding the development of genotypes that are phenotypically heat stress-responsive. High temperatures throughout the rice-growing season are becoming an increasing concern in Pakistan, especially in heat-prone areas where exposure during the reproductive stage can lower spikelet fertility, grain filling, and final yield. As a result, advanced mutant generations may provide valuable breeding material for determining phenotypic variation linked to yield stability and heat-stress response. This was achieved through the implementation of a lines x tester mating design using mutation-derived, advanced and morphologically uniform M₆ parental genotypes resulting in 32 F₁ hybrids from 12 parental genotypes, which were then evaluated in both normal and heat-stressed conditions. Through gamma-ray-induced mutagenesis at 150 Gy, the parental genotypes were developed from a Basmati-type fragrant rice background and progressed to the M₆ generation. However, limited information is reported regarding the application of mutation-derived M₆ rice accessions in line × tester analysis under field-induced reproductive-stage heat stress, especially when combining ability, heterosis, stress indices, and multivariate analysis are assessed together. Plant height, panicle length, number of tillers per plant, thousand grain weight, days to flowering, spikelet fertility, flag leaf area, number of grains per panicle, and harvest index were recorded. Furthermore, genotypes were assessed using principal component analysis (PCA), the Stress Tolerance Index (STI), Heat Tolerance Index (HTI), and the Stress Susceptibility Index (SSI). There is significant variation (p < 0.01) in yield-related traits between genotypes and their partition, including general combining ability (GCA) and specific combining ability (SCA), parents, crosses, and their interaction. In contrast to normal conditions, heat stress significantly reduced grain production and all traits in parents and hybrids. The phenotypic heat-stress response was further supported by targeted qRT-PCR analysis of selected HSP70 and HSFA2 gene family members in selected M₆ parental genotypes, which revealed differential regulation under heat-stress conditions. Additionally, for grain yield and all traits, the majority of parents and hybrids strongly prefer GCA and SCA effects respectively. Accessions 6511, 6543, and 7201 exhibit the best mean performances, stress tolerance index (STI), and desired GCA effects. These parents can be utilized for developing superior rice hybrids under heat stress, based on principal component analysis (PCA). For grain yield and majority traits under heat stress, the hybrid combinations 6511 × 7201, 6543 × 7218, 6561 × 7218, and 6696 × 7218 were found to be good specific combiners. Future breeding programs could make use of the identified genotypes and their traits to increase rice grain yield and heat-stress response.